[Institutional delivery rate in minority inhabited areas of China from 1996 to 2017].

Objective: To describe the secular trends of institutional delivery (ID) rate in minority inhabited areas of China from 1996 to 2017 according to national health policies. Methods: The number of live births and IDs for each county/district in 31 provinces of China were derived from the datasets collected by the Office for National Maternal & Child Health Statistics of China. Information on health policies and ethnical areas was derived from official governmental websites. The calendar years were divided into three periods: pre-program period (1996 to 1999), program implementation period (2000 to 2008) and post-program period (2009 to 2017). Minority autonomous regions, autonomous prefectures, and autonomous counties were defined as minority inhabited areas. The ethnic that a county was classified into was determined by a principle of close proximity to the name of the county or its next higher level administrative division. A total of 700 counties in minority inhabited areas were included in the analysis. Results: A total of 45 684 265 live births including 35 098 855 delivered in institutions were analyzed. The ID rate in minority inhabited areas was 37.5% (696 221/1 856 164) in 1996 and 99.2% (2 371 209/2 390 131) in 2017, with an annual growth rate of 4.7%. During the 22-years period, the ID rates in the eastern, central and western regions increased simultaneously, with the annual growth rates of 3.1%, 4.2% and 4.9% respectively. The difference between the eastern and western regions decreased steadily from 16% in 1996 to <1% in 2017 and the difference between the urban and rural areas decreased from 32.1% in 1996 to <1% in 2017. Besides, the ID rates in Tibetan and Yi inhabited areas with lower baseline levels increased 73 and 63 percentage points respectively. The number of counties with the ID rate of <96% were substantially reduced from 589 in 1996 to 72 in 2017; the 71 counties were all located in national deep poverty-stricken areas named Three Districts and Three States, predominantly involving Tibetan (58), Yi (6), Uygur (2) and Lisu (2) ethnics. Conclusion: During the past 22 years, the ID rate in minority inhabited areas in China has dramatically increased, achieving the goal of 2 020 ahead of schedule, but there remains a few western counties where ID rates are still<96%, indicating that minority inhabited western areas should be focused in developing national policies concerning institutional delivery.

[HIV epidemic among pregnant women in China, 2016: trend and spatial analysis].

Objective: To describe the spatial distribution characteristics of the HIV prevalence among pregnant women in mainland China in 2016, providing scientific evidence for the prevention of mother-to-child transmission of HIV. Methods: Data on pregnant women and those living with HIV in 2016 for all counties in mainland China is from the National Maternal & Child Health Statistics dataset. To obtain robust estimates, 2 964 counties were merged into 344 cities. Spatial autocorrelation analysis and trend analysis were performed based on the city-level dataset to detailedly describe the characteristics of the spatial distribution. Results: A total of 14 879 082 pregnant women were included in the analysis, among whom 5 051 were diagnosed to be infected with HIV, giving an overall prevalence of 34.0 per 100 000 pregnant women. The prevalence was higher in the south than in the north, and decreased from the west (93.5/100 000) to the east(8.6/100 000 ), more specifically, the prevalence in the West region was 11 times as high as that in the East region(χ(trend)(2)=68.61, P<0.01). Stratified analysis by provinces showed that there were 6 provinces whose prevalence was >50.0 per 100 000, and they (Yunnan, Xinjiang, Sichuan, Guangxi, Guizhou and Chongqing) were all located in the West Region; pregnant women in these provinces accounted for 21% of all pregnant women, but the HIV cases accounted for 76% of all cases diagnosed in mainland China. Stratified analysis by cities showed that there were 30 cities whose prevalence was >100.0 per 100 000, and 28 of these cities were also located in the western provinces above. Furthermore, the global Moran's I (0.5, P<0.01) indeed indicated a strong clustered distribution across mainland China; 2 hot spots were observed in the Midwest of Xinjiang, and Yunnan and its bordering areas (Sichuan, Guizhou, Guangxi and Chongqing), while 1 cold spot in the central and east China. The HIV prevalence in the hot spots (183.6/100 000) was 23 times as much as that in the cold spot (8.1/100 000). Conclusion: The overall HIV prevalence for pregnant women who lived in mainland China in 2016 (34.0/100 000) ranked at low-level worldwide, but varied markedly across the whole country with 2 high-prevalence-clustered areas: the Midwest of Xinjiang Uygur Autonomous Region, and Yunnan province along with its bordering areas, indicating comprehensive intervention strategies especially targeted to the areas with high HIV prevalence should be developed.

