Safety and immunogenicity of booster vaccination and fractional dosing with Ad26.COV2.S or BNT162b2 in Ad26.COV2.S-vaccinated participants.

We report the safety and immunogenicity of fractional and full dose Ad26.COV2.S and BNT162b2 in an open label phase 2 trial of participants previously vaccinated with a single dose of Ad26.COV2.S, with 91.4% showing evidence of previous SARS-CoV-2 infection. A total of 286 adults (with or without HIV) were enrolled >4 months after an Ad26.COV2.S prime and randomized 1:1:1:1 to receive either a full or half-dose booster of Ad26.COV2.S or BNT162b2 vaccine. B cell responses (binding, neutralization and antibody dependent cellular cytotoxicity-ADCC), and spike-specific T-cell responses were evaluated at baseline, 2, 12 and 24 weeks post-boost. Antibody and T-cell immunity targeting the Ad26 vector was also evaluated. No vaccine-associated serious adverse events were recorded. The full- and half-dose BNT162b2 boosted anti-SARS-CoV-2 binding antibody levels (3.9- and 4.5-fold, respectively) and neutralizing antibody levels (4.4- and 10-fold). Binding and neutralizing antibodies following half-dose Ad26.COV2.S were not significantly boosted. Full-dose Ad26.COV2.S did not boost binding antibodies but slightly enhanced neutralizing antibodies (2.1-fold). ADCC was marginally increased only after a full-dose BNT162b2. T-cell responses followed a similar pattern to neutralizing antibodies. Six months post-boost, antibody and T-cell responses had waned to baseline levels. While we detected strong anti-vector immunity, there was no correlation between anti-vector immunity in Ad26.COV2.S recipients and spike-specific neutralizing antibody or T-cell responses post-Ad26.COV2.S boosting. Overall, in the context of hybrid immunity, boosting with heterologous full- or half-dose BNT162b2 mRNA vaccine demonstrated superior immunogenicity 2 weeks post-vaccination compared to homologous Ad26.COV2.S, though rapid waning occurred by 12 weeks post-boost. Trial Registration: The study has been registered to the South African National Clinical Trial Registry (SANCTR): DOH-27-012022-7841. The approval letter from SANCTR has been provided in the up-loaded documents.

Safety and immunogenicity of booster vaccination and fractional dosing with Ad26.COV2.S or BNT162b2 in Ad26.COV2.S-vaccinated participants.

Background: We report the safety and immunogenicity of fractional and full dose Ad26.COV2.S and BNT162b2 in an open label phase 2 trial of participants previously vaccinated with a single dose of Ad26.COV2.S, with 91.4% showing evidence of previous SARS-CoV-2 infection. Methods: A total of 286 adults (with or without HIV) were enrolled >4 months after an Ad26.COV2.S prime and randomized 1:1:1:1 to receive either a full or half-dose booster of Ad26.COV2.S or BNT162b2 vaccine. B cell responses (binding, neutralization and antibody dependent cellular cytotoxicity-ADCC), and spike-specific T-cell responses were evaluated at baseline, 2, 12 and 24 weeks post-boost. Antibody and T-cell immunity targeting the Ad26 vector was also evaluated. Results: No vaccine-associated serious adverse events were recorded. The full- and half-dose BNT162b2 boosted anti-SARS-CoV-2 binding antibody levels (3.9- and 4.5-fold, respectively) and neutralizing antibody levels (4.4- and 10-fold). Binding and neutralizing antibodies following half-dose Ad26.COV2.S were not significantly boosted. Full-dose Ad26.COV2.S did not boost binding antibodies but slightly enhanced neutralizing antibodies (2.1-fold). ADCC was marginally increased only after a full-dose BNT162b2. T-cell responses followed a similar pattern to neutralizing antibodies. Six months post-boost, antibody and T-cell responses had waned to baseline levels. While we detected strong anti-vector immunity, there was no correlation between anti-vector immunity in Ad26.COV2.S recipients and spike-specific neutralizing antibody or T-cell responses post-Ad26.COV2.S boosting. Conclusion: In the context of hybrid immunity, boosting with heterologous full- or half-dose BNT162b2 mRNA vaccine demonstrated superior immunogenicity 2 weeks post-vaccination compared to homologous Ad26.COV2.S, though rapid waning occurred by 12 weeks post-boost. Trial Registration: South African National Clinical Trial Registry (SANCR): DOH-27-012022-7841. Funding: South African Medical Research Council (SAMRC) and South African Department of Health (SA DoH).

