Human JC virus-like particles as a gene delivery vector

Introduction: As a viral gene delivery vector, the recombinant JC virus-like particles (VLPs) can be easily generated in large quantities and at low cost. Exogenous genes of interest can be packaged by the VLP without the involvement of viral genetic material and then delivered into any tissue susceptible to JC virus (JCV) to allow gene transduction. Therefore, it should be possible in the future to develop a gene delivery vector using the human JC VLPs that will allow gene therapy.

Areas covered: Development of a gene delivery vector using the polyomavirus VLPs is reviewed in this article. The advantages and disadvantages of using JC VLP for gene delivery are discussed.

Expert opinion: Human JC VLPs are readily produced and can be engineered with ease; they allow specific targeting without the presence of any viral genetic material. For therapeutic purposes, gene(s) of interest or other compounds can be packaged into the VLP and delivered to JCV-susceptible cells at high efficiency.     

Safety and immunogenicity studies in animal models support clinical development of a bivalent norovirus-like particle vaccine produced in plants

Noroviruses (NoV) are the leading cause of epidemic acute gastroenteritis in humans worldwide. A safe and effective vaccine that prevents NoV infection or minimizes NoV disease burden is needed, especially for children and the elderly who are particularly susceptible to NoV disease. A plant-based expression system (magnICON®) was used to manufacture two different virus-like particle (VLP) immunogens derived from human NoV genogroups I and II, genotype 4 (GI.4 and GII.4), which were subsequently blended 1:1 (w/w) into a bivalent vaccine composition (rNV-2v). Here, we report on the safety and immunogenicity of rNV-2v from one pilot and two GLP-compliant toxicity studies in New Zealand White rabbits administered the vaccine subcutaneously (SC) or intramuscularly (IM). Strong genogroup-specific immune responses were induced by vaccination without adjuvant at various doses (200 to 400 μg VLP/administration) and administration schedules (Days 1 and 7; or Days 1, 15 and 29). The results showed sporadic local irritation at the injection site, which resolved over time, and was non-adverse and consistent with expected reactogenicity. There were no signs of systemic toxicity related to vaccine administration relative to vehicle-treated controls with respect to clinical chemistry, haematology, organ weights, macroscopic examinations, or histopathology. In a 3-administration regimen (n + 1 the clinical regimen), the NOAEL for rNV-2v via the SC or IM route was initially determined to be 200 μg. An improved GI.4 VLP variant mixed 1:1 (w/w) with the wild-type GII.4 VLP was subsequently evaluated via the IM route at a higher dose in the same 3-administration model, and the NOAEL was raised to 300 µg. Serology performed in samples of both toxicity studies showed significant and substantial anti-VLP-specific antibody titers for rNV-2v vaccines administered via the IM or SC route, as well as relevant NoV blocking antibody responses. These results support initiation of clinical development of the plant-made NoV vaccine.     

Multi-site analytical validation of an assay to detect anti-JCV antibodies in human serum and plasma

Background

JC virus (JCV) infection is a prerequisite for development of progressive multifocal leukoencephalopathy (PML). We previously described the development of a novel, two-step enzyme-linked immunosorbent assay (ELISA) that detects anti-JCV antibodies in human serum or plasma, and the potential clinical utility of anti-JCV antibody status for PML risk stratification.

Objectives

To validate the anti-JCV antibody ELISA at multiple clinical laboratories in order to demonstrate the robustness of the method.

Study design

Analytical validation of the ELISA was performed at four laboratories by evaluation of intra- and inter-assay precision, analytical specificity and sensitivity, matrix interference, robustness, sample and reagent stability.

Results

Analytical validation demonstrated that the assay is sensitive, specific, and precise. The assay sensitivity was estimated at 1.7 ng/mL using a humanized anti-JCV monoclonal antibody control. The sensitivity to detect JCV infection was estimated to be 97.5%. The specificity of the assay to discriminate JCV-specific antibodies from antibodies directed to BK virus, a related polyomavirus, was also demonstrated. The inter- and intra-assay precision was ≤6.0% and 9.8% for the screening step and 2.6% and 11.3% for the confirmation step. Results obtained for plasma and serum were highly congruent, and assay robustness was demonstrated by the highly concordant results generated by four laboratories testing a common panel of 100 blinded samples.

Conclusions

The novel, two-step ELISA to detect anti-JCV antibodies in human serum and plasma is robust, and assay performance is consistent and reproducible in multiple laboratories, making it suitable to support testing for global clinical studies.     

Active immunization against IL-23p19 improves experimental arthritis

Introduction

IL-23 is a pro-inflammatory cytokine essential for the differentiation of Th17 lymphocytes, a subtype of T lymphocyte implied in auto-immunity. IL-23 shares a subunit with IL-12, IL-12/23p40, and comprises a specific subunit, IL-23p19. We previously demonstrated that active immunization against entire TNF-α and against peptides of IL-1β was protective in animal models of rheumatoid arthritis. The aim of this study was to evaluate the effect of peptide-based vaccines targeting the IL-23p19 subunit in collagen-induced arthritis (CIA).

