Establishment of a vimentin knockout and HIV-1 gp120 transgenic mouse model / 南方医科大学学报

OBJECTIVE@#To construct a HIV-1 gp120 transgenic mice (gp120 Tg) with vimentin (VIM) gene knockout.@*METHODS@#Female HIV-1 gp120 Tg mice were mated to VIM heterozygote mice (F0). All the offspring mice were derived from these original founders so that both genotypes had the same mixed genetic background. The F1 mice were bred to generate of VIM, VIM, VIM/gp120 Tg and VIM/gp120 Tg mice. PCR was performed for genotyping of the mice, and the expressions of VIM and gp120 in the brain tissues were examined using immunoblotting.@*RESULTS@#The results of PCR showed the presence of the target bands in VIM, VIM, VIM/gp120 Tg and VIM/gp120 Tg mice. In VIM/gp120 Tg mice, gp120 expression was detected throughout the brain regions while no VIM expression was detected.@*CONCLUSIONS@#We generated gp120 transgenic mouse models with VIM gene knockout, which facilitate the exploration of the role of VIM in gp120-induced neurotoxicity.

SBi4211 alleviates gp120-induced central nervous system injury / 南方医科大学学报

OBJECTIVE@#To explore the protective effect of SBi4211 (heptamidine), an inhibitor of S100B, against central nervous system injury induced by HIV-1 envelope protein gp120.@*METHODS@#In an @*RESULTS@#In the cell co-culture system, SBi4211 treatment significantly inhibited gp120-induced expression of S100B, RAGE and GFAP in U251 cells (@*CONCLUSIONS@#SBi4211 can protect neurons from gp120-induced neurotoxicity possibly by inhibiting the S100B/ RAGE-mediated signaling pathway.

Antiviral Activity of Dual-acting Hydrocarbon-stapled Peptides against HIV-1 Predominantly Circulating in China / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>New rationally designed i,i+7-hydrocarbon-stapled peptides that target both HIV-1 assembly and entry have been shown to have antiviral activity against HIV-1 subtypes circulating in Europe and North America. Here, we aimed to evaluate the antiviral activity of these peptides against HIV-1 subtypes predominantly circulating in China.</p><p><b>METHODS</b>The antiviral activity of three i,i+7-hydrocarbon-stapled peptides, NYAD-36, NYAD-67, and NYAD-66, against primary HIV-1 CRF07_BC and CRF01_AE isolates was evaluated in peripheral blood mononuclear cells (PBMCs). The activity against the CRF07_BC and CRF01_AE Env-pseudotyped viruses was analyzed in TZM-bl cells.</p><p><b>RESULTS</b>We found that all the stapled peptides were effective in inhibiting infection by all the primary HIV-1 isolates tested, with 50% inhibitory concentration toward viral replication (IC50) in the low micromolar range. NYAD-36 and NYAD-67 showed better antiviral activity than NYAD-66 did. We further evaluated the sensitivity of CRF01_AE and CRF07_BC Env-pseudotyped viruses to these stapled peptides in a single-cycle virus infectivity assay. As observed with the primary isolates, the IC50s were in the low micromolar range, and NYAD-66 was less effective than NYAD-36 and NYAD-67.</p><p><b>CONCLUSION</b>Hydrocarbon-stapled peptides appear to have broad antiviral activity against the predominant HIV-1 viruses in China. This finding may provide the impetus to the rational design of peptides for future antiviral therapy.</p>

IL15 DNA adjuvant enhances cellular and humoral immune responses induced by DNA and adenoviral vectors encoding HIV-1 subtype B gp160 gene / 病毒学报

To enhance the immunogenicity of DNA and adenoviral vector vaccines expressing HIV-1 subtype B gp160, human interleukin 15 (hIL15) DNA adjuvant (pVR-hIL15) was constructed. BALB/c mice received DNA prime/protein boost immunization with pVR-HIVgp160/Ad5-HIVgp160 alone or combined with pVR-hIL15. Cellular and humoral immune responses were evaluated by IFN-gamma enzyme-linked immunosorbent spot assay and enzyme-linked immunosorbent assay, respectively. Compared with those immunized with vaccines alone, the mice immunized with vaccines combined with pVR-hIL15 had significantly increased specific cellular response and antibody titer (P < 0.05). It suggests that the IL15 DNA adjuvant can enhance the immune responses induced by prime-boost regimen using DNA and adenoviral vector encoding HIV-1 subtype B gp160.