Novavax NVX-COV2373 triggers neutralization of Omicron sub-lineages.

The SARS-CoV-2 Omicron (B.1.1.529) Variant of Concern (VOC) and its sub-lineages (including BA.2, BA.4, BA.5, BA.2.12.1) contain spike mutations that confer high level resistance to neutralizing antibodies induced by vaccination with ancestral spike or infection with previously circulating variants. The NVX-CoV2373 vaccine, a protein nanoparticle vaccine containing the ancestral spike sequence, has value in countries with constrained cold-chain requirements. Here we report neutralizing titers following two or three doses of NVX-CoV2373. We show that after two doses, Omicron sub-lineages BA.1 and BA.4/BA.5 were resistant to neutralization by 72% (21/29) and 59% (17/29) of samples respectively. However, after a third dose of NVX-CoV2373, we observed high titers against Omicron BA.1 (GMT: 1,197) and BA.4/BA.5 (GMT: 582), with responses similar in magnitude to those triggered by three doses of an mRNA vaccine. These data are of particular relevance as BA.4/BA.5 is dominating in multiple locations, and highlight the potential utility of the NVX-CoV2373 vaccine as a booster in resource-limited environments.

Production and Immunogenicity of Soluble Plant-Produced HIV-1 Subtype C Envelope gp140 Immunogens.

The development of effective vaccines is urgently needed to curb the spread of human immunodeficiency virus type 1 (HIV-1). A major focal point of current HIV vaccine research is the production of soluble envelope (Env) glycoproteins which reproduce the structure of the native gp160 trimer. These antigens are produced in mammalian cells, which requires a sophisticated infrastructure for manufacture that is mostly absent in developing countries. The production of recombinant proteins in plants is an attractive alternative for the potentially cheap and scalable production of vaccine antigens, especially for developing countries. In this study, we developed a transient expression system in Nicotiana benthamiana for the production of soluble HIV Env gp140 antigens based on two rationally selected virus isolates (CAP256 SU and Du151). The scalability of the platform was demonstrated and both affinity and size exclusion chromatography (SEC) were explored for recovery of the recombinant antigens. Rabbits immunized with lectin affinity-purified antigens developed high titres of binding antibodies, including against the V1V2 loop region, and neutralizing antibodies against Tier 1 viruses. The removal of aggregated Env species by gel filtration resulted in the elicitation of superior binding and neutralizing antibodies. Furthermore, a heterologous prime-boost regimen employing a recombinant modified vaccinia Ankara (rMVA) vaccine, followed by boosts with the SEC-purified protein, significantly improved the immunogenicity. To our knowledge, this is the first study to assess the immunogenicity of a near-full length plant-derived Env vaccine immunogen.

AAV-Mediated Expression of Broadly Neutralizing and Vaccine-like Antibodies Targeting the HIV-1 Envelope V2 Region.

HIV-1 infection continues to be a global health challenge and a vaccine is urgently needed. Broadly neutralizing antibodies (bNAbs) are considered essential as they inhibit multiple HIV-1 strains, but they are difficult to elicit by conventional immunization. In contrast, non-neutralizing antibodies that correlated with reduced risk of infection in the RV144 HIV vaccine trial are relatively easy to induce, but responses are not durable. To overcome these obstacles, adeno-associated virus (AAV) vectors were used to provide long-term expression of antibodies targeting the V2 region of the HIV-1 envelope protein, including the potent CAP256-VRC26.25 bNAb, as well as non-neutralizing CAP228 antibodies that resemble those elicited by vaccination. AAVs mediated effective antibody expression in cell culture and immunocompetent mice. Mean concentrations of human immunoglobulin G (IgG) in mouse sera increased rapidly following a single AAV injection, reaching 8-60 µg/mL for CAP256 antibodies and 44-220 µg/mL for CAP228 antibodies over 24 weeks, but antibody concentrations varied for individual mice. Secreted antibodies collected from serum retained the expected binding and neutralizing activity. The vectors generated here are, therefore, suitable for the delivery of V2-targeting HIV antibodies, and they could be used in a vectored immunoprophylaxis (VIP) approach to sustain the level of antibody expression required to prevent HIV infection.

