Induction of Long-Term Protective Immune Responses by Influenza H5N1 Virus-Like Particles

Background

Recurrent outbreaks of highly pathogenic H5N1 avian influenza virus pose a threat of eventually causing a pandemic. Early vaccination of the population would be the single most effective measure for the control of an emerging influenza pandemic.

Methodology/Principal Findings

Influenza virus-like particles (VLPs) produced in insect cell-culture substrates do not depend on the availability of fertile eggs for vaccine manufacturing. We produced VLPs containing influenza A/Viet Nam1203/04 (H5N1) hemagglutinin, neuraminidase, and matrix proteins, and investigated their preclinical immunogenicity and protective efficacy. Mice immunized intranasally with H5N1 VLPs developed high levels of H5N1 specific antibodies and were 100% protected against a high dose of homologous H5N1 virus infection at 30 weeks after immunization. Protection is likely to be correlated with humoral and cellular immunologic memory at systemic and mucosal sites as evidenced by rapid anamnestic responses to re-stimulation with viral antigen in vivo and in vitro.

Conclusions/Significance

These results provide support for clinical evaluation of H5N1 VLP vaccination as a public health intervention to mitigate a possible pandemic of H5N1 influenza.     

Latent Adeno-Associated Virus Infection Elicits Humoral but Not Cell-Mediated Immune Responses in a Nonhuman Primate Model

Latent infection with wild-type (wt) adeno-associated virus (AAV) was studied in rhesus macaques, a species that is a natural host for AAV and that has some homology to humans with respect to the preferred locus for wt AAV integration. Each of eight animals was infected with an inoculum of 10(10) IU of wt AAV, administered by either the intranasal, intramuscular, or intravenous route. Two additional animals were infected intranasally with wt AAV and a helper adenovirus (Ad), while one additional animal was inoculated with saline intranasally as a control. There were no detectable clinical or histopathologic responses to wt AAV administration. Molecular analyses, including Southern blot, PCR, and fluorescence in situ hybridization, were performed 21 days after infection. These studies indicated that AAV DNA sequences persisted at the sites of administration, albeit at low copy number, and in peripheral blood mononuclear cells. Site-specific integration into the AAVS1-like locus was observed in a subset of animals. All animals, except those infected by the intranasal route with wt AAV alone, developed a humoral immune response to wt AAV capsid proteins, as evidenced by a >/=fourfold rise in anti-AAV neutralizing titers. However, only animals infected with both wt AAV and Ad developed cell-mediated immune responses to AAV capsid proteins. These findings provide some insights into the nature of anti-AAV immune responses that may be useful in interpreting results of future AAV-based gene transfer studies.     

携带HBV包膜大蛋白DNA疫苗的减毒沙门菌在小鼠诱导免疫应答

探索一种简便、有效的乙型肝炎病毒DNA疫苗免疫方法。将编码绿色荧光蛋白的真核表达质粒pEGFPN1转化到减毒鼠伤寒沙门菌SL7207,灌胃饲服BALB/c小鼠,流式细胞术检测出小鼠脾细胞内表达的绿色荧光蛋白;构建编码HBV包膜大蛋白的DNA疫苗pCIS1S2S,分别以SL7207为载体的口服途径或直接肌肉注射途径免疫BALB/c小鼠,检测小鼠的血清抗体、T细胞增殖和细胞毒性T淋巴细胞反应,结果表明两种免疫途径均能在小鼠体内诱生细胞和体液免疫应答,但口服途径诱导免疫应答的强度明显强于肌肉注射途径。口服携带HBV DNA疫苗的减毒伤寒沙门菌可能代表一种简便、有效的治疗乙型肝炎的新方法。      

信号肽和辅助性T细胞表位增强HBV核心抗原DNA疫苗诱导的免疫应答

为增强HBVDNA疫苗的免疫效率 ,于HBV核心抗原 (HBcAg)基因 5′末端引入人IL 2信号肽和一个通用型辅助性T淋巴细胞表位基因 ,并构建成DNA疫苗 ,转染COS7细胞后经ELISA检测出分泌型HBcAg。通过肌肉注射途径分别将这种DNA疫苗和编码天然HBcAg的DNA疫苗免疫BALB/c小鼠 ,检测小鼠的血清抗体、T细胞增殖和细胞毒性T淋巴细胞反应 ,结果表明前者诱导细胞和体液免疫应答的强度均明显超过后者 ,且更趋向于T辅助细胞 1(Th1)型免疫应答 ,故其对慢性HBV感染的治疗可能有潜在的应用价值     