Design of small molecules with HIV fusion inhibitory property based on Gp41 interaction assay

Gp41 of HIV [Human Immunodeficiency Virus] is a protein that mediates fusion between viral and cellular membranes. The agent, T-20, which has been approved for HIV inhibition, can restrain Gp41 function in the fusion process; nevertheless, it has disadvantages like instability, high cost of production and injection form to be delivered twice a day. Several molecules like NB-2 and NB-64 have been discovered that can inhibit HIV infection. These molecules were used as template compounds to design and develop more effective small molecules functioning as HIV-1 fusion inhibitors targeting Gp41. The process included in silico docking protocols using HEX and ArgusLab applications. A multisource database was created, after choosing the best molecules; they were tested in vitro for inhibitory activity by HIV-1 single-cycle model, transfected in HEK cells [293T]. Computational analysis and experimental data were combined to explore molecular properties and the most potent ones were found, with the best suitable criteria for interaction with Gp41. Several examples [DAA-6, DAA-9 and DAA-12] could inhibit infection in vitro as effective as NB-2, NB- 64. Since disadvantages of available fusion inhibitor [T-20], it seems necessary to find similar molecules to be approved and have small size providing suitable bioactivity profile. The molecules explored in this study can be good candidates for further investigations to be used as oral HIV fusion inhibitors in the future

Virtual screening of small molecular HIV-1 entry inhibitor NC-2 targeting gp120 and its action mechanism / 南方医科大学学报

<p><b>OBJECTIVE</b>To screen the HIV-1 entry inhibitors targeting HIV-1 gp120 from the IBS natural product database by virtual screening based on the binding mode of the neutralizing antibody VRC01 with HIV-1 gp120 and investigate the anti-viral activities of the inhibitors and their action mechanisms.</p><p><b>METHODS</b>The binding interaction of the candidate molecules binding gp120 and changes of the binding free energy were analyzed by MM-PBSA calculation. The anti-HIV-1 activities of the tested compounds were detected by HIV-1 pseudotyped virus, laboratory-adapted HIV-1 and a cell-cell fusion assay. The cytotoxicity of the studied molecules was examined by XTT colorimetric assay. The mechanisms of the anti-viral activities of the candidate molecules were analyzed using enzyme-linked immunosorbent assay.</p><p><b>RESULTS</b>A total of 19 molecules with distinct reduction of the binding free energy after binding with gp120 were screened from 40000 molecules. Among them, NC-2 showed anti-HIV-1 activities against HIV-1 pseudotyped virus and laboratory-adapted HIV-1, and was capable of blocking HIV-1 envelope-mediated cell-cell fusion. The IC50 of NC-2 for inhibiting HIV-1IIIB and pseudotyped HIV-1JRFL infection were 1.95∓0.44 µmol/L and 10.58∓0.13 µmol/L, respectively. The results of ELISA suggested that NC-2 could inhibit the binding of HIV-1 gp120 to CD4 without blocking the formation of gp41 six-helix bundle in vitro.</p><p><b>CONCLUSION</b>This computer-based virtual screening method can be used to screen HIV-1 entry inhibitors targeting gp120. Using this virtual screening approach combined with anti-viral activity screening, we obtained a potent HIV-1 entry inhibitor NC-2 with novel structure.</p>

Identification of Env-specific monoclonal antibodies from Chinese HIV-1 infected person by magnetic beads separating B cells and single cell RT-PCR cloning / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To establish a simple and practical method for screening of Env-specific monoclonal antibodies from HIV-1 infected individuals.</p><p><b>METHODS</b>Human B cells were purified by negative sorting from PBMCs and memory B cells were further enriched using anti-CD27 microbeads. Gp120 antigen labbled with biotin was incubated with memory B cells to specifically bind IgG on cells membrane. The memory B cells expressing the Env-specific antibody were harvested by magnetic beads separating, counted and diluted to the level of single cell in each PCR well that loading with catch buffer containing RNase inhibitor to get RNAs. The antibody genes were amplified by single cell RT-PCR and nested PCR, cloned into eukaryotic expression vectors and transfected into 293T cells. The binding activity of recombinant antibodies to Env were tested by ELISA.</p><p><b>RESULTS</b>Three monocolonal Env-specific antibodies were isolated from one HIV-1 infected individual.</p><p><b>CONCLUSION</b>We can obtain Env-specific antibody by biotin labbled antigen, magnetic beads separating technique coupled with single cell RT-PCR and expression cloning.</p>

