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2008 Vol.23(4)

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Research Article

Comparison of the Immunogenicities of HIV-1 Mutants Based on Structural Modification of env

Jian-hui NIE, Chun-tao ZHANG, Hui-hui CHONG, Xue-ling WU, Chun-yu LIU, Yu WU, Chen-yan ZHAO, Lin-qi ZHANG, You-chun WANG

2008, 23(4): 233 doi: 10.1007/s12250-008-2949-4

Received: 20 February 2008 Accepted: 01 April 2008
Eleven env mutants were designed and generated by site-directed mutagenesis of the regions around NAb epitopes and deletions of variable regions in env. The immunogenicities of the generated mutants were evaluated using single-cycle infection neutralization assays with two pseudoviruses and IFN-γ ELISPOT. Overall, five mutants (dWt, M2, M5-2, M5-1 and dM7) induced higher neutralization activities for both pseudoviruses than plasmid Wt, while only two of the mutants (dWt and M5-2) showed significant differences (P<0.05). Two mutants (M2 and dM2) induced more Env-specific T cells than plasmid Wt. Statistically however, significance was only reached for mutant M2. Thus, properly modified HIV-1 Env may have the potential to induce potent cellular and humoral immune responses.

Expression of Major Antigen Domains of E2 Gene of CSFV and Analysis of its Immunological Activity *

Hong TIAN, Xiang-tao LIU, Jing-yan WU, You-jun SHANG, Tao JIANG, Hai-xue ZHENG, Qing-ge XIE

2008, 23(4): 247 doi: 10.1007/s12250-008-2956-5

Received: 21 March 2008 Accepted: 30 May 2008
E2 is an envelope glycoprotein of Classical swine fever virus (CSFV) and contains sequential neutralizing epitopes to induce virus-neutralizing antibodies and mount protective immunity in the natural host. In this study, four antigen domains (ABCD) of the E2 gene was cloned from CSFV Shimen strain into the retroviral vector pBABE puro and expressed in eukaryotic cell (PK15) by an retroviral gene expression system, and the activity of recombinant E2 protein to induce immune responses was evaluated in rabbits. The results indicated that recombinant E2 protein can be recognized by fluorescence antibodies of CSFV and CSFV positive serum (Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China) using Western blot, indirect immunofluorescence antibody test (IFAT) and ELISA, Furthermore, anti-CSFV specific antibodies and lymphocyte proliferation were elicited and increased by recombinant protein after vaccination. In the challenge test, all of rabbits vaccinated with recombinant protein and Chinese vaccine strain (C-strain) were fully protected from a rabbit spleen virus challenge. These results indicated that a retroviral-based epitope-vaccine carrying the major antigen domains of E2 is able to induce high level of epitope-specific antibodies and exhibits similar protective capability with that induced by the C-strain, and encourages further work towards the development of a vaccine against CSFV infection.

HIV-1 Vif Protein Mediates the Degradation of APOBEC3G in Fission Yeast When Over-expressed Using Codon Optimization

Lin LI, Jing-yun LI, Hong-shuai SUI, Richard Y. Zhao, Yong-jian LIU, Zuo-yi BAO, Si-yang LIU, Dao-min ZHUANG

2008, 23(4): 255 doi: 10.1007/s12250-008-2957-4

Received: 17 March 2008 Accepted: 04 May 2008
Interaction between the HIV-1 Vif protein and the cellular host APOBEC3G protein is a promising target for inhibition of HIV-1 replication. Considering that human cells are a very complicated environment for the study of protein interactions, the goal of this study was to check whether fission yeast could be used as a model cell for studying the Vif-APOBEC3G interaction. Vif and APOBEC3G were expressed in fusion with GFP protein in the S. pombe SP223 strain. Subcellular localizations of Vif and APOBEC3G were observed with fluorescent microscopy. Codon optimization was used to over express the Vif protein in S. pombe cells. The degradation of APOBEC3G mediated by Vif was tested through expressing Vif and GFP-APOBEC3G proteins in the same cell. Western Blot analysis was used to measure the corresponding protein levels under different experimental conditions. The results showed that the Vif protein was predominantly localized in the nucleus of S. pombe cells, APOBEC3G was localized in the cytoplasm and concentrated at punctate bodies that were often in close proximity to the nucleus but were not necessarily restricted from other regions in the cytoplasm. Vif protein expression levels were increased significantly by using codon optimization and APOBEC3G was degraded when Vif was over-expressed in the same S. pombe cells. These results indicate that fission yeast is a good model for studying the interaction between the Vif and APOBEC3G proteins.