[Secular trends of institutional delivery rate in China from 1996 to 2015].

Objective: To describe the secular trends of institutional delivery (ID) rate in China from 1996 to 2015, and to assess the impacts of national health policies on the ID rate. Methods: Data on the number of live births and IDs for districts/counties in 31 provinces of China was annually collected by the Office for National Maternal & Child Health Statistics of China. Information concerning the relevant policies was from official governmental websites, including the programme to reduce maternal mortality and eliminate neonatal tetanus (2000 to 2008), and ID subsidy programme in rural China (2009 to present). According to the programme to reduce maternal mortality and eliminate neonatal tetanus, the calendar years were categorized into three periods: pre-programme period (1996 to 1999), programme implementation period (2000 to 2008) and post-programme period (2009 to 2015). Results: A total of 244 398 010 live births were included in the analysis, in which 211 605 727 were delivered in institutions. During the 20 years, the ID rate steadily increased from 58.7% (6 309 255/10 739 816) in 1996 to 99.7% (13 583 658/13 626 948) in 2015, with a compound annual growth rate of 2.8%. Analyses stratified by economic regions or urban-rural areas showed notably consistent increases in ID rates, and the regional and urban-rural differences became nearly disappeared by 2015. The largest regional difference between East (71.6%, 2 540 896/3 547 423) and West (44.6%, 1 675 305/3 752 873) was 27% in 1996 and <1% in 2015 (East 99.9%[5 177 865/5 180 636]and West 99.0%[3 925 766/3 964 622]). The urban-rural difference was 22.7% in 1996 (urban 73.5%[2 756 531/3 748 703], rural 50.8%[3 552 724/6 991 113]) and 0.4% in 2015(urban 99.9%[6 257 853/6 262 763], rural 99.5%[7 325 805/7 364 185]). During the programme implementation period and the post-programme period, the ID rates in rural area increased faster than those in urban area, and the corresponding compound annual growth rates in rural area were 2.4 and 2.8 times of those in urban area; the ID rates in Middle and West regions increased faster than those in East region, and the corresponding compound annual growth rates in West region were 3.6 and 6.3 times of those in East region. By 2015, the ID rates in all provinces other than Tibet (90.5%[48 445/53 505]) and Qinghai (97.2%[60 836/62 600]) reached or were close to 100%. However, there were still 112 districts/counties with ID rates <96%, of which 39 with ID rates <80%; the 39 districts/counties were all located in four western provinces (Tibet 19, Sichuan 15, Qinghai 3, and Xinjiang 2). Conclusions: During the past 20 years, the ID rate in China has steadily increased and achieved the goal of the year 2020 ahead of schedule; the regional and urban-rural inequality in ID has nearly disappeared. Given universal two-child policy, it is of significance to strengthen existing achievements, focus on complicated pregnancies and comprehensively improve the capability and quality of ID services; meanwhile, it is also of significance to develop particular policies and explore the medical-aid model for the minority-inhabited western regions with lower ID rates.

Enhanced antitumor immunotherapeutic effect of B-cell-based vaccine transduced with modified adenoviral vector containing type 35 fiber structures.