Identification of human monoclonal HIV-1-neutralizing antibodies from phage antibody library by cell-based screening / 病毒学报

To identify human monoclonal HIV-l-neutralizing antibodies from an HIV-1 CRF07BC specific phage display antibody library by cell-based screening. 293T cells were transfected by pCH064. 2-Env plas mid and then used to biopan the phage antibody library. The positive phage clones were screened by cell based ELISA and sequenced for the variable region of heavy (VH) and light (VL) chains. The expressed Fabs were purified by Ni(+2) -NTA column and analyzed by SDS-PAGE. The cell- and gp120 protein-based ELISA as well as flow cytometry were used to measure Fab's binding activity. The neutralizing activity of Fabs was assessed by HIV-1 pseudoviruses. After 4-round biopanning, the binding phages to transfected cells were enriched about 650-folds. A total of 28 positive clones were screened out by cell ELISA and sequence analysis identified 5 different Fabs possessing unique VH and VL (2801, 2837, 2863, 2870 and 2920). Interestingly, these Fabs reacted with the Env-transfected 293T cells but not soluble gp120 proteins, suggesting that they might target conformation-dependent epitopes presenting on viral Env complex. We found that three Fabs (2801, 2863, 2870) exhibited potent neutralizing activity against CRF07_BC isolate CH120. 6 with IC50 of 2.24, 0.89 and 3.09 microg/mL respectively, and that 2801 and 2863 cross-neutral ized the subtype B isolate SF162 at IC50 of 0.69 and 3.52 microg/mL respectively. In conclusion, the HIV-1 Env-transfected 293T cells can be used to efficiently enrich and screen the phage antibody library and isolate human monoclonal HIV-1-neutralizing Fabs that target the Env complex-dependent conformational epitopes. Therefore, our studies provide a powerful platform for exploring the mechanism of HIV-1 neu tralizing response and for designing AIDS vaccines.

Cloning and eukaryotic expression of HIV-1B gp120 genes from a patient with AIDS dementia complex / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To clone and express the HIV-1B gp120 genes isolated at different organizations from a patient died of AIDS dementia complex (ADC) in eukaryotic cells.</p><p><b>METHODS</b>Using the genomic DNA isolated from peripheral lymphnodes, choroid plexus and occipital white matter from a patient died of ADC as the template, HIV-1B gp120 gene was amplified with PCR. After sequenced, HIV-1B gp120 was inserted into pcDNA3.1 (+) and recombinant expressing vector gp120/pcDNA3.1 (+) was constructed succeffuly confirming with sequencing. Then expressing vector was transfected into eukaryotic cells U87 using liposome transfection and expression of HIV-1B gp120 gene was assayed with indirect immunofluorescence.</p><p><b>RESULTS</b>HIV-1B gp120 genes isolated from peripheral lymphnodes, choroid plexus and occipital white matter of the ADC patient were successfully cloned and recombinant expressing vector gp120/pcDNA3; 1 (+) could express envelope glycoprotein HIV-1B gp120 in U87 cells.</p><p><b>CONCLUSION</b>All the HIV-1B gp120 gene isolated at the different organizations of the same ADC patient could express in U87 cells, which may supply a valuable basis for studying the neurotoxicity and neurotoxic mechanism of HIV-1 gp120 protein.</p>

Design and expression of an inhibitor for HIV-1 targeting dendritic cell / 生物工程学报

Human immunodeficiency virus (HIV) infects the host cells by the fusion of viral and cell membranes. Blocking the combining between HIV and the receptors can prevent HIV from entering the host cells. We designed an invasion-inhibitor for HIV-1 targeting dendritic cell (DC), including 2 important HIV-1 receptors CD4 and CCR5, and 2 molecules Flt3-L and Mip-3alpha. With the synthetic gene of the inhibitor, 2 eukaryotic expression vectors pABK-CKR5-CD4/Flt3L-Mip3alpha (pABK-HIV-MF) and pABK-CKR5-CD4 (pABK-HIV-MT) were constructed and transfected into HEK 293 cells for expression. Results from RT-PCR, immunofluorescent assay, ELISA and Western blot approved that the invasion-inhibitor for HIV-1 was successfully and exactly expressed in the eukaryotic cells. Current study formed a solid base for the further research on the function of inhibitors for HIV-1 and elimination targeting DC.