Measuring the ability of HIV-specific antibodies to mediate trogocytosis.

Antibody Fc effector functions contribute to HIV control and have been implicated in the partial efficacy seen in the RV144 vaccine trial. Fc-mediated trogocytosis has been previously described for anti-cancer antibodies and results in the removal of membrane fragments from target cells. Here we developed a flow cytometry-based assay which measures the transfer of membrane fragments from a gp120-coated CD4+ lymphocytic cell line (CEM.NKR-CCR5 cells stained with a membrane dye PKH26) to monocytic cells (THP-1 cells stained with CFSE). We showed that this transfer occurred rapidly, within 1 h, and was mediated through engagement of the FcγRIIa/b receptors on the THP-1 cells. HIV-specific IgG as well as gp120 and CD4 could be detected on the surface of THP-1 cells in a process that we demonstrated was distinct from phagocytosis. Furthermore, while the THP-1 effector cells remained intact following the receipt of new membrane proteins, the viability of the target CEM.NKR-CCR5 cells decreased over time. Analysis of HIV-specific plasma revealed that antibodies with trogocytic activity were common in acute and chronic HIV infection but were higher in individuals with broadly neutralizing antibody responses We also examined trogocytosis mediated by broadly neutralizing antibodies (bNAbs) targeting multiple epitopes on the BG505.SOSIP.664 trimer and show that levels of binding correlated with the trogocytosis score. Overall, our data describe a new antiviral Fc effector function mediated by HIV-specific antibodies that could be harnessed for vaccination and cure strategies.

Nonprogressing HIV-infected children share fundamental immunological features of nonpathogenic SIV infection.

Disease-free infection in HIV-infected adults is associated with human leukocyte antigen-mediated suppression of viremia, whereas in the sooty mangabey and other healthy natural hosts of simian immunodeficiency virus (SIV), viral replication continues unabated. To better understand factors preventing HIV disease, we investigated pediatric infection, where AIDS typically develops more rapidly than in adults. Among 170 nonprogressing antiretroviral therapy-naïve children aged >5 years maintaining normal-for-age CD4 T cell counts, immune activation levels were low despite high viremia (median, 26,000 copies/ml). Potent, broadly neutralizing antibody responses in most of the subjects and strong virus-specific T cell activity were present but did not drive pediatric nonprogression. However, reduced CCR5 expression and low HIV infection in long-lived central memory CD4 T cells were observed in pediatric nonprogressors. These children therefore express two cardinal immunological features of nonpathogenic SIV infection in sooty mangabeys-low immune activation despite high viremia and low CCR5 expression on long-lived central memory CD4 T cells-suggesting closer similarities with nonpathogenetic mechanisms evolved over thousands of years in natural SIV hosts than those operating in HIV-infected adults.

Hepatic Delivery of Artificial Micro RNAs Using Helper-Dependent Adenoviral Vectors.

The potential of RNA interference (RNAi)-based gene therapy has been demonstrated in many studies. However, clinical application of this technology has been hampered by a paucity of efficient and safe methods of delivering the RNAi activators. Prolonged transgene expression and improved safety of helper-dependent adenoviral vectors (HD AdVs) makes them well suited to delivery of engineered artificial intermediates of the RNAi pathway. Also, AdVs' natural hepatotropism makes them potentially useful for liver-targeted gene delivery. HD AdVs may be used for efficient delivery of cassettes encoding short hairpin RNAs and artificial primary microRNAs to the mouse liver. Methods for the characterization of HD AdV-mediated delivery of hepatitis B virus-targeting RNAi activators are described here.

Inhibition of hepatitis B virus replication by helper dependent adenoviral vectors expressing artificial anti-HBV pri-miRs from a liver-specific promoter.

