Xianghui Yu

Controlled release of PEI/DNA complexes from mannose-bearing chitosan microspheres as a potent delivery system to enhance immune response to HBV DNA vaccine

n Abstractn n A novel approach involving the preparation of mannose-bearing chitosan microspheres with entrapping complexes of HBV DNA and PEI was developed to improve the delivery of DNA into antigen-presenting cells (APCs) after intramuscular (i.m.) injection. Compared with the traditional chitosan microspheres, the microspheres could quickly release intact and penetrative PEI/DNA complexes. Whats more, chitosan was modified with mannose to target the primary APCs such as dendritic cells (DCs) owing to the high density of mannose receptors expressing on the surface of immature DCs. After i.m. immunization, the microspheres induced significantly enhanced serum antibody and cytotoxic T lymphocyte (CTL) responses in comparison to naked DNA.n n

Identification of a Cullin5-ElonginB-ElonginC E3 Complex in Degradation of Feline Immunodeficiency Virus Vif-Mediated Feline APOBEC3 Proteins

ABSTRACT Various feline APOBEC3 (fA3) proteins exhibit broad antiviral activities against a wide range of viruses, such as feline immunodeficiency virus (FIV), feline foamy virus (FFV), and feline leukemia virus (FeLV), as well as those of other species. This activity can be counteracted by the FIV Vif protein, but the mechanism by which FIV Vif suppresses fA3s is unknown. In the present study, we demonstrated that FIV Vif could act via a proteasome-dependent pathway to overcome fA3s. FIV Vif interacted with feline cellular proteins Cullin5 (Cul5), ElonginB, and ElonginC to form an E3 complex to induce degradation of fA3s. Both the dominant-negative Cul5 mutant and a C-terminal hydrophilic replacement ElonginC mutant potently disrupted the FIV Vif activity against fA3s. Furthermore, we identified a BC-box motif in FIV Vif that was essential for the recruitment of E3 ubiquitin ligase and also required for FIV Vif-mediated degradation of fA3s. Moreover, despite the lack of either a Cul5-box or a HCCH zinc-binding motif, FIV Vif specifically selected Cul5. Therefore, FIV Vif may interact with Cul5 via a novel mechanism. These finding imply that SOCS proteins may possess distinct mechanisms to bind Cul5 during formation of the Elongin-Cullin-SOCS box complex.

A novel disulfide-stabilized single-chain variable antibody fragment against rabies virus G protein with enhanced in vivo neutralizing potency

Rabies is a fatal infectious disease requiring efficient protection provided by post-exposure prophylaxis (PEP) with rabies immunoglobulin (RIG). The single-chain Fv fragment (scFv) is a small engineered antigen binding protein derived from antibody variable heavy (V(H)) and light (V(L)) chains. This novel antibody format may potentially replace the current application of RIG to detect and neutralize rabies virus (RV). However, the broad use of scFvs is confined by their generally low stability. In this study, a scFv (FV57) was constructed based on the monoclonal antibody, MAB57, against RV. To enhance its stability and neutralizing potency, a disulfide-stabilized scFv, ds-FV57, was also derived by introduction of cysteines at V(H)44 and V(L)100. Furthermore, the cysteine at V(L)85 of ds-FV57 was mutated to serine to construct ds-FV57(VL85Ser) in order to avoid potential mis-formed disulfide bonds which would alter the affinity of the scFv. The stability and activity of all three proteins expressed in Escherichia coli were evaluated. All of the constructed scFvs could provide efficient protection against RV infection both in vivo and in vitro. However, the stability of ds-FV57(VL85Ser) was notably improved, and its in vitro neutralizing potency against RV infection was enhanced. Our findings from these stabilization modifications support the feasibility of developing scFvs for PEP treatment of rabies.

Seroprevalence of neutralizing antibodies to human adenovirus type 5 in healthy adults in China

Human adenoviruses have been used to develop candidate HIV vaccine vectors, but a major obstacle to the use of vectors derived from Ad5 and other common serotypes is the high prevalence of neutralizing antibodies (NAbs) in humans. Prior studies have reported a prevalence of Ad5 between 60% and 70% in Europe and US, and up to 98% in surveyed African and Asian (Thai) tropical countries. However, few studies have reported on the Ad5 prevalence in Chinese populations. In this study, a total of 1,250 healthy adult serum samples from six administratively separate regions of China were screened by high‐throughput luciferase‐based virus neutralization assays. Results showed that overall 72% of healthy adults were Ad5 seropositive, and 46.4% with baseline Ad5 NAb titers of >200 in China. Comprehensive analysis by geographical region and age showed that the seroprevalence of Ad5 in southern China such as Guangxi was higher than that in other regions. Geographical differences and climate were considered as the major factors affecting the titer levels of Ad5 in healthy adults. In addition, no apparent gender and ethnic difference was found in any group classified according to region, age, or NAb titer. The present study may provide useful insights for the future development of Ad5‐based vaccines and gene therapy. J. Med. Virol. 84:1408–1414, 2012.