PCR Detection and Sequence Analysis of Duck Circovirus in Sick Muscovy Ducks

Shi-jin JIANG, Xing-xiao ZHANG, Shao-ning LIU, Yu WANG, Yi-bo KONG, Xiu-li WEI, Ya-ni SUN, Qin ZHAO

2008, 23(4): 265 doi: 10.1007/s12250-008-2934-y

Received: 15 January 2008 Accepted: 28 April 2008
The duck circovirus (DuCV) infection in sick ducks from Fujian Province was investigated. The liver samples of 43 sick Muscovy ducks with infectious serositis were collected from 12 duck farms in Fujian Province. Based on the published sequences of DuCV, two primers were designed for the detection of DuCV and four pairs of primers were designed to amplify four overlapping fragments that cover the complete genome of DuCV. The specific PCR products were amplified from positive samples. The fragments were then cloned into pMD18-T vector and sequenced, and the full length genomic sequence of the FJ0601 isolate of DuCV was obtained. PCR analysis showed that the proportion of ducks which were positive for circovirus was 79% and 10 out of the 12 farms were positive. Sequence analysis showed that the complete genome of DuCV-FJ0601 was 1988 bp and possessed features common to the family Circoviridae which included a stem-loop structure and the Rep protein motifs. Homology analysis showed that FJ0601 isolate of DuCV had 97.3%~97.5% nucleotide sequence identity to all the four Taiwan isolates (TC1/2002, TC2/2002, TC3/2002, TC4/2002), 82.9% identity to the America (33753-52) isolate and 82.3% identity to the Germany isolate. Phylogenetic analysis with ClustalW, however, showed that FJ0601 isolate of DuCV was on a common branch with Taiwan isolates, and Germany and America isolates belonged to the other branch .

Biological Analysis of HSV-1 Immediate-early Proteins ICP0, ICP22, and ICP27 in Neuroblastoma Cells

Lei WANG, Yan-chun CHE, Wei CUN, Wei-zhong LI, Yun LIAO, Long-ding LIU, Qi-han LI

2008, 23(4): 272 doi: 10.1007/s12250-008-2937-8

Received: 29 January 2008 Accepted: 10 June 2008
The three immediate-early proteins of HSV-1, ICP0, ICP22, and ICP27, have specific and pivotal functions in transcriptional activation and inhibition, multiple regulatory and control processes of viral genes. In this paper, the expression and localization of these three proteins were studied in neuroblastoma cells using biochemical assays, and their possible and potential interactive functions are discussed. The data show that the three proteins are localized in different structures, specifically in the PML-NB-associated structure, which is a specific nuclear structure composed of many protein molecules and bound tightly to the nuclear matrix in neuroblastoma cells. The results suggest that the activating and suppressive functions of ICPs are mostly dependent on their transcriptional and regulatory roles, including the PML-NB-associated structure.