Research on applying RNA interference (RNAi) to counter HBV replication has led to identification of potential therapeutic sequences. However, before clinical application liver-specific expression and efficient delivery of these sequences remain an important objective. We recently reported short-term inhibition of HBV replication in vivo by using helper dependent adenoviral vectors (HD Ads) expressing anti-HBV sequences from a constitutively active cytomegalovirus (CMV) promoter. To develop the use of liver-specific transcription regulatory elements we investigated the utility of the murine transthyretin (MTTR) promoter for expression of anti-HBV primary microRNAs (pri-miRs). HD Ads containing MTTR promoter effected superior expression of anti-HBV pri-miRs in mice compared to HD Ads containing the CMV promoter. MTTR-containing HD Ads resulted in HBV replication knockdown of up to 94% in mice. HD Ads expressing trimeric anti-HBV pri-miRs silenced HBV replication for 5 weeks. We previously showed that the product of the codelivered lacZ gene induces an immune response, and the duration of HBV silencing in vivo is likely to be attenuated by this effect. Nevertheless, expression of anti-HBV pri-miRs from MTTR promoter is well suited to countering HBV replication and development of HD Ads through attenuation of their immunostimulatory effects should advance their clinical utility.

Inhibition of HBV replication in vivo using helper-dependent adenovirus vectors to deliver antiviral RNA interference expression cassettes.

BACKGROUND: HBV is hyperendemic to southern Africa and parts of Asia, but licensed antivirals have little effect on limiting life-threatening complications of the infection. Although RNA interference (RNAi)-based gene silencing has shown therapeutic potential, difficulties with delivery of anti-HBV RNAi effectors remain an obstacle to their clinical use. To address concerns about the transient nature of transgene expression and toxicity resulting from immunostimulation by recombinant adenovirus vectors (Ads), utility of RNAi-activating anti-HBV helper-dependent (HD) Ads were assessed in this study. METHODS: Following intravenous administration of 5×10(9) unmodified or pegylated HD Ad infectious particles to HBV transgenic mice, HBV viral loads and serum HBV surface antigen levels were monitored for 12 weeks. Immunostimulation of HD Ads was assessed by measuring inflammatory cytokines, hepatic function and immune response to the co-delivered LacZ reporter gene. RESULTS: Unmodified and pegylated HD Ads transduced 80-90% of hepatocytes and expressed short hairpin RNAs (shRNAs) were processed to generate intended HBV-targeting guides. Markers of HBV replication were decreased by approximately 95% and silencing was sustained for 8 weeks. Unmodified HD Ads induced release of proinflammatory cytokines and there was evidence of an adaptive immune response to ß-galactosidase. However the HD Ad-induced innate immune response was minimal in preparations that were enriched with infectious particles. CONCLUSIONS: HD Ads have potential utility for delivery of therapeutic HBV-silencing sequences and alterations of these vectors to attenuate their immune responses may further improve their efficacy.

Efficient silencing of hepatitis B virus by helper-dependent adenovirus vector-mediated delivery of artificial antiviral primary micro RNAs.

Hepatitis B virus (HBV) infection is endemic to southern Africa and parts of Asia where approximately 350 million individuals are chronically infected. Persistent infection increases risk for the serious complications of cirrhosis and hepatocellular carcinoma. Licensed HBV treatments rarely eradicate the virus, which makes developing new strategies for the treatment of chronic HBV a priority. Pol II-transcribed mono- and trimeric primary micro RNAs (primiRNAs) have previously been used to activate RNA interference (RNAi) and inhibit HBV gene expression, indicating that this approach holds promise for HBV therapy. Nevertheless, achieving safe and efficient delivery of anti-HBV RNAi expression cassettes remains an important objective before therapeutic application of this gene silencing technology is realized. Recombinant adenoviruses (Ads) are amongst the most efficient hepatotropic gene delivery vehicles, but a drawback of their use is transient transgene expression and toxicity that results from induction of host immune responses. To diminish immunostimulation of anti-HBV RNAi-activating vectors, helper-dependent (HD) Ads with all viral proteinencoding sequences removed from their genomes, were generated. A CMV Pol II promoter element was used to transcribe antiviral pri-miRNAs that target HBV. Processing of the anti-HBV pri-miRNA RNAi activators occurred according to intended design. Assessment in cultured cells and in a HBV transgenic model of the infection demonstrated that HD Ads delivered the silencing sequences efficiently and replication of the virus was inhibited without causing overt toxic effects. Collectively these data augur well for clinical use of HD Ads to deliver Pol II HBV-silencing cassettes to counter the persistent infection.

Therapeutic potential of adenoviral vectors for delivery of expressed RNAi activators.