DNA and adenovirus tumor vaccine expressing truncated survivin generates specific immune responses and anti-tumor effects in a murine melanoma model

Survivin is overexpressed in major types of cancer and is considered an ideal “universal” tumor-associated antigen that can be targeted by immunotherapeutic vaccines. However, its anti-apoptosis function raises certain safety concerns. Here, a new truncated human survivin, devoid of the anti-apoptosis function, was generated as a candidate tumor vaccine. Interleukin 2 (IL-2) has been widely used as an adjuvant for vaccination against various diseases. Meanwhile, the DNA prime and recombinant adenovirus (rAd) boost heterologous immunization strategy has been proven to be highly effective in enhancing immune responses. Therefore, the efficacy of a new cancer vaccine based on a truncated form of survivin, combined with IL-2, DNA prime, and rAd boost, was tested. As prophylaxis, immunization with the DNA vaccine alone resulted in a weak immune response and modest anti-tumor effect, whereas the tumor inhibition ratio with the DNA vaccine administered with IL-2 increased to 89xa0% and was further increased to nearly 100xa0% by rAd boosting. Moreover, complete tumor rejection was observed in 5 of 15 mice. Efficacy of the vaccine administered therapeutically was enhanced by nearly 300xa0% when combined with carboplatin. These results indicated that vaccination with a truncated survivin vaccine using DNA prime–rAd boost combined with IL-2 adjuvant and carboplatin represents an attractive strategy to overcoming immune tolerance to tumors and has potential therapeutic benefits in melanoma cancer.

Improvement of enzymatic stability and intestinal permeability of deuterohemin-peptide conjugates by specific multi-site N-methylation

The deuterohemin-peptide conjugate, DhHP-6 (Dh-β-AHTVEK-NH2), is a microperoxidase mimetic, which has demonstrated substantial benefits in vivo as a scavenger of reactive oxygen species (ROS). In this study, specific multi-site N-methylated derivatives of DhHP-6 were designed and synthesized to improve metabolic stability and intestinal absorption, which are important factors for oral delivery of therapeutic peptides and proteins. The DhHP-6 derivatives were tested for (1) scavenging potential of hydrogen peroxide (H2O2); (2) permeability across Caco-2 cell monolayers and everted gut sacs; and (3) enzymatic stability in serum and intestinal homogenate. The results indicated that the activities of the DhHP-6 derivatives were not influenced by N-methylation, and that tri-N-methylation of DhHP-6 could significantly increase intestinal flux, resulting in a two- to threefold higher apparent permeability coefficient. In addition, molecules with N-methylation at selected sites (e.g., Glu residue) showed high resistance against proteolytic degradation in both diluted serum and intestinal preparation, with 50- to 140-fold higher half-life values. These findings suggest that the DhHP-6 derivatives with appropriate N-methylation could retain activity levels equivalent to that of the parent peptide, while showing enhanced intestinal permeability and stability against enzymatic degradation. The tri-N-methylated peptide Dh-β-AH(Me)T(Me)V(Me)EK-NH2 derived from this study may be developed as a promising candidate for oral administration.

Polarity Changes in the Transmembrane Domain Core of HIV-1 Vpu Inhibits Its Anti-Tetherin Activity

Tetherin (BST-2/CD317) is an interferon-inducible antiviral protein that restricts the release of enveloped viruses from infected cells. The HIV-1 accessory protein Vpu can efficiently antagonize this restriction. In this study, we analyzed mutations of the transmembrane (TM) domain of Vpu, including deletions and substitutions, to delineate amino acids important for HIV-1 viral particle release and in interactions with tetherin. The mutants had similar subcellular localization patterns with that of wild-type Vpu and were functional with respect to CD4 downregulation. We showed that the hydrophobic binding surface for tetherin lies in the core of the Vpu TM domain. Three consecutive hydrophobic isoleucine residues in the middle region of the Vpu TM domain, I15, I16 and I17, were important for stabilizing the tetherin binding interface and determining its sensitivity to tetherin. Changing the polarity of the amino acids at these positions resulted in severe impairment of Vpu-induced tetherin targeting and antagonism. Taken together, these data reveal a model of specific hydrophobic interactions between Vpu and tetherin, which can be potentially targeted in the development of novel anti-HIV-1 drugs.

