Evaluation of protection induced by a dengue virus serotype 2 envelope domain III protein scaffold/DNA vaccine in non-human primates

We describe the preclinical development of a dengue virus vaccine targeting the dengue virus serotype 2 (DENV2) envelope domain III (EDIII). This study provides proof-of-principle that a dengue EDIII protein scaffold/DNA vaccine can protect against dengue challenge. The dengue vaccine (EDIII-E2) is composed of both a protein particle and a DNA expression plasmid delivered simultaneously via intramuscular injection (protein) and gene gun (DNA) into rhesus macaques. The protein component can contain a maximum of 60 copies of EDIII presented on a multimeric scaffold of Geobacillus stearothermophilus E2 proteins. The DNA component is composed of the EDIII portion of the envelope gene cloned into an expression plasmid. The EDIII-E2 vaccine elicited robust antibody responses to DENV2, with neutralizing antibody responses detectable following the first boost and reaching titers of greater than 1:100,000 following the second and final boost. Vaccinated and naïve groups of macaques were challenged with DENV2. All vaccinated macaques were protected from detectable viremia by infectious assay, while naïve animals had detectable viremia for 2–7 days post-challenge. All naïve macaques had detectable viral RNA from day 2–10 post-challenge. In the EDIII-E2 group, three macaques were negative for viral RNA and three were found to have detectable viral RNA post challenge. Viremia onset was delayed and the duration was shortened relative to naïve controls. The presence of viral RNA post-challenge corresponded to a 10–30-fold boost in neutralization titers 28 days post challenge, whereas no boost was observed in the fully protected animals. Based on these results, we determine that pre-challenge 50% neutralization titers of >1:6000 correlated with sterilizing protection against DENV2 challenge in EDIII-E2 vaccinated macaques. Identification of the critical correlate of protection for the EDIII-E2 platform in the robust non-human primate model lays the groundwork for further development of a tetravalent EDIII-E2 dengue vaccine.     

D2-43病毒E蛋白在酵母细胞表面的展示

目的：在酵母细胞表面展示登革2型病毒43株(D2—43)的E基因，探索利用酵母表面展示系统建立DNA改组筛选平台的可行性。方法：通过RT-PCR扩增获得D2-43的E基因，将该基因亚克隆至T载体后，再克隆至酵母表面展示载体pYDI，于酿酒酵母EBY100中利用半乳糖进行诱导表达。表达产物采用间接免疫荧光法和FACS进行检测。结果：酵母表面展示产物可与D2-43的腹水抗体特异性地结合；在半乳糖诱导后24h，展示E蛋白的酵母细胞百分数达22．07％。结论：本研究为建立基于酵母表面展示系统的DNA改组筛选平台奠定了基础。     

我国登革2型病毒43株包膜E蛋白基因的特征

对我国1987年流行的登革2型病毒43株包膜E蛋白基因的核苷酸序列进行了分析。结果表明登革2型病毒43株包膜E蛋白基因核苷酸序列含1485个核苷酸，编码495个氨基酸，并就其核苷酸序列及其相应的氨基酸序列与其它的登革2型病毒株进行了比较，发现核苷酸序列与我国1985年分离的登革2型病毒04株，新几内亚C株（NGC），牙买加株1409（JAM）和马来西亚当地流行株M1（登革出血热）、血2（登革休克综合征）、M3（登革热）同源性分别是95.8%、94.6%、97.5%、925%、92.7%和939%，氨基酸序列的同源性分别是94.3%、94.3%、96.0%、93.7%、93.7%和91.5%，推断出的氨基酸序列显示出12个保守的半胱氨酸残基和两个潜在的糖基化位点，分别位于Asn-67和Asn-153位。     