Methods

Using bioinformatics, the murine IL-23p19 subunit was modeled and two peptides were defined in the receptor interacting domain. Each peptide was coupled to keyhole limpet hemocyanin (KLH) to obtain two vaccines IL23-K1 and IL23-K2. Both vaccines were used for immunizations in incomplete Freund adjuvant (IFA) in groups of DBA/1 mice. Control groups received KLH or PBS at the same dates. CIA was induced by two subcutaneous injections of bovine type II collagen (CIIb), and the development of disease assessed during the next two months. Anti-CIIb and anti-IL-23 antibody levels were assessed by ELISA. Pro- and anti-inflammatory cytokines mRNA were quantified by qRT-PCR in the spleen and the synovium. T-cell populations in the spleen were evaluated by FACS analysis.

Results

The clinical scores showed that mice treated with IL23-K1 developed less arthritis than negative controls (p < 0.05). Mice immunized with IL23-K1 produced more anti-IL-23 antibodies than those immunized with IL23-K2 (p < 0.001). mRNA quantification showed that the IL23-K1 immunization led to an increase of IL-10 in the spleen (p < 0.05 vs KLH), without any effect on IL-17 level. Histological examination showed that IL23-K1 strongly protected against joint destruction and inflammation (p < 0.01 vs KLH and p < 0.001 vs PBS). T-cell populations in the spleen were not modified by IL-23 modulation.

Conclusion

These data show that targeting IL-23p19 through a vaccination strategy is protective in CIA. This specific targeting of IL-23 might constitute a promising therapeutic approach to explore in rheumatoid arthritis.     

Aerosol delivery of virus-like particles to the genital tract induces local and systemic antibody responses

The induction of mucosal immune responses in the genital tract may be important for increasing the effectiveness of vaccines for sexually transmitted infections (STIs). In this study, we asked whether direct immunization of the mouse genital tract with a non-replicating virus-like particle (VLP)-based vaccine could induce local mucosal as well as systemic antibody responses. Using VLPs derived from two bacteriophages, Qβ and PP7, and from a mammalian virus that normally infects the genital tract, human papillomavirus (HPV), we show that intravaginal aerosol administration of VLPs can induce high titer IgG and IgA antibodies in the female genital tract as well as IgG in the sera. Using a mouse model for HPV infection, we show that intravaginal immunization with either HPV type 16 VLPs or with PP7 bacteriophage VLPs displaying a peptide derived from the HPV minor capsid protein L2 could protect mice from genital infection with an HPV16 pseudovirus. These results provide a general method for inducing genital mucosal and systemic antibody responses using VLP-based immunogens.     

含Aβ1-15 的嵌合型HBcAg 颗粒抗原的制备及其免疫原性分析

目的: 原核表达并纯化含β- 淀粉样肽(β-amyloid peptide, Aβ) 氨基段15 肽(Aβ1-15) 和删除c/e1表位的截断型HBcAg 的融合蛋白,观察其形成的病毒样颗粒,检测其免疫原性,为阿尔茨海默病(Alzheimer’sdisease, AD) 基因工程疫苗的研究提供基础。方法:将合成的Aβ1-15 基因连接于HBcAg 的1~71 的3′端,再将HBcAg 的88~144 位氨基酸的基因片断连接于Aβ_1-15 的基因的3′端,构建重组质粒pUC/c-Aβ₁₅-c,将重组基因亚克隆于原核表达载体pET-28a(+) 中,构建表达质粒pET/ c-Aβ₁₅-c。异丙基-β-D- 硫代吡喃半乳糖苷(isopropyl β-D-1-thiogalactopyranoside, IPTG) 诱导、SDS-PAGE 和考马斯亮蓝染色检测重组基因的表达。表达的融合蛋白( 命名为CA15C) 纯化后,透射电镜观察病毒样颗粒的形成。以病毒颗粒抗原CA15C 腹腔注射免疫昆明小鼠,间接ELISA 法检测小鼠血清中抗-Aβ 抗体的滴度。结果:经酶切鉴定、DNA 序列测定证实,目的基因重组于表达质粒之中,与理论设计相符。诱导表达后,在细菌裂解液的上清和沉淀中均可见表达蛋白CA15C,以沉淀中为多,约占细菌沉淀总蛋白的40%。电镜下可见纯化后的CA15C 形成直径约30 nm 的病毒样颗粒。昆明小鼠经CA15C 免疫5 次后,其血清中抗-Aβ 抗体的滴度可达1:10000,且检测不到抗-HBc 抗体。结论:原核表达制备的含Aβ_1-15 和HBcAg 的融合蛋白CA15C,可形成病毒样颗粒,有较强的免疫原性。     

Recent developments in human papillomavirus diagnosis and therapy in genital neoplasia