HIV-1B gp120 genes from one patient with AIDS dementia complex can affect the secretion of tumor necrosis factor and interleukin 1β in glial cells / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>HIV-1 infected and immune-activated macrophages and microglia secrete neurotoxins, such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), which play major role in the neuronal death. It has been shown that different HIV-1 variants have varying abilities to elicit secretion of TNF-α by peripheral blood mononuclear cell (PBMC); however, whether the difference of gp120 gene could affect the secretion of TNF-α and IL-1β by glial cells is unknown. The aim of this study was to explore the association between gene diversity and induction of neurotoxic cytokines.</p><p><b>METHODS</b>In this study, we constructed retroviral vectors MSCV-IRES-GFP/gp120 using HIV-1 gp120 genes isolated from four different tissues of one patient who died of AIDS dementia complex (ADC). Recombinant retroviruses produced by cotransfection of MSCV-IRES-GFP/gp120, pCMV-VSV-G and pUMVC into 293T cells were collected and added into U87 glial cells. Concentrations of TNF-α and IL-1β secreted by transduced U87 cells were assayed with ELISA separately.</p><p><b>RESULTS</b>The four HIV-1 gp120 were in the different branch of the neighbor-joining tree. Compared to the pMIG retrovirus (gp120-negative) or U87 cells, all the gp120-positive recombinant retroviruses induced more TNF-α (P < 0.01) and IL-1β (P < 0.01). In addition, compared with the L/MIG retrovirus, all the three brain gp120-positive recombinant retroviruses induced less TNF-α (P < 0.01) and IL-1β (P < 0.01).</p><p><b>CONCLUSIONS</b>HIV-1 gp120 could induce U87 cells secret more TNF-α and IL-1β again. The more important is that difference of HIV-1 gp120, especially cell-tropism may account for the different ability in eliciting secretion of TNF-α and IL-1β, which might supply a novel idea helping understand the pathogenesis of ADC.</p>

HIV entry inhibitors: progress in development and application / 药学学报

This review discusses recent progress in the development of anti-HIV agents, with emphasis on small molecule HIV-1 entry inhibitors. The entry inhibitors primarily target HIV-1 envelope glycoproteins or the cellular receptors, CD4 and chemokine receptors. Two of the entry inhibitors, enfuvirtide and maraviroc, have been approved by the US FDA for AIDS therapy. The drug resistance associated with some of the entry inhibitors will also be discussed.

Immunogenicity of recombinant adeno-asscociated virus type 1 expressing HIV-1 gp120 gene in mice and Rhesus macaques / 病毒学报

To study the immunogenicity of recombinant adeno-asscociated virus type 1 expressing HIV-1 gp120 gene (rAAV2/1-gp120) in BALB/c mice and Rhesus macaques. The gp120 gene derived from Chinese HIV-1 isolates was constructed into rAAV2/1 and rAd5 vectors. Firstly, the immunogenicity of rAAV2/1-gp120 was compared with rAd5-gp120 in BALB/c mice when used once or twice in 3 weeks interval. Then the monkeys were immunized with rAAV2/1-gp120 once. The HIV-1 specific IgG levels and neutralization activity to pseudotyped HIV-1 virus were tested using ELISA and neutralization assay, and the cellular immune responses were analyzed by IFN-gamma enzyme-linked immunospot (ELISPOT) and in vivo CTL assays. Compared with rAd5-gp120 immunized mice, mice immunized with rAAV2/1-gp120 once in duced stronger gp120-specific IgG and were sustained for at least 21 weeks. rAd5-gp120 immunized mice generated stronger cellular immune responses than rAAV2/1-gp120 in spleen and draining lymph node. But only moderate gp120-specific in vivo CTL activity was observed in both rAAV2/1-gp120 and rAd5-gp120 immunized mice. Four of five monkeys vaccinated with rAAV2/1-gp120 generated gp120 specific IgG, the titer ranged from 1:100 to 1:400 with end-point dilution. Gp120 specific IgG could be detected 4 weeks after immunization and reached the peak at 10 weeks after immunization. No neutralization activity against pseudotyped HIV-1 virus expressing NL4-3 Env antigen was detected. In Conclusion, rAAV2/1-gp120 induced high level of HIV-1 specific IgG antibody and moderate cellular immune responses. No neutralizing antibody was elicited. It indicates that the env gene and immunization strategy should be optimized to elicit neutralizing antibody against HIV-1 in further studies.