IMPORTANCE OF THE FIELD: Harnessing RNA interference (RNAi) to silence pathology-causing genes has shown promise as a mode of therapy. The sustained gene inhibition that may be achieved with expressed sequences is potentially useful for treatment of chronic viral infections, but efficient and safe delivery of these sequences remains a challenge. It is generally recognized that there is no ideal vector for all therapeutic RNAi applications, but recombinant adenovirus vectors are well suited to hepatic delivery of expressed RNAi activators. AREAS COVERED IN THIS REVIEW: Adenoviruses are hepatotropic after systemic administration, and this is useful for delivering expressed RNAi activators that silence pathology-causing genes in the liver. However, drawbacks of adenoviruses are toxicity and diminished efficacy, which result from induction of innate and adaptive immune responses. In this review, the advantages and hurdles facing therapeutic application of adenoviral vectors for liver delivery of RNAi effectors are covered. WHAT THE READER WILL GAIN: Insights into adenovirus vectorology and the methods that have been used to make these vectors safer for advancing clinical application of RNAi-based therapy. TAKE HOME MESSAGE: Adenoviruses are very powerful hepatotropic vectors. To make adenoviruses more effective for clinical use, polymer conjugation and deletion of viral vector sequences have been used successfully. However, further modifications to attenuate immunostimulation as well as improvements in large-scale production are necessary before the therapeutic potential of adenovirus-mediated delivery of RNAi activators is realized.

Inhibition of hepatitis B virus replication in vivo using lipoplexes containing altritol-modified antiviral siRNAs.

Chronic infection with the hepatitis B virus (HBV) occurs in approximately 6% of the world's population and carriers of the virus are at risk for complicating hepatocellular carcinoma. Current treatment options have limited efficacy and chronic HBV infection is likely to remain a significant global medical problem for many years to come. Silencing HBV gene expression by harnessing RNA interference (RNAi) presents an attractive option for development of novel and effective anti HBV agents. However, despite significant and rapid progress, further refinement of existing technologies is necessary before clinical application of RNAi-based HBV therapies is realized. Limiting off target effects, improvement of delivery efficiency, dose regulation and preventing reactivation of viral replication are some of the hurdles that need to be overcome. To address this, we assessed the usefulness of the recently described class of altritol-containing synthetic siRNAs (ANA siRNAs), which were administered as lipoplexes and tested in vivo in a stringent HBV transgenic mouse model. Our observations show that ANA siRNAs are capable of silencing of HBV replication in vivo. Importantly, non specific immunostimulation was observed with unmodified siRNAs and this undesirable effect was significantly attenuated by ANA modification. Inhibition of HBV replication of approximately 50% was achieved without evidence for induction of toxicity. These results augur well for future application of ANA siRNA therapeutic lipoplexes.

DODAG; a versatile new cationic lipid that mediates efficient delivery of pDNA and siRNA.

We report the syntheses of novel cationic lipids comprised of cholesteryl-moieties linked to guanidinium functional groups, and also cationic lipids comprising a dialkylglycylamide moiety conjugated with a polyamine or a guanidinium functional group. In plasmid DNA (pDNA) transfection studies, these cationic lipids were formulated into cationic liposomes with the neutral co-lipid dioleoyl-L-alpha-phosphatidylethanolamine (DOPE) or with a recently reported neutral lipophosphoramidate derivative of histamine (MM27). We observe that cationic liposomes prepared from the cationic lipid N',N'-dioctadecyl-N-4,8-diaza-10-aminodecanoylglycine amide (DODAG) and DOPE frequently mediate the highest levels of transfection in vitro in all three different cell lines studied (OVCAR-3, IGROV-1 and HeLa) both in the presence or absence of serum. In addition, in vitro cellular toxicity was found to be minimal. Alternatively, we observe that DODAG alone forms lipoplex nanoparticles with small interfering RNA (siRNA) that are able to mediate the functional delivery of two previously validated anti-hepatitis B virus (HBV)--siRNAs to murine liver in vivo with minimal observable liver toxicity and immune stimulation. Specific knock-down of HBV infection parameters (virion and hepatic mRNA levels) is observed that is at least equivalent to the impact of extensive treatment with lamivudine (a licensed antiviral drug).

Controlling HBV replication in vivo by intravenous administration of triggered PEGylated siRNA-nanoparticles.