CpG对乙型肝炎基因重组(CHO细胞)疫苗免疫效果的影响

为了研究CpG-寡脱氧核苷酸(CpG-OPN)作为佐剂对乙型肝炎基因重组(CHO细胞)疫苗(简称乙肝疫苗)免疫效果的影响,以乙肝疫苗加Al(OH)3、疫苗加CpG和疫苗加Al(OH)3与CpG3三种配伍方式,通过腹腔、皮下或肌内3种不同途径免疫Balb/c小鼠,观察不同免疫途径和不同配伍的免疫效果.同时又将疫苗与CpG混合后在4℃存放6个月再免疫小鼠,观察CpG的稳定性.结果表明:①3种免疫途径中以肌内注射效果最好,这在使用CpG的实验组尤为明显,在该组肌内免疫的ED50比腹腔的低了10倍,而诱发的抗体滴度提高了3倍;②疫苗与CpG、Al(OH)3联合使用的免疫效果最好,在肌内免疫时联合使用的免疫效果比疫苗+Al(OH)3提高4倍,比疫苗+CpG提高7倍;③疫苗+Al(OH)3免疫时,表现为IgG1抗体亚型占优势,而再加入CpG后则IgG1和IgG2a均升高,以IgG2a最显著;④疫苗与CpG混合后4℃保存半年,不影响其活性.     

Heterotypic Humoral and Cellular Immune Responses following Norwalk Virus Infection