Infection with certain types of human papillomavirus (HPV) is casually associated with the development of cervical cancer and other anogenital neoplasia. There is increasing evidence of a wider association with other epithelial cancers. The availability of recombinant molecular biological techniques has circumvented the difficulties in studying the virus life cycle that is critically dependent on the differentiation of the epithelial target cell. From an understanding of the virology and cell biology of HPV infection, prospects for diagnostic and screening procedures in cervical neoplasia are being tested. The prize is the cost effective estimation of risk from HPV infection. New approaches need to be compared to existing and proven, although not perfect, smear test screening methods for prevention of cervical cancer. Novel research and development activities aimed at treating HPV infections in early premalignant lesions are utilising aspects of interference with early viral gene expression, or their co-ordinate cellular targets. Many immunological approaches with the goal of prevention of infection (prophylactic vaccination) or therapy of early cervical lesions are being tested. A virus-like particle composed of HPV L1 or L1/L2 capsid proteins, formulated to generate neutralising antibodies, is a key technology. Chimeric variations delivering other target antigens that can stimulate cytotoxic T-lymphocyte (CTL) activity may have wider applications in cancer treatments. Immunotherapeutic strategies aimed at treating late stage disease by optimising the stimulation of CTL versus HPV oncogenes is another major area of activity. Clinical trials testing the safety and efficacy of some of these inventions are in progress.     

Off-target effects of an insect cell-expressed influenza HA-pseudotyped Gag-VLP preparation in limiting postinfluenza Staphylococcus aureus infections

Clinical and historical data underscore the ability of influenza viruses to ally with Staphylococcus aureus and predispose the host for secondary bacterial pneumonia, which is a leading cause of influenza-associated mortality. This is fundamental because no vaccine for S. aureus is available and the number of antibiotic-resistant strains is alarmingly rising. Hence, this leaves influenza vaccination the only strategy to prevent postinfluenza staphylococcal infections. In the present work, we assessed the off-target effects of a Tnms42 insect cell-expressed BEI-treated Gag-VLP preparation expressing the HA of A/Puerto Rico/8/1934 (H1N1) in preventing S. aureus superinfection in mice pre-infected with a homologous or heterologous H1N1 viral challenge strain. Our results demonstrate that matched anti-hemagglutinin immunity elicited by a VLP preparation may suffice to prevent morbidity and mortality caused by lethal secondary bacterial infection. This effect was observed even when employing a single low antigen dose of 50 ng HA per animal. However, induction of anti-hemagglutinin immunity alone was not helpful in inhibiting heterologous viral replication and subsequent bacterial infection. Our results indicate the potential of the VLP vaccine approach in terms of immunogenicity but suggest that anti-HA immunity should not be considered as the sole preventive method for combatting influenza and postinfluenza bacterial infections.     

Virus-like particles of hepatitis B virus core protein containing five mimotopes of infectious bursal disease virus (IBDV) protect chickens against IBDV

Current infectious bursal disease virus (IBDV) vaccines suffer from maternal antibody interference and mimotope vaccines might be an alternative. Previously we demonstrated an IBDV VP2 five-mimotope polypeptide, 5EPIS, elicited protective immunity in chickens. In the current study, the 5epis gene was inserted into a plasmid carrying human hepatitis B virus core protein (HBc) gene at its major immunodominant region site. The recombinant gene was efficiently expressed in Escherichia coli to produce chimeric protein HBc-5EPIS which self-assembles to virus-like particles (VLP). Two-week old specific-pathogen-free chickens were immunized intramuscularly with HBc-5EPIS VLP or 5EPIS polypeptide without adjuvant (50 μg/injection) on day 0, 7, 14 and 21. Anti-5EPIS antibody was first detected on day 7 and day 21 in HBc-5EPIS and 5EPIS groups, respectively; on day 28, anti-5EPIS titers reached 12,800 or 1600 by ELISA, and 3200 or 800 by virus neutralization assay in HBc-5EPIS and 5EPIS groups, respectively. No anti-5EPIS antibody was detected in the buffer control group throughout the experiment. Challenge on day 28 with a virulent IBDV strain (GX8/99) resulted in 100%, 40.0% and 26.7% survival for chickens immunized with HBc-5EPIS, 5EPIS and buffer, respectively. These data suggest epitope presentation on chimeric VLP is a promising approach for improving mimotope vaccines for IBDV.     

Vaccine synergy with virus-like particle and immune complex platforms for delivery of human papillomavirus L2 antigen

Diverse HPV subtypes are responsible for considerable disease burden worldwide, necessitating safe, cheap, and effective vaccines. The HPV minor capsid protein L2 is a promising candidate to create broadly protective HPV vaccines, though it is poorly immunogenic by itself. To create highly immunogenic and safe vaccine candidates targeting L2, we employed a plant-based recombinant protein expression system to produce two different vaccine candidates: L2 displayed on the surface of hepatitis B core (HBc) virus-like particles (VLPs) or L2 genetically fused to an immunoglobulin capable of forming recombinant immune complexes (RIC). Both vaccine candidates were potently immunogenic in mice, but were especially so when delivered together, generating very consistent and high antibody titers directed against HPV L2 (>1,000,000) that correlated with virus neutralization. These data indicate a novel immune response synergy upon co-delivery of VLP and RIC platforms, a strategy that can be adapted generally for many different antigens.