For successful clinical tumor immunotherapy outcomes, strong immune responses against tumor antigens must be generated. Cell-based vaccines compromise one strategy with which to induce appropriate strong immune responses. Previously, we established a natural killer T-cell (NKT) ligand-loaded, adenoviral vector-transduced B-cell-based anticancer cellular vaccine. To enhance tumor antigen delivery to B cells, we established a modified adenoviral vector (Ad-k35) that encoded a truncated form of the breast cancer antigen Her2/neu (Ad-k35HM) in which fiber structure was substituted with adenovirus serotype 35. We observed increased tumor antigen expression with Ad-k35HM in both human and murine B cells. In addition, an Ad-k35HM-transduced B-cell vaccine elicited strong antigen-specific cellular and humoral immune responses that were further enhanced with the additional loading of soluble NKT ligand KBC009. An Ad-k35HM-transduced, KBC009-loaded B-cell vaccine efficiently suppressed the in vivo growth of established tumors in a mouse model. Moreover, the vaccine elicited human leukocyte antigen (HLA)-A2 epitope-specific cytotoxic T-cell responses in B6.Cg (CB)-Tg (HLA-A/H2-D) 2Enge/Jat mice. These findings indicated that the Ad-k35 could be appropriate for the preclinical and clinical development of B-cell-based anticancer immunotherapies.

Tuberculous biliary stricture.

Tuberculosis (TB) of the hepatobiliary system is not uncommon, but as a cause of biliary strictures, it is very rare. It poses difficulty in diagnosis and often requires surgical intervention to exclude underlying malignancy. To our knowledge, there are fewer than 20 reported cases in the English literature. We report a 35-year-old Filipino woman who presented with a 3-day history of obstructive jaundice, associated with significant weight loss and anorexia. Computed tomography (CT) revealed dilated intrahepatic biliary system secondary to distal stricture at the confluence of the left and right bile ducts. Magnetic resonance cholangiopancreatography characterised the lesion as an irregular stricturing at several sites in the common bile duct. Incidentally, the scans also showed indeterminate pulmonary nodules in the right lower lobes. CT thorax confirmed bilateral involvement of the lungs. She required percutaneous transhepatic drainage for biliary decompression. Tests on tissue from the lung lesions, the blood, and the bile all confirmed the presence of TB. She was treated with anti-TB medication. This report emphasizes the importance of considering TB as a possibile cause of biliary stricture, especially in South-East Asia.

Anti-allergic effects of PG102, a water-soluble extract prepared from Actinidia arguta, in a murine ovalbumin-induced asthma model.

BACKGROUND: Asthma is a chronic inflammatory disease of the lung and its incidence has been increasing around the world. We previously reported that oral administration of a water-soluble extract prepared from Actinidia arguta, code-named PG102, could modulate the level of Th1 and Th2 cytokines and suppress the production of immunoglobulin E (IgE) in the ovalbumin (OVA)-immunized murine model as well as in the in vitro cell culture system, and furthermore could significantly improve dermatitis conditions in the NC/Nga murine model. These data suggested that PG102 might have therapeutic effects in a broad range of allergic diseases. OBJECTIVE: To assess the possible anti-allergic effects of PG102 in the OVA-induced murine asthma model. METHODS: The quality of PG102 was standardized, using its effects on the production of IgE, IL-5, and IL-13, in in vitro cell culture systems. To test effects on asthma, BALB/c mice were orally administrated with PG102, followed by OVA sensitization and challenge to induce asthmatic symptoms. Airway hyperresponsiveness (AHR), bronchoalveolar lavage fluid, serum, and lung tissue were analysed by using various methods. RESULTS: PG102 could decrease the level of IgE, IL-5, and IL-13 in in vitro cell culture systems with IC(50) being 1.12-1.43 mg/mL. PG102 could ameliorate asthmatic symptoms, including AHR and eosinophilia in the lungs. Such improvement of asthmatic symptoms by PG102 was accompanied by the down-regulation of IL-5 and IgE. In PG102-treated mice, high level expression of heme oxygenase-1, a potent anti-inflammatory enzyme, was observed in alveolar inflammatory cells, while the mRNA levels of foxp3, TGF-beta1, and IL-10, important markers for regulatory T cells, were also up-regulated in the lung tissue. CONCLUSIONS: PG102 may have potential as a safe and effective reagent for the prevention or treatment of asthma.