Norovirus immunity is poorly understood as the limited data available on protection after infection are often contradictory. In contrast to the more prominent GII noroviruses, GI norovirus infections are less frequent in outbreaks. The GI noroviruses display very complex patterns of heterotypic immune responses following infection, and many individuals are highly susceptible to reinfection. To study the immune responses and mechanisms of GI.1 persistence, we built structural models and recombinant virus-like particles (VLPs) of five GI strains: GI.1-1968, GI.1-2001, GI.2-1999, GI.3-1999, and GI.4-2000. Structural models of four GI genotype capsid P domain dimers suggested that intragenotype structural variation is limited, that the GI binding pocket is mostly preserved between genotypes, and that a conserved, surface-exposed epitope may allow for highly cross-reactive immune responses. GI VLPs bound to histo-blood group antigens (HBGAs) including fucose, Lewis, and A antigens. Volunteers infected with GI.1-1968 (n = 10) had significant increases between prechallenge and convalescent reactive IgG for all five GI VLPs measured by enzyme immunoassay. Potential cross-neutralization of GI VLPs was demonstrated by convalescent-phase serum cross-blockade of GI VLP-HBGA interaction. Although group responses were significant for all GI VLPs, each individual volunteer demonstrated a unique VLP blockade pattern. Further, peripheral blood mononuclear cells (PBMCs) were stimulated with each of the VLPs, and secretion of gamma interferon (IFN-γ) was measured. As seen with blockade responses, IFN-γ secretion responses differed by individual. Sixty percent responded to at least one GI VLP, with only two volunteers responding to GI.1 VLP. Importantly, four of five individuals with sufficient PBMCs for cross-reactivity studies responded more robustly to other GI VLPs. These data suggest that preexposure history and deceptive imprinting may complicate PBMC and B-cell immune responses in some GI.1-1968-challenged individuals and highlight a potential complication in the design of efficacious norovirus vaccines.Noroviruses are the second-most important cause of severe viral gastroenteritis in young children and cause approximately 20% of endemic familial diarrheal disease and traveler''s diarrhea in all ages (reviewed in references 45 and 70). Noroviruses are genetically grouped into five different genogroups (GI to GV). GI and GII genogroups are responsible for the majority of human infections and are subdivided into more than 25 different genotypes (for example, GI.1 is genogroup I genotype 1). Most norovirus outbreaks are caused by the GII.4 genotype (65). Although genogroup I strains are associated with fewer reported outbreaks, they are frequently identified in environmental samples and in children (7, 21, 33, 58, 74, 82). The severity of norovirus disease is usually moderate although infection can be especially virulent, even fatal, in the elderly (14, 24, 31, 38, 46, 67). An effective vaccine would be particularly advantageous to vulnerable older populations, food handlers, child and health care providers, and military personnel. One major obstacle to norovirus vaccine development is the lack of understanding of the extensive antigenic relationships between heterogenic norovirus family members and of how this antigenic heterogeneity affects host protective immunity. Norovirus heterogeneity can be examined through sequence, structural, ligand binding, and host immune studies.Structurally, noroviruses are ∼38-nm icosahedral viruses with an ∼7.5 kb single-stranded, positive-sense RNA genome that encodes three large open reading frames (ORFs). ORF1 encodes the replicase polyprotein, while ORFs 2 and 3 encode the major and minor capsid proteins, respectively. The ORF2 major capsid protein sequence can vary by up to 60% between genogroups and by ∼20 to 30% between the genotypes (91). Expression of the major capsid protein (ORF2) in baculovirus and Venezuelan equine encephalitis (VEE) results in formation of virus-like particles (VLPs) composed of 180 copies of the monomeric protein (72). The monomer is structurally divided into the shell domain (S) that forms the structural core of the particle and the protruding domain (P) that protrudes away from the core. The P domain is further subdivided into the P1 subdomain (residues 226 to 278 and 406 to 520) and the P2 subdomain (residues 279 to 405) (72). P2 represents the most exposed surface of the viral particle and determines interaction with both potential neutralizing antibody recognition sites and putative cellular receptors, the histo-blood group antigens (HBGAs) (13, 16, 54, 57).The P domain has been shown to independently form dimers and P particles comprised of 12 monomers (85). Dimers and P particles share structural and HBGA binding similarities with the VLP generated with the same monomers (9, 85, 87). Three norovirus-HBGA binding profiles have been identified: (i) those that bind A/B and/or H epitopes, (ii) those that bind Lewis and/or H epitopes, and (iii) those that do not bind any available HBGA (86). Elegant structural analyses of Norwalk virus VLPs in complex with synthetic HBGAs identified a highly conserved binding site within the G1 noroviruses and predicted that structural constraints within the GI strains would restrict HBGA binding patterns to either a terminal Gal-Fuc or GalNAc (18, 88).Norwalk virus (NV; GI.1-1968) is the prototypic GI strain and typically infects individuals who encode a functional FUT2 α-1,2-fucosyltransferase enzyme resulting in expression of HBGAs on mucosal surfaces (secretor-positive phenotype) (53). Individuals who do not encode a functional FUT2 enzyme have a secretor-negative phenotype, do not express ABH HBGAs on mucosal surfaces, and are resistant to NV infection. Outbreak investigations have confirmed the association between HBGA expression and norovirus infection for some GI and GII strains (37, 39, 43, 49, 89). It remains likely that enzymes other than FUT2 may function as norovirus susceptibility factors because secretor-negative individuals have low-level norovirus-reactive antibodies (49, 52, 53) and can become infected after challenge with a GII.2 strain (52); in addition, some norovirus strains bind to FUT2-independent HBGAs in vitro (35, 54, 79).Early challenge studies (reviewed in reference 50) suggested that short-term protective immunity may occur following NV challenge (96). Demonstration of long-term protective immunity has been more complex. One early rechallenge study found that 50% of NV-challenged volunteers experienced repeat infections after ∼3 years while the other 50% remained well initially and after repeated challenge (69). Whether these volunteers remained disease free because of acquired immunity or genetic resistance could not be ascertained (69). However, contemporary norovirus challenge studies suggest that an early mucosal IgA response is associated with protection from NV infection (53). Further, strong gamma interferon (IFN-γ) secretion from CD4⁺ T cells (52) was identified in some uninfected GII.2-1976-challenged volunteers.In the absence of additional rechallenge studies, the most compelling evidence for a long-term protective immune response comes from the growing number of reports from around the world indicating that periods of “high norovirus activity” correlated with the emergence of new GII.4 strains (1, 10, 42, 66, 75, 90). Subsequently, the years following the high activity were characterized by decreased numbers of outbreaks, indicating that herd immunity may be an important regulator of GII.4 noroviruses (54, 80, 81). Clearly, the molecular basis for differential protective immunity/susceptibility following repeat norovirus infection is complex and a major challenge for the field.In this report, we compare the VLP phenotypes of the prototypical norovirus strain NV to an extant GI.1 strain isolated 33 years after NV and to a panel of VLPs representing strains GI.2, GI.3, and GI.4. In the results, we evaluate sequence conservation, carbohydrate (CHO) binding patterns, and antigenic relatedness at the antibody and T-cell levels. In contrast to earlier predictions (19), these data suggest that the GI noroviruses can bind many different HBGAs and that individuals infected with norovirus usually mount robust B- and T-cell responses against homologous strains. Surprisingly, some individuals appear to preferentially mount immune responses against heterologous GI strains.     