Establishment of stable cell line and expression and purification of HIV gp120 in Drosophila S2 cells / 病毒学报

Constructing eukaryotic expressing vector of pMT/Bip/V5-His A-HIV gp120 and transfecting S2 cells to establish stable gp120-expressing S2 cell line. The gp120 gene of HIV CRF07-BC epidemic in China was amplified by polymerase chain reaction from the recombinant vector PRSV-gp120 and confirmed by DNA sequence analysis. The DNA fragment of HIV gp120 was digested with the restriction endonucleases NcoI and XhoI, then inserted into eukaryotic expressing vector pMT/BiP/V5-His A. A selection vector containing the streptomyces griseochromogenes bsd gene conferring blasticidin resistance was cotransfected into drosophila S2 cells by Cellfectin II reagent. The stable cell line was established following repeated screening by blasticidin. HIV gp120 was purified by a Ni-NTA affinity column followed by molecular sieve chromatography. Recombinant protein was characterized by SDS-PAGE, Western blot and ELISA. The eukaryotic expressing vector pMT/BiP/V5-His A-HIV gp120 was constructed, stable expressing cell line was established, and protein was expressed and purified successfully. This result will contribute to functional study of gp120 and vaccine design against AIDS.

A non-infectious and quantitative cell-based bioassay for screening HIV entry inhibitors targeting HIV envelope proteins / 南方医科大学学报

<p><b>OBJECTIVE</b>To develop an objective bioassay for quantitative detection of HIV-induced cell-cell fusion for screening HIV entry inhibitors.</p><p><b>METHODS</b>HL2/3 cells expressing HIV envelope proteins gp120/gp41, Tat, and other HIV proteins were co-cultured with HeLa-CD4-LTR-beta-gal cells expressing CD4 receptor and HIV LTR triggered reporter gene beta-galactosidase. The enzyme activities of beta-galactosidase were detected by a chromogenic substrate, chlorophenol red-beta-galactopyranoside (CPRG). Specific HIV entry inhibitors were used to validate the established detecting method.</p><p><b>RESULTS</b>No syncytium was formed by mixing HL2/3 and HeLa-CD4-LTR-beta-gal cells. However, the membrane could be fused and the Tat expressed by HL2/3 cells could bind to HIV LTR on HeLa-CD4-LTR-beta-gal cells and trigger the expression of beta-galactosidase. CPRG allowed quantitative and sensitive detection of the activity of beta-galactosidase. Further studies showed that HIV entry inhibitors could inhibit the activity of beta-galactosidase in a dose-dependent manner.</p><p><b>CONCLUSION</b>We have developed a simple, cheap, objective and quantitative non-infectious cell-cell fusion bioassay that can be used to screen for anti-HIV agents targeting the virus entry from natural and synthetic compound libraries.</p>

Immunogenicity of plasmid DNA and adenoviral vectors encoding HIV-1 subtype B env gene / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To construct DNA and recombinant adenovirus vector vaccines containing an env gene from the prevalent subtype B strain in China and try to use them for therapeutic and prophylactic vaccines.</p><p><b>METHODS</b>The candidate plasmid DNA vaccine pVR-gp160 and recombinant adenovirus vaccine rAdV-gp160 were constructed separately. BALB/c mice were immunized with these two vaccines in different administration schemes. HIV-1 Gp120-specific cellular responses and antibody levels were detected by ELISPOT and ELISA respectively.</p><p><b>RESULTS</b>DNA vaccine alone and combined vaccines in a DNA prime/rAdV-gp160 boost vaccination regimen induced high level of Gp120-specific cellular responses. While low level of Gp120-specific antibodies were elicited in all groups.</p><p><b>CONCLUSION</b>DNA and rAdV vaccines could efficiently express Gp160 protein and activate specific cellular responses.</p>

Analysis of genetic diversity and biological functional site of HIV-1 gp120 from a patient with AIDS dementia complex / 病毒学报