How good are we at advising appropriate patients with glaucoma to inform the DVLA? A closed audit loop.

PURPOSE: To establish how good we are as clinicians at advising glaucoma patients with bilateral visual field defects of their legal responsibility to inform the Driver and Vehicle Licensing Agency (DVLA). By using a sticker placed in the patients' notes to highlight driving status and visual fields, we sought to improve our success in providing and documenting this advice. METHODS: We interviewed and examined the notes of two groups of 100 consecutive glaucoma patients before and after the introduction of a 'driver sticker' placed into patients' notes at the time of visual field testing. We examined the documentation of driving status, and the provision and documentation of advice regarding the DVLA. RESULTS: In the first audit, we found only 9% of patients had driving status documented. Only 20% of drivers with bilateral field defects were advised to inform the DVLA with 11.4% documentation of this advice. After the introduction of the sticker, we succeeded in improving the documentation of driving status to 99%. We advised and documented the advice to inform the DVLA in 97% of drivers with bilateral field defects. CONCLUSIONS: We found that as a unit we were poor at documenting driving status and advising glaucoma patients with bilateral field defects to inform the DVLA. By the simple measure of introducing a sticker into patients' notes, we were able to highlight this critical group and improve our provision and documentation of appropriate advice regarding informing the DVLA.

Immunogenicity and safety profiles of genetic vaccines against human Her-2/neu in cynomolgus monkeys.

Her-2/neu is a well-characterized tumor-associated antigen, the overexpression of which in human carcinomas correlates with a poor prognosis. Here, we evaluated Her-2/neu-specific humoral and cellular immune responses in immunized monkeys after immunization with nonreplicating adenovirus (AdHM) expressing the extracellular and transmembrane domain of human Her-2/neu (HM) and/or naked DNA vaccine (pHM-hGM-CSF) expressing human granulocyte-macrophage colony-stimulating factor together with HM. Priming of monkeys with AdHM generated Her-2/neu-specific long-lasting antibody production. Furthermore, these Her-2/neu-specific antibodies produced by AdHM immunization, some of which shared epitope specificity with Herceptin, were able to induce antibody-dependent cellular cytotoxicity against Her-2-expressing target cells. Cellular immune responses were elicited in all monkeys immunized with Her-2/neu-expressing vaccine; interferon-gamma was secreted when these splenocytes were restimulated with Her-2/neu-expressing autologous cells, and immunization with AdHM induced Her-2/neu-specific lymphoproliferative responses. Further, immunization with pHM-hGM-CSF before AdHM immunization noticeably enhanced cytotoxic T-lymphocyte activity. In addition, we observed no abnormalities that would indicate that the genetic vaccines had toxic effects in the immunized monkeys. Thus, we can conclude that our genetic vaccines efficiently elicited Her-2/neu-specific humoral and cellular immune responses without causing severe adverse effects in nonhuman primates and that as such they warrant further clinical investigation.

Sentinel node detection with radiocolloid lymphatic mapping in early invasive cervical cancer.

We assessed the feasibility of sentinel lymph node detection using technicium-99 radiocolloid lymphatic mapping for predicting lymph node metastases in early invasive cervical cancer. Thirty patients with cervical cancer (stages IA2-IIA) underwent preoperative lymphoscintigraphy using technicium-99 intracervical injection and intraoperative lymphatic mapping with a handheld gamma probe. After dissection of the sentinel nodes, the standard procedure of pelvic lymph node dissection and radical hysterectomy was performed as usual. The sentinel node detection rate was 100% (30/30). There were seven (23.3%) cases of microscopic lymph node metastases on pathologic analysis. All of them had sentinel node involvement. Therefore, the sensitivity of sentinel node identification for prediction of lymph node metastases was 100%, and no false negative was found. Preoperative lymphoscintigraphy, coupled with intraoperative lymphatic mapping, located the sentinel nodes accurately in our study patients. This sentinel node detection method appears to be feasible for predicting lymph node metastases.