中国人丙型肝炎病毒囊膜蛋白2HVR1 cDNA的序列分析

目的：了解中国ＨＣＶ流行株高变区１（ＨＶＲ１）的特点。方法：对来自山东（ＳＤ）、上海（ＳＨ）两地患者血清ＲＮＡ进行逆转录－巢式ＰＣＲ，扩增ＨＣＶ囊膜蛋白２基因片段，用ＰＣＲ直接测序法对该片段进行序列测定。结果：（１）扩增片段（命名为Ｐ３８８）对应于日本ＨＣＶ－Ｊ株序列核苷酸１４３９￣１８２６位（氨基酸位３７１￣４９８）；（２）中国人ＨＣＶ ＨＶＲ１位于氨基酸位３８４￣４１０，与发表的ＨＣＶⅡ型ＨＶ     

Immunoglobulin mimicry by Hepatitis C Virus envelope protein E2

Hepatitis C virus (HCV) establishes persistent infection in the majority of infected individuals. The currently accepted hypothesis of immune evasion by antigenic variation in hypervariable region 1 (HVR1) of glycoprotein E2 does not however, explain the lack of subsequent immune recognition. Here, we show that the N-terminal region of E2 is antigenically and structurally similar to human immunoglobulin (Ig) variable domains. E2 is recognized by anti-human IgG antibodies and also possesses common amino acid (aa) sequence features of the conserved v-gene framework regions of human Ig light chains in particular but also heavy chains and T cell receptors. Using a position specific scoring system, the degree of similarity of HVR1 to Ig types correlated with immune escape and persistence in humans and experimentally infected chimpanzees. We propose a unique role for threshold levels of Ig molecular mimicry in HCV biology that not only advances our concept of viral immune escape and persistent infection but also provides insight into host-dependent disease patterns.     

Immunolocalization of lamins and nuclear pore complex proteins by atomic force microscopy

The nuclear envelope functions as a selective barrier separating the nuclear from the cytosolic compartment. Nuclear pore complexes (NPCs) mediate nuclear import and export of macromolecules and, therefore, are potential regulators of gene expression. In this study we applied atomic force microscopy (AFM) to visualize the three dimensional (3D) structure of individual NPCs in the absence and presence of two different antibodies, one directed against a pore protein (gp62) and another directed against Xenopus lamin LIII, a component of the nuclear lamina, a filament meshwork localized on the nucleoplasmic side of the nuclear envelope (NE) adjacent to and interacting with NPCs. Using 12-nm gold-labelled secondary antibodies and transmission electron microscopy we could clearly localize the primary single anti-gp62 antibody on NPCs and the primary single anti-LIII antibody between NPCs. Using AFM, the secondary antibodies against anti-gp62 could be detected as particles 7 nm in height on the nucleoplasmic face of NPCs. The secondary antibodies against anti-LIII could be clearly identified between NPCs. The secondary antibodies, attached to a 12-nm colloidal gold particle and visualized on glass, revealed similar shapes and heights as found on NEs. According to the 3D images, the volume of a single gold particle conjugated with secondary antibodies was 10 203 nm³. This volume is equivalent to the volume of 38 IgG molecules associated with one individual gold particle. A similar volume of 11 987 nm³ was calculated from a model assuming that the 150-kDa IgG molecules perfectly cover the spherical gold particle. We conclude that AFM can be used for identifying antibodies or other macromolecules associated with biomembranes.     

Recombinant gp120 induces IL-10 in resting peripheral blood mononuclear cells; correlation with the induction of other cytokines.

Immunological abnormalities present in HIV-1-infected individuals often reflect an imbalance of cytokine production. The HIV-1 gp120 has the ability to induce a number of cytokines, and to enhance immunoglobulin release by normal peripheral blood mononuclear cells (PBMC) in vitro, in the absence of IL-2 production and of lymphoproliferation. This study provides evidence that gp120 is a potent IL-10 inducer in normal PBMC cultures. The pattern of other cytokines induced by gp120 includes interferon-alpha (IFN-alpha) and IFN-gamma, tumour necrosis factor-alpha (TNF-alpha), IL-6, IL-1 alpha and -beta, and not IL-2 and IL-4. These findings further define the pattern of cytokine release induced by gp120 on human resting PBMC. Furthermore, the present findings roughly parallel those observed both in the sera of patients and in the mononuclear cells from HIV+ individuals early after infection, suggesting that gp120 could be a good candidate as one of the agents responsible for cytokine dysregulation observed in HIV-1-infected individuals.     