HCV/HBV复合抗原基因的克隆、表达及免疫应答研究

为探讨ＨＣＶ／ＨＢＶ 复合疫苗的可行性,将合成的丙型肝炎病毒（ＨＣＶ）复合多表位抗原基因ＰＣＸ与ＨＢｓＡｇ 基因连接成ＰＣＸＳ基因,与β－半乳糖苷酶（ＧＺ）基因融合后在大肠杆菌及减毒鼠伤寒沙门氏菌中获得表达．目的蛋白ＧＺ－ＰＣＸＳ可被抗－ＨＢｓ 及抗－ＨＣＶ 抗体所特异识别．ＧＺ－ＰＣＸＳ抗原皮下注射免疫ＩＣＲ小鼠后,诱发了较高水平的抗－ＧＺ－ＰＣＸＳＩｇＧ反应．构建的重组减毒鼠伤寒沙门氏菌ＳＬ３２６１（ｐＷＲ／ＰＣＸＳ）口服免疫小鼠后,诱发了高水平的ＣＤ８＋ Ｔ细胞增殖反应及抗ＧＺ－ＰＣＸＳＩｇＧ反应．所有免疫小鼠均未见明显的毒副作用．该研究揭示,ＨＣＶ／ＨＢＶ 复合抗原可诱发特异性体液免疫及细胞免疫应答,而活菌苗口服可能是理想的免疫途径,为ＨＣＶ／ＨＢＶ 双价疫苗研究提供了一定的理论及实验依据．     

Induction and Evasion of Innate Antiviral Responses by Hepatitis C Virus

Persistent hepatitis C virus infection is associated with progressive hepatic fibrosis and liver cancer. Acute infection evokes several distinct innate immune responses, but these are partially or completely countered by the virus. Hepatitis C virus proteins serve dual functions in replication and immune evasion, acting to disrupt cellular signaling pathways leading to interferon synthesis, subvert Jak-STAT signaling to limit expression of interferon-stimulated genes, and block antiviral activities of interferon-stimulated genes. The net effect is a multilayered evasion of innate immunity, which negatively influences the subsequent development of antigen-specific adaptive immunity, thereby contributing to virus persistence and resistance to therapy.     

Enhancement of Hepatitis B Virus Infection by Noninfectious Subviral Particles

The biological function of the huge excess of subviral particles over virions in hepatitis B virus infections is unknown. Using the duck hepatitis B virus as a model, we unexpectedly found that subviral particles strongly enhance intracellular viral replication and gene expression. This effect is dependent on the multiplicity of infection, the ratio of virions over subviral particles, and the time point of addition of subviral particles. Most importantly, we show that the pre-S protein of the subviral particles triggers enhancement and requires the presence of the binding regions for putative cell-encoded virus receptor proteins. These data suggest that enhancement is due either to the recently described transactivation function of the pre-S protein or to signalling pathways which become activated upon binding of subviral particles to cellular receptors. The findings are of clinical importance, since they imply that infectivity of sera containing hepadnaviruses depends not only on the amount of infectious virions but also decisively on the number of particles devoid of nucleic acids. A similarly dramatic enhancing effect of noninfectious particles in other virus infections is well conceivable.     