Three-dimensional power Doppler imaging of early-stage cervical cancer.

OBJECTIVES: To characterize intratumoral vascularization in early-stage cervical cancer by three-dimensional (3D) power Doppler ultrasound. METHODS: One hundred and forty-one patients with carcinoma of the uterine cervix and 30 normal controls were studied by transvaginal 3D power Doppler ultrasound. The tumor volume of the cervical cancer was determined. The blood flow within the tumor or normal cervix was measured and expressed as the vascularization index (VI), flow index (FI) and vascularization flow index (VFI). RESULTS: Of the 141 patients with cervical cancer, 44 patients had undergone prior cervical conization. Eighty-seven patients had measurable cervical tumors, of whom five had had prior conization. Abundant intratumoral power Doppler signals could be detected, and the VI, FI and VFI were significantly elevated in cervical cancer patients compared with women with a normal cervix and patients in whom no cervical tumor could be detected (P < 0.05, one-way ANOVA). We observed four types of intratumoral vascularity patterns, which did not significantly differ in VI, FI and VFI: localized, peripheral, scattered and single-vessel types. Cervical tumor volume was positively correlated with FI (linear regression, r = 0.373, P = 0.001), but not with VI or VFI. CONCLUSIONS: 3D power Doppler ultrasound provides a useful tool to investigate intratumoral vascularization and volume of cervical cancer.

Mapping B-cell epitopes of hepatitis C virus E2 glycoprotein using human monoclonal antibodies from phage display libraries.

Clinical and experimental evidence indicates that the hepatitis C virus (HCV) E2 glycoprotein (HCV/E2) is the most promising candidate for the development of an effective anti-HCV vaccine. Identification of the human epitopes that are conserved among isolates and are able to elicit protective antibodies would constitute a significant step forward. This work describes the mapping of the B-cell epitopes present on the surface of HCV/E2, as recognized by the immune system during infection, by the analysis of the reciprocal interactions of a panel of human recombinant Fabs derived from an HCV-infected patient. Three unrelated epitopes recognized by antibodies with no neutralization-of-binding (NOB) activity were identified; a fourth, major epitope was defined as a clustering of minor epitopes recognized by Fabs endowed with strong NOB activity.

A broad-spectrum caspase inhibitor blocks concanavalin A-induced hepatitis in mice.

Fulminant hepatic failure (FHF) is a clinical syndrome resulting from massive death of liver cells or sudden and severe impairment of liver function. The causes of FHF are diverse and the overall mortality is very high. Recently, it became clear that apoptosis of hepatocytes is the critical cause of acute hepatic failure in FHF. It is well known that a family of cysteine proteases called caspase is one of the key mediators of the apoptotic pathway. Thus, caspases are attractive potential targets for the treatment of disorders resulting from excessive apoptosis. In this report, we examined the activity of a new caspase inhibitor, Xyz 033 mp. This nonpeptide inhibitor showed broad-spectrum caspase-inhibiting activity and protected primary rat hepatocytes from apoptotic death. In a mouse model of FHF induced by concavalin A (Con A), Xyz 033 mp suppressed elevated AST and ALT and specifically reduced IL-1 beta concentration. Also, Xyz 033 mp rescued mice from lethal experimental hepatitis induced by Con A. In addition, histological examinations indicated that Xyz 033 mp protected hepatocytes from the fatal apoptogenic effect of Con A. These results suggest that Xyz 033 mp may be a candidate therapeutic agent for FHF caused by massive apoptotic death of hepatocytes.

The HIV-1 Env protein signal sequence retards its cleavage and down-regulates the glycoprotein folding.