Hepatitis B surface antigen particles of subtypes adw and adr, and compound subtype (adwr) in symptom-free carriers in Japan.

Of sera from 1,878 Japanese blood donors who carried hepatitis B surface antigen (HBsAg), 420 were subtyped as adw (22.4%) and 1,443 as adr (76.8%); only 15 (0.8%) contained HBsAg of subtype ayw or ayr. Sera with HBsAg/adr had higher HBsAg titres than those with HBsAg/adw (geometric mean of haemagglutination titre: 10.1 +/- 2.4 vs. 9.7 +/- 2.4, p less than 0.01), and a higher prevalence of hepatitis B e antigen (24% vs. 13%, p less than 0.001). Carriers of HBsAg/adr progressively predominated over those of HBsAg/adw with increasing age. Of sera from 1,863 carriers of HBsAg/adw or HBsAg/adr, 182 (9.8%) contained HBsAg particles with both subtypic determinants in the w/r allele. The presence of w and r determinants on the same particles was ascertained by sandwiching them between monoclonal antibody with the specificity for w and that with the specificity for r. HBsAg particles of compound subtype (adwr) were found more often in sera with hepatitis B e antigen than those without it (145/403 [36.0%] vs. 37/1,460 [2.5%], p less than 0.001). Sera with HBsAg/adwr particles had HBsAg titres higher than those without them (12.4 +/- 1.9 vs. 9.7 +/- 2.3, p less than 0.001). HBsAg/adwr particles arise from phenotypic mixing of the S-gene product of wild-type virus and that of mutants with point mutations for subtypic changes. The results obtained indicated that HBV strains of subtype adr have a higher replicative activity than those of adw, and suggested that mutations in the S gene for subtypic changes would be associated with an active replication of hepatitis B virus.     

Sequence variation within the human immunodeficiency virus V3 loop at seroconversion

Analysis of the HIV-1 V3 quasispecies present in an individual at the time of seroconversion was carried out. The polymerase chain reaction (PCR) was used to amplify proviral HIV-1 DNA extracted from peripheral blood mononuclear cells from a patient who was viraemic (p24 = 15 pg/ml) and had an equivocal HIV-1 antibody status. The PCR products were cloned and the DNA sequence determined for 15 clones. These data showed that the V3 region contained only limited sequence heterogeneity with a major variant accounting for 66% of the protein quasispecies present. The protein sequence of the principal neutralising domain on all species contained the relatively rare GPGKTL motif rather than GPGRAF. The relevance of these data for early stages of HIV infection are discussed.     

Dissociated antibody responses to the S and pre-S2 regions of the hepatitis B virus after vaccination in hemophiliacs

The membranes of hepatocytes and the pre-S2 envelope protein of the hepatitis B virus (HBV) contain binding sites for polymerized human albumin, which is thought to act as a link between HBV and hepatocytes. Hence, anti-pre-S2 antibodies should prevent HBV uptake by the liver, and there is indeed preliminary evidence that they protect chimpanzees from HBV infection. To evaluate whether a plasma-derived vaccine containing the pre-S2 sequence induced an anti-pre-S2 response in 105 vaccinated hemophiliacs, anti-pre-S2 was measured in parallel with antibody to hepatitis B surface antigen (anti-HBs). Eighty-five percent of the hemophiliacs had both anti-pre-S2 and anti-HBs when vaccination was completed, 13% had anti-HBs alone, and 2% (two cases) had anti-pre-S2 alone. Eighty-seven percent of anti-pre-S2-positive hemophiliacs compared with only 50% of anti-pre-S2-negative hemophiliacs (P less than 0.001) developed high anti-HBs titers (greater than or equal to 1,000 mlU/ml). This study demonstrates, therefore, that the antibody responses to the S and pre-S2 regions of HBV may be dissociated after vaccination in hemophiliacs and that higher anti-HBs titers are attained in anti-pre-S2-positive hemophiliacs.