HCV编码病毒结构蛋白的DNA免疫研究

应用HCVC (pC)和HCVCE1E2 (pCE1E2 )重组体转染真核细胞并且通过肌注免疫BALB/C小鼠 ,对其体液免疫和细胞免疫进行检测。所用的 pC和 pCE1E2 均可在真核细胞内表达出特异性HCVC蛋白 ;肌注DNA免疫后均可诱导出BALB/C小鼠的体液和细胞免疫反应 ,抗体反应的A值 :pC组为 0 .358± 0 .0 96 ,pCE1E2 组为 0 .4 15± 0 .12 7;CTL活力 pC组为 18.6 5%± 5.72 % ,pCE1E2 组为 2 0 .0 7%± 11.11% ;通过免疫鼠荷瘤检测体内CTL反应 ,可观察到免疫组鼠发瘤时间滞后 ,发瘤部位减少和存活时间延长。在开发和研制HCV疫苗的过程中 ,DNA免疫是在快速构建、评价和筛选免疫原方面的有效办法。     

HCV编码病毒结构蛋白的DNA免疫研究

应用HCV C (pC)和HCV CE     

Silica Nanoparticles as the Adjuvant for the Immunisation of Mice Using Hepatitis B Core Virus-Like Particles

Advances in nanotechnology and nanomaterials have facilitated the development of silicon dioxide, or Silica, particles as a promising immunological adjuvant for the generation of novel prophylactic and therapeutic vaccines. In the present study, we have compared the adjuvanting potential of commercially available Silica nanoparticles (initial particles size of 10–20 nm) with that of aluminium hydroxide, or Alum, as well as that of complete and incomplete Freund''s adjuvants for the immunisation of BALB/c mice with virus-like particles (VLPs) formed by recombinant full-length Hepatitis B virus core (HBc) protein. The induction of B-cell and T-cell responses was studied after immunisation. Silica nanoparticles were able to adsorb maximally 40% of the added HBc, whereas the adsorption capacity of Alum exceeded 90% at the same VLPs/adjuvant ratio. Both Silica and Alum formed large complexes with HBc VLPs that sedimented rapidly after formulation, as detected by dynamic light scattering, spectrophotometry, and electron microscopy. Both Silica and Alum augmented the humoral response against HBc VLPs to the high anti-HBc level in the case of intraperitoneal immunisation, whereas in subcutaneous immunisation, the Silica-adjuvanted anti-HBc level even exceeded the level adjuvanted by Alum. The adjuvanting of HBc VLPs by Silica resulted in the same typical IgG2a/IgG1 ratios as in the case of the adjuvanting by Alum. The combination of Silica with monophosphoryl lipid A (MPL) led to the same enhancement of the HBc-specific T-cell induction as in the case of the Alum and MPL combination. These findings demonstrate that Silica is not a weaker putative adjuvant than Alum for induction of B-cell and T-cell responses against recombinant HBc VLPs. This finding may have an essential impact on the development of the set of Silica-adjuvanted vaccines based on a long list of HBc-derived virus-like particles as the biological component.     

HBV包膜-核心蛋白融合基因DNA疫苗诱导小鼠持久的细胞免疫应答

构建编码HBV包膜-核心蛋白融合基因的DNA疫苗pSC、pSS1S2C和编码HBV包膜蛋白或核心蛋白基因的DNA疫苗pHBs、pHBc,分别肌肉注射免疫BALB/c小鼠,检测小鼠的血清抗体、T细胞增殖和细胞毒性T淋巴细胞反应,比较融合基因DNA疫苗与单基因DNA疫苗诱生免疫应答的强度,发现融合基因DNA疫苗诱生抗体的效率明显不及单基因DNA疫苗,但其能诱导更强、更持久的细胞免疫应答,表明HBV包膜-核心蛋白融合基因DNA疫苗对于治疗慢性乙型肝炎可能比单基因DNA疫苗更为有效.     

IL-12增强HBV融合抗原DNA疫苗在小鼠诱导的细胞免疫应答

乙型肝炎病毒(hepatitis B virus,HBV)极易形成慢性感染,主要机制在于感染者不能产生强有力的细胞免疫应答以清除病毒[1].慢性HBV感染者体内虽然存在HBV抗原特异性T淋巴细胞,但对HBV抗原的反应性较低.研究发现,增强这类T淋巴细胞的反应性,可以促进HBV的清除[2].