Secretory proteins and most membrane proteins are synthesized with a signal sequence that is usually cleaved from the nascent polypeptide chain, during its transport, into the lumen of the endoplasmic reticulum (ER). We have analyzed the kinetics of the cleavage of the HIV-1 Env protein signal sequence from gp160 and gp120 in HeLa, BHK, and Jurkat cells. Furthermore, we have determined the effects of this cleavage on the association of the gp160 and gp120 glycoproteins with the ER protein calnexin and the effects of the signal sequence cleavage on protein folding. The cleavage of the HIV-1 Env protein signal sequence on both gp160 and gp120 occurred very slowly in all three cell lines with a t(1/2) of 45-60 min. The core glycosylated and signal-sequence-retained forms of gp160 and gp120 associated with calnexin while the signal-sequence-cleaved forms of gp160 and gp120 had disassociated from calnexin and correctly folded as determined by their ability to associate with the CD4 cellular receptor. Further analysis of the folding state of gp160 and gp120 in nonreducing SDS-PAGE revealed that the signal-sequence-retained and calnexin-associated forms of gp160 and gp120 migrated as broad, diffuse bands, whereas the signal-sequence-cleaved or CD4-associated forms of gp160 and gp120 migrated as single sharper bands. The cause of this retardation in the rate of folding and intracellular transport of HIV-1 glycoproteins was localized to their signal sequences by fusing the vesicular stomatitis virus G protein with the HIV-1 Env protein signal sequence and expressing this chimeric protein in mammalian cells. The HIV-1 Env protein signal sequence on the VSV-G protein also confers a reduced rate of cleavage and slow intracellular transport and folding of the chimeric G protein. These results provide direct evidence that in vivo the HIV-1 glycoprotein signal sequence inhibits the folding of HIV-1 Env protein. Our data also suggest a direct correlation between the rate of the signal sequence cleavage and protein folding.

A humanized anti--4-1BB monoclonal antibody suppresses antigen-induced humoral immune response in nonhuman primates.

The interaction of 4-1BB and its ligand plays an important role in the regulation of T-cell-mediated immune responses. In this study, the authors examined the effect of a humanized anti--4-1BB monoclonal antibody (H4B4) on ovalbumin-induced immune responses in baboons. Previously, a mouse monoclonal antibody, 4B4 against the human 4-1BB molecule, was generated and characterized. Based on this antibody, a humanized version of 4B4 monoclonal antibody was constructed and the resultant antibody, H4B4, showed full recovery of the binding activity of the original antibody 4B4: a 1.5-fold increase in affinity for 4-1BB. In addition, H4B4 mediated antibody-dependent cellular cytotoxicity of activated human peripheral blood T cells and CEM cells in a dose-dependent manner. Weekly administration of H4B4 at doses of 1 or 4 mg/kg could suppress immunoglobulin G production against ovalbumin. This was not a result of the overall immune suppression, because the numbers of B and T cells and the total immunoglobulin G production were not altered during treatment with H4B4. These findings suggest that treatment with H4B4 may be a valid therapeutic approach to control unwanted immune responses in persons with autoimmune diseases.

Kinetic analysis of oral tolerance: memory lymphocytes are refractory to oral tolerance.

Oral administration of soluble Ag before immunization induces peripheral tolerance and is effective in suppressing animal models of autoimmune diseases. Although tolerance induction in primed animals is more clinically relevant, it is not well studied. Therefore, this study was designed to examine the feeding effects on different phases of the immune response. We observed that feeding a single high dose (250 mg) of OVA to OVA-primed BALB/c mice could induce OVA-specific suppression in the Ab production and T cell proliferation only at the naive and the activation phases of the immune response, whereas multiple high doses (100 mg/feed for 10 days) were effective at the effector phase. OVA-specific IL-4 production in culture supernatant was also suppressed in the tolerized groups. However, when the mice had resting memory lymphocytes, even multiple feeding regimens were not effective in tolerance induction, although multiple low doses (1 mg/feed for 10 days) partially suppressed Ab production. This phenomenon was confirmed by adoptive transfer study. Nevertheless, the reactivated memory response was suppressed partially by multiple high doses. Our findings have an important implication for understanding the mechanism of oral tolerance and for the therapeutic applications of oral tolerance to autoimmune diseases.