Harnessing RNA interference (RNAi) to inhibit hepatitis B virus (HBV) gene expression has promising application to therapy. Here we describe a new hepatotropic nontoxic lipid-based vector system that is used to deliver chemically unmodified small interfering RNA (siRNA) sequences to the liver. Anti HBV formulations were generated by condensation of siRNA (A component) with cationic liposomes (B component) to form AB core particles. These core particles incorporate an aminoxy cholesteryl lipid for convenient surface postcoupling of polyethylene glycol (PEG; C component, stealth/biocompatibility polymer) to give triggered PEGylated siRNA-nanoparticles (also known as siRNA-ABC nanoparticles) with uniform small sizes of 80-100 nm in diameter. The oxime linkage that results from PEG coupling is pH sensitive and was included to facilitate acidic pH-triggered release of nucleic acids from endosomes. Nanoparticle-mediated siRNA delivery results in HBV replication knockdown in cell culture and in murine hydrodynamic injection models in vivo. Furthermore repeated systemic administration of triggered PEGylated siRNA-nanoparticles to HBV transgenic mice results in the suppression of markers of HBV replication by up to 3-fold relative to controls over a 28 day period. This compares favorably to silencing effects seen during lamivudine treatment. Collectively these observations indicate that our PEGylated siRNA-nanoparticles may have valuable applications in RNAi-based HBV therapy.

Expressed anti-HBV primary microRNA shuttles inhibit viral replication efficiently in vitro and in vivo.

The use of RNA interference (RNAi) to inhibit gene expression is potentially applicable in the treatment of viral infections such as hepatitis B virus (HBV) persistence. Although efficient HBV gene silencing by short hairpin RNA (shRNA) expressed from RNA polymerase (Pol) III promoters has been reported, constitutive high-level transcription may cause harmful side effects. Here, we report an approach that allows the use of a Pol II promoter to improve transcription regulation of expressed RNAi effecters. Pol II [cytomegalovirus (CMV)] or Pol III (U6) promoter cassettes that transcribe anti-HBV primary microRNA (pri-miR)-122 and pri-miR-31 shuttles were generated. In cultured cells both types of pri-miR-like sequences effected knockdown of markers of viral replication (>80%) and were processed to form intended 21-nucleotide guides. The concentration of CMV-expressed miRs was approximately 85-fold lower than the U6 shRNA-derived guide RNA. When cells were co-transfected with pri-miR expression cassettes, attenuation of independent RNAi-mediated gene silencing was not observed, which is in contrast to the action of U6 shRNA expression cassettes. The efficacy of the anti-HBV pri-miR shuttles in vivo was verified using the murine hydrodynamic injection model. Employing Pol II-expressed pri-miR mimics may be useful in the treatment of HBV infection, and potentially also for generic application in RNAi-based therapy.

A valine to phenylalanine mutation in the precore region of hepatitis B virus causes intracellular retention and impaired secretion of HBe-antigen.

AIM: Hepatitis B virus (HBV) e antigen (HBeAg) is translated from precore mRNA as a precore/core protein, which is post-translationally modified to give rise to the protein that is secreted into the serum. The G1862T mutation in HBV occurs in the bulge of the encapsidation signal within the pregenomic RNA. When the precore mRNA is translated, this mutation results in a valine to phenylalanine substitution at the -3 position to the signal peptide cleavage site at the amino end of the precursor protein. The aim of this study was to determine whether this mutation could affect HBV replication and/or HBeAg expression. METHODS: Following transfection of Huh 7 cells, HBV replication was followed using real time polymerase reaction (PCR) and expression of HBeAg expression was monitored using confocal microscopy. RESULTS: HBV replication was reduced when this mutation was introduced into genotype D but not into genotype A replication-competent constructs. Using mutant HBeAg-expressing plasmids, we demonstrated a 54% reduction in HBeAg secretion relative to the wild type. Confocal microscopy demonstrated that the mutant HBeAg accumulated in the endoplasmic reticulum, endoplasmic reticulum intermediate compartment and Golgi. These aggregates of mutant protein increased in size following treatment of the cells with a proteasome inhibitor, MG132, and had the hallmark features of aggresomes. They attracted ubiquitin, heat shock proteins and proteasomes and were isolated from the cytosol by the intermediate filaments, vimentin and cytokeratin. CONCLUSION: The formation of aggresomes, as a result of the G1862T mutation, may play a contributory role in HBV-induced liver disease.