Profile of physical status and gene variation of human papillomavirus 58 genome in cervical cancer

Epidemiological studies have shown that human papillomavirus 58 (HPV 58) is found at a relatively high frequency in east Asia and some regions of Central and South America. To investigate the physical status of HPV 58 and analyse sequence variations of HPV 58 in cervical cancer patients, the HPV 58 genome in 37 HPV 58-positive cervical cancer specimens collected from China were investigated by a mapping analysis based on nested PCR and nucleotide sequencing. A pure integrated genome was found in 78.4 % (29/37) of specimens, which is much higher than that found in previous studies. Multiple disruptions were first found among the integrated HPV 58 genomes in 51.7 % (15/29) of specimens. Among the 7824 bp of the HPV 58 genome, 119 (1.52 %) nucleotide positions were found to be variable, and 45 of them lead to amino acid changes. Phylogenetic analyses, based on partial L1 sequences of 14 variants isolated in previous studies and this study, show that two main groups were observed in HPV 58 variants, the prototype or prototype-like group and the non-prototype-like group.

Evaluation of high-risk Human papillomaviruses type distribution in cervical cancer in Sichuan province of China

BackgroundInfection with high-risk human papillomavirus is an important factor associated with cervical cancer, and the distribution of HPV types varies greatly worldwide. Determination of type-specific HPV prevalence constitutes an important step towards the development of vaccines for the prevention of cervical cancer.MethodsThe human papillomavirus (HPV) genotypes in 190 cervical cancer specimens taken from the Sichuan province, the most populous province of Southwest China, were detected by a combination of MY09/11 consensus primers PCR (MY09/11 PCR), type-specific primers one-step PCR (One-step TS PCR) and E6/E7 gene type-specific primers nested PCR (Nested TS PCR). The prevalence and distribution of HPV in patients with cervical cancer, especially for HPV types 16, 18, 52, 58 and 59, suspected to be most common in certain parts of China, was investigated.ResultsThe HPV infection rates detected by MY09/11 PCR, One-step TS PCR and Nested TS PCR were 159 (83.7%), 145 (76.3%) and 172 (90.5%), respectively. The overall HPV prevalence was 93.2% (177/190). The positive specimens for HPV16, 18, 52, 58 and 59 detected by One-step TS-PCR were 111 (58.4%), 14 (7.4%), 6 (3.2%), 13 (6.8%) and 4 (2.1%), respectively. By Nested TS-PCR analysis, the detection rates of HPV16, 52, 58 and 59 were increased to 140 (73.7%), 30 (15.8%), 37 (19.5%) and 25 (13.2%), while only 4 (2.1%) additional specimens were found to be infected with HPV18.ConclusionOur data demonstrate that, besides HPV 16, which was found to be the most prevalent type, HPV types 58, 52 and 59 are more prevalent than HPV18 in women with cervical cancer in the Sichuan area of China.

Co-expression of herpes simplex virus thymidine kinase and Escherichia coli nitroreductase by an hTERT-driven adenovirus vector in breast cancer cells results in additive anti-tumor effects.

Breast cancers especially in the late and metastatic stages remain refractory to treatment despite advances in surgical techniques and chemotherapy. Tumor-specific promoter-directed suicide gene therapy and adenoviral technology can be promising strategies for such advanced disease. Previous studies suggested that combining herpes simplex virus thymidine kinase (HSV-TK) and ganciclovir (GCV) with Escherichia coli nitroreductase (Coli.NTR) and 5-(azaridin-1-yl)-2, 4-dinitrobenzamide (CB1954) by a recombinant retrovirus delivery system resulted in a co-operative killing effect in vitro. We constructed a bicistronic adenovirus type 5 (Ad5)-based vector which co-expresses herpes HSV-TK and Coli.NTR under the control of the human telomerase reverse transcriptase (hTERT) promoter and SV40 enhancer. NTR gene expression mediated by an internal ribosome entry site (IRES) was inserted after the hTERT and HSV-TK sequences. Anti-tumor activities of the novel vector, Ad-hT-TK/NTR-enh, combined with prodrugs were evaluated in human breast cancer cells (ZR-75-30, MCF-7) in vitro and inxa0vivo. We showed that expression of HSV-TK and NTR genes by Ad-hT-TK/NTR-enh in combination with GCV and CB1954 resulted in specific and significant cytotoxic effects in breast cancer cells in vitro. The anti-tumor activity of this system was more efficient than that from a single suicide gene, and only slightly lower than by HSV-TK and NTR driven from separate hTERT promoters in vitro and in vivo while the total amount of adenovirus of Ad-hT-TK/NTR-enh was half that of Ad-hT-TK-enh+Ad-hT-NTR-enh. These results suggest that suicide genes HSV-TK and NTR mediated by a single adenovirus vector under the control of an enhanced hTERT promoter results in additive anti-tumor effects and may provide a relatively safe strategy for the treatment of breast cancer by tumor-specific targeting.