Oral tolerance by a high dose OVA in BALB/c mice is more pronounced and persistent in Th2-mediated immune responses than in Th1 responses.

Oral administration of antigen induces an antigen-specific immunologic tolerance and many studies are being carried out to apply this phenomenon to the treatment of autoimmune diseases. In this study, we investigated long-term Th1 and Th2 tolerance in mice given a high dose of orally administered Ovalbumin (OVA). Feeding OVA to BALB/c mice suppressed OVA-specific IgG response and the degree of inhibition was dose-dependent in the range of 2.5-250 mg. Moreover, the state of tolerance established by prior feeding of high dose of OVA was present after 26 weeks. Interestingly, even though both Th subsets were tolerized significantly for a short period, the tolerizing effect was more pronounced and persistent in Th2-mediated immune responses. Thus we speculate that oral administration of a single high dose of OVA induces Th1- and Th2-tolerance by different mechanisms. Our findings could be important in the development of therapeutics for the treatment of autoimmune disease and allergy.

Development of HIV/AIDS vaccine using chimeric gag-env virus-like particles.

We attempted to develop a candidate HIV/AIDS vaccine, by using unprocessed HIV-2 gag pr45 precursor protein. We found that a 45 kDa unprocessed HIV-2 gag precursor protein (pr45), with a deletion of a portion of the viral protease, assembles as virus-like particles (VLP). We mapped the functional domain of HIV-2 gag VLP formation in order to find the minimum length of gag protein to form VLP. A series of deletion mutants was constructed by sequentially removing the C-terminal region of HIV-2 gag precursor protein and expressed truncated genes in Spodoptera frugiperda (SF) cells by infecting recombinant baculoviruses. We found that deletion of up to 143 amino acids at the C-terminus of HIV-2 gag, leaving 376 amino acids at the N-terminus of the protein, did not affect VLP formation. There is a proline-rich region at the amino acid positions 373 to 377 of HIV-2 gag, and replacement of these proline residues by site-directed mutagenesis completely abolished VLP assembly. Our data demonstrate that the C-terminal p12 region of HIV-2 gag precursor protein, and zinc finger domains, are dispensable for gag VLP assembly, but the presence of at least one of the three prolines at amino acid positions 373, 375 or 377 of HIV-2NIH-Z is required for VLP formation. Animals immunized with these gag particles produced high titer antibodies and Western blot analyses showed that anti-gag pr45 rabbit sera react with p17, p24 and p55 gag proteins of HIV-1. We then constructed chimeric gag genes, which carry the hypervariable V3 region of HIV-1 gp120, because the V3 loop is known to interact with chemokine receptor as a coreceptor, and known to induce the major neutralizing antibodies and stimulate the cytoxic T lymphocyte responses in humans and mice. We expressed chimeric fusion protein of HIV-2 gag with 3 tandem copies of consensus V3 domain that were derived from 245 different isolates of HIV-1. In addition, we also constructed and expressed chimeric fusion protein that contains HIV-2 gag with V3 domains of HIV-1IIIB, HIV-1MN, HIV-1SF2 and HIV-1RF. The chimeric gag-env particles had a spherical morphology, and the size was slightly larger than that of a gag particle. Immunoprecipitation and Western blot analyses show that these chimeric proteins were recognized by HIV-1 positive human sera and antisera raised against V3 peptides, as well as by rabbit anti-gp120 serum. We obtained virus neutralizing antibodies in rabbits by immunizing these gag-env VLPs. In addition, we found that gag-env chimeric VLPs induce a strong CTL activity against V3 peptide-treated target cells. Our results indicate that V3 peptides from all major clades of HIV-1 carried by HIV-2 gag can be used as a potential HIV/AIDS vaccine.

Production and characterization of monoclonal antibodies specific to atrazine group compounds: effects of coating ligand structure on the variation of sensitivity and specificity.