Efficient inhibition of hepatitis B virus replication in vivo, using polyethylene glycol-modified adenovirus vectors.

Achieving safe delivery of anti-hepatitis B virus (HBV) RNA interference (RNAi) effectors is an important objective of this gene-silencing technology. Adenoviruses (Ads) have a natural tropism for the liver after systemic administration, and are useful for delivery of expressed anti-HBV RNAi sequences. However, a drawback of Ad vectors is diminished efficacy and toxicity that results from stimulation of innate and adaptive immunity. To attenuate these effects we used monomethoxy polyethylene glycol-succinimidyl propionate (mPEG-SPA) to modify first-generation vectors that express an anti-HBV RNAi effector. Efficient hepatocyte transduction and knockdown of HBV replication were achieved after intravenous administration of 5 x 10(9) PEGylated or native recombinant Ads to HBV transgenic mice. After the first injection, circulating HBV viral particle equivalents (VPEs) remained low for 3 weeks and began to increase after 5 weeks. A second dose of PEGylated anti-HBV Ad caused a less sustained decrease in circulating VPEs, but no silencing after a second dose was observed in animals treated with unmodified vector. Release of inflammatory cytokines, including monocyte chemoattractant protein-1 (MCP-1), interferon-gamma, interleukin-6, and tumor necrosis factor-alpha, was elevated in animals receiving unmodified vectors. However, only a modest increase in MCP-1 was observed in mice that received a second dose of PEG Ads. Also, polymer-conjugated vectors induced a weaker adaptive immune response and were less hepatotoxic than their unmodified counterparts. Collectively, these observations show that PEG modification of Ads expressing RNAi effectors improves their potential for therapeutic application against HBV infection.

Specific inhibition of HBV replication in vitro and in vivo with expressed long hairpin RNA.

Activating RNA interference to achieve specific gene silencing has shown promise for the development of RNA-based treatment of chronic hepatitis B virus (HBV) infection. To further this approach, we assessed the efficacy of expressed long hairpin RNAs (lhRNAs) that target the conserved HBx open reading frame of HBV. As substrates for Dicer, lhRNAs have the potential to generate multiple short interfering RNAs (siRNAs) to enable simultaneous targeting of different sites. Two U6 Pol III vectors were constructed that encode anti-HBV lhRNAs with a 62 base pair stem sequence containing multiple G:U pairings. Assessment in transfected cultured cells and also in vivo using the murine hydrodynamic injection model showed that one of the lhRNA vectors (lhRNA 1) diminished markers of virus replication by 70-90% without evidence of interferon response induction. Greatest silencing efficacy was observed for targets that are complementary to sequences located at the base of the hairpin stem and this correlated with a higher concentration of siRNAs derived from this region of the lhRNA. Although lhRNA 1 has the advantage of targeting a greater viral sequence, incomplete cellular processing may result in unequal silencing across the span of the viral target RNA.

Effective inhibition of HBV replication in vivo by anti-HBx short hairpin RNAs.

Exploiting the RNA interference pathway has shown promise for developing novel and effective treatment of hepatitis B virus (HBV) infection. To advance this approach, we analyzed the antiviral efficacy of a panel of 10 Pol III U6 promoter-encoded short hairpin RNAs (shRNAs) that target conserved sequences of the oncogenic HBx open reading frame. To facilitate intracellular processing, the shRNAs included mismatches in the 25-bp stem region and a terminal loop of miRNA-23. Two shRNAs (shRNA 5 and shRNA 6) showed knockdown of HBV markers by 80-100% in transfected hepatocytes and also in a murine hydrodynamic injection model of HBV replication. Intracellular processing of hairpin RNA with the intended strand bias correlated with antiviral efficacy. Moreover, markers of HBV replication were inhibited without inducing genes associated with the nonspecific interferon response. To assess the antiviral efficacy of the shRNAs in a context that is similar to natural HBV infection, shRNA-encoding cassettes were tested against the virus in a HBV transgenic murine model. When delivered using recombinant adenovirus vectors, U6 shRNA 5 and U6 shRNA 6 mediated significant HBV knockdown. Collectively, these observations indicate that U6 shRNA 5 and U6 shRNA 6 are promising candidates for therapy of chronic HBV infection.