Bioinformatic Analysis of Structural Proteins of Paramyxovirus Tianjin Strain

Li-ying SHI, Mei LI, Xiao-mian LI, Li-jun YUAN, Qing WANG

2008, 23(4): 279 doi: 10.1007/s12250-008-2947-6

Received: 19 February 2008 Accepted: 04 June 2008
The amino acid sequences of the NP, P, M, F, HN and L proteins of the paramyxovirus Tianjin strain were analyzed by using the bioinformatics methods. Phylogenetic analysis based on 6 structural proteins among the Tianjin strain and 25 paramyxoviruses showed that the Tianjin strain belonged to the genus Respirovirus, in the subfamily Paramyxovirinae, and was most closely related to Sendai virus (SeV). Phylogenetic analysis with 14 known SeVs showed that Tianjin strain represented a new evolutionary lineage. Similarities comparisons indicated that Tianjin strain P protein was poorly conserved, sharing only 78.7%~91.9% amino acid identity with the known SeVs, while the L protein was the most conserved, having 96.0%~98.0% amino acid identity with the known SeVs. Alignments of amino acid sequences of 6 structural proteins clearly showed that Tianjin strain possessed many unique amino acid substitutions in their protein sequences, 15 in NP, 29 in P, 6 in M, 13 in F, 18 in HN, and 29 in L. These results revealed that Tianjin strain was most likely a new genotype of SeV. The presence of unique amino acid substitutions suggests that Tianjin strain maybe has a significant difference in biological, pathological, immunological, or epidemiological characteristics from the known SeVs

Antigenicity and Hemaglutination Activity of a Recombinant Hemagglutinin-Neuraminidase of Paramyxovirus Tianjin Strain

Mei LI, Li-jun YUAN, Li-ying SHI, Xiao-mian LI, Qing WANG, Wen-xiu WANG

2008, 23(4): 287 doi: 10.1007/s12250-008-2965-4

Received: 31 March 2008 Accepted: 26 April 2008
Paramyxovirus Tianjin strain, a new genotype of Sendai virus, was isolated from the lungs of common cotton-eared marmoset that died of severe respiratory infection in the marmoset colonies. The 19.28% IgM positive rate in the young children with acute respiratory tract infection suggested a close relationship between Tianjin strain and humans. Hemagglutinin-neuraminidase (HN) is its major transmembrane glycoprotein responsible for viral attachment, penetration and release. To clear the relationship between HN structure and function of paramyxovirus Tianjin strain, rHN1, rHN2 and rHN3 overlapping the ectodomain of HN protein were expressed. Their antigenicity and hemaglutination activity, as well as cross reactivity to standard antisera against influenza virus type A, type B were analyzed. The results indicated expressed rHNs have the natural antigenicity. The segment rHN2 possesses more linear epitopes exposed on the surface of the native HN protein than found in segments rHN3 and rHN1. The hemagglutination activity of segment rHN3 is higher than that of segments rHN2 and rHN1, and partially dependent on the three-dimensional conformation of HN3 protein. Cross-reactivity between rHNs and standard antisera against influenza virus type A, type B suggested that rHNs might not be the best alternative as specific antigens to detect virus in clinical serum specimens.

Immunogenicity of DNA and Recombinant Sendai Virus Vaccines Expressing the HIV-1 gag Gene

Xia FENG, Shuang-qing YU, Tsugumine Shu, Tetsuro Matano, Mamoru Hasegawa, Xiao-li WANG, Hong-tao MA, Hong-xia LI, Yi ZENG

2008, 23(4): 295 doi: 10.1007/s12250-008-2946-y

Received: 19 February 2008 Accepted: 24 June 2008
Combinations of DNA and recombinant-viral-vector based vaccines are promising AIDS vaccine methods because of their potential for inducing cellular immune responses. It was found that Gag-specific cytotoxic lymphocyte (CTL) responses were associated with lowering viremia in an untreated HIV-1 infected cohort. The main objectives of our studies were the construction of DNA and recombinant Sendai virus vector (rSeV) vaccines containing a gag gene from the prevalent Thailand subtype B strain in China and trying to use these vaccines for therapeutic and prophylactic vaccines. The candidate plasmid DNA vaccine pcDNA3.1(+)-gag and recombinant Sendai virus vaccine (rSeV-gag) were constructed separately. It was verified by Western blotting analysis that both DNA and rSeV-gag vaccines expressed the HIV-1 Gag protein correctly and efficiently. Balb/c mice were immunized with these two vaccines in different administration schemes. HIV-1 Gag-specific CTL responses and antibody levels were detected by intracellular cytokine staining assay and enzyme-linked immunosorbant assay (ELISA) respectively. Combined vaccines in a DNA prime/rSeV-gag boost vaccination regimen induced the strongest and most long-lasting Gag-specific CTL and antibody responses. It maintained relatively high levels even 9 weeks post immunization. This data indicated that the prime-boost regimen with DNA and rSeV-gag vaccines may offer promising HIV vaccine regimens.