Hybridoma cells were prepared by immunizing mice with carboxylic derivatives of atrazine conjugate to bovine serum albumin. After the screening of culture supernatant of hybridomas, five cell lines producing monoclonal antibodies were established and 1.8-5.3 ml of ascitic fluid per mouse was obtained from each cell line. The protein A affinity purification yielded 0.35-0.65 mg per ml of ascitic fluid from each cell line. The characterization studies in terms of sensitivity and specificity indicate that MAb 2F9 and MAb 4B9 showed the best responses with atrazine and its group of ametryne and cyanazine, using microtiter plate coated with simazine derivative of 6-amino hexanoic acid; no cross-reactivity was shown with simazine and cyanuric chloride.

Identification of a domain containing B-cell epitopes in hepatitis C virus E2 glycoprotein by using mouse monoclonal antibodies.

Evidence from clinical and experimental studies of human and chimpanzees suggests that hepatitis C virus (HCV) envelope glycoprotein E2 is a key antigen for developing a vaccine against HCV infection. To identify B-cell epitopes in HCV E2, six murine monoclonal antibodies (MAbs), CET-1 to -6, specific for HCV E2 protein were generated by using recombinant proteins containing E2t (a C-terminally truncated domain of HCV E2 [amino acids 386 to 693] fused to human growth hormone and glycoprotein D). We tested whether HCV-infected sera were able to inhibit the binding of CET MAbs to the former fusion protein. Inhibitory activity was observed in most sera tested, which indicated that CET-1 to -6 were similar to anti-E2 antibodies in human sera with respect to the epitope specificity. The spacial relationship of epitopes on E2 recognized by CET MAbs was determined by surface plasmon resonance analysis and competitive enzyme-linked immunosorbent assay. The data indicated that three overlapping epitopes were recognized by CET-1 to -6. For mapping the epitopes recognized by CET MAbs, we analyzed the reactivities of CET MAbs to six truncated forms and two chimeric forms of recombinant E2 proteins. The data suggest that the epitopes recognized by CET-1 to -6 are located in a small domain of E2 spanning amino acid residues 528 to 546.

Induction of V3-specific cytotoxic T lymphocyte responses by HIV gag particles carrying multiple immunodominant V3 epitopes of gp120.

Effort to develop a vaccine to prevent infection of human immunodeficiency virus (HIV) have focused on the induction of neutralizing antibodies. In our previous study, we reported that chimeric gag-env virus-like particles (VLPs) induce neutralizing antibodies which block HIV infection. In addition to the neutralizing antibodies, the cytotoxic T-lymphocyte (CTL) response is considered to be another major immune defense mechanism required for recovery from many different viral infections. In the present study, we have constructed chimeric fusion proteins using HIV-2 gag precursor protein with (1) four neutralizing epitopes from HIV-1 gp160; (2) three tandem copies of consensus V3 domain, which have been derived from 245 different isolates of HIV-1 and carries both the principal neutralizing determinant (PND) and CTL epitopes; and (3) V3 domains from HIV-1IIIB, HIV-1MN, HIV-1RF, and HIV-1SF2. These chimeric fusion proteins were expressed in a large quantity within insect cells, and released as VLPs into the cell culture medium. The purified gag-env VLPs from all three constructs appear to be spherical particles similar to immature HIV but slightly larger than the gag VLPs. Immunoprecipitation analysis showed that the chimeric proteins were recognized not only by HIV-1 positive patient sera, but also by monoclonal and polyclonal antisera raised against V3 peptides of HIV-1IIIB, HIV-1MN, HIV-1RF, and the gp120 antiserum against HIV-1SF2. Balb/C mice immunized with these chimeric VLPs successfully induced CTL activity against V3 peptide-stimulated target cells. In addition, a high degree of cross-reactivity was observed among the four different strains of HIV-1 V3 domain, indicating that the tandem multiple consensus V3 peptide sequence carried by HIV-2 gag can be used as a potential HIV vaccine against various HIVs.