To explore the relationship between the genetic diversity and biological functional site of human immunodeficiency virus HIV-1 gp120 and the pathogenesis of AIDS dementia complex (ADC), the full length sequences of gp120 gene was amplified with PCR from genomic DNA which was extracted from lymphoid and different brain department (periaortic lymphoid, temporal gray/white matter junction, periventricular tissue, choroids plexus, occipital white matter and occipital gray/white matter junction.) of a patient who died of ADC. PCR products were cloned into the pGEM-T vector and positive clones were sequenced. The analysis of neighbor-joining tree, N-glycosylation sites, values of ds/dn, and loop were then all performed. The samples were all identified as HIV-1 B and genetic variation existed in HIV-1 gp120 isolated from different tissues. Compared with standard HIV-1B gp120, biological functional sites of HIV-1 gp120 isolated from the patient changed to some extent. In addition, there were differences in some biological functional sites of HIV-1 gp120 between lymphoid and brain. Therefore, genetic diversity and alterations of some biological functional sites of HIV-1 gp120 might be associated with the pathogenesis of ADC.

Genetic analysis of the complete env genes of HIV-1 from paid blood donors in Henan province / 病毒学报

Complete HIV-1 env genes were amplified by nested PCR from uncultured peripheral blood mononuclear cells (PBMCs) DNA of 60 HIV-1 positive paid blood donors in Henan province, and the amplified full-length genes were sequenced. Twenty one full-length env genes were obtained, sequence analysis found that 15 of them had intact open reading frame (ORF). Fourteen sequences conformed to subtype B', their average genetic distance with the international reference sequence RL42 was 4.87% +/- 0.31%. One was subtype B, its genetic distance with the international reference sequence HXB2 was 5.43%. The amino acid sequences of these env genes were deduced according to their nucleotide sequences and extensive analysis and comparison of important structural motifs were performed. The results indicated that there was no drastic alteration in the number and position of potential N-linked glycosylation sites among these 15 sequences. And the residues involved in forming the CD4 binding site were highly conserved. Genotype prediction of coreceptor usage based on V3 sequence and net charge suggested that most samples use CCR5 coreceptor. GPGR motif at the tetrapeptide crown in the V3 loop was most common in these samples and it was detected in 40% sequences. The cleavage site of gp120/gp41 was highly conserved, so Gp160 precursor of all isolates would be efficiently cleaved into the Gp120 and Gp41 subunits. The known neutralizing antibody binding sites for 2G12, IgG1b12, 4E10 and 2F5 were also highly conserved, it is expected that most of these isolates will be sensitive to neutralization by these antibodies. Further study to elucidate the correlation of the env genotype to functionally relevant motifs is necessary and that will aid vaccine and novel drug design.

Immunogenicities of Env glycoproteins from circulating HIV-1 isolates in China focusing on the strategy of "DNA prime plus protein boost" / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The adenovirus-based HIV-1 vaccine developed by Merck Company suffered from an unexpected failure in September 2007. This generated a big shift in the strategy of HIV vaccine development with renewed focus on the induction of neutralizing antibodies. A major challenge in developing an HIV-1 vaccine is to identify immunogens and adopt delivery methods that can elicit broadly neutralizing antibodies against primary isolates of different genetic subtypes.</p><p><b>METHODS</b>Most circulating HIV-1 isolates in China are composed of clades Thai-B, CRF_BC and CRF01_AE. In order to construct DNA vaccines against these 3 HIV-1 subtypes, DNA vaccines carrying the gp120 regions from HIV-1 isolates of GX48(AE), GX79(AE), NX22(BC), GS22(BC), HN24(Thai-B) were constructed. Expression of gp120 from these DNA vaccines was detected by Western blotting in transiently transfected 293T cells. Pilot immunizations of New Zealand white rabbits were performed using the strategy of "DNA prime plus protein boost" and the neutralizing antibody response was detected in a Tzm-bl cell based assay against different HIV-1 strains.</p><p><b>RESULTS</b>Response of gp120-specific antibody was relatively low after DNA primes (mean titer = 10(4.72)); however, the titer of gp120-specific antibody went up with 2 protein boosts (mean titer = 10(6.81)). Above all, neutralizing antibody (Nab) titers induced by this combined approach were much better than those elicited by DNA or protein used alone (P < 0.01). Neutralizing activities of immunized rabbit sera against several pseudoviruses and laboratorial strains were evaluated, most rabbit sera primed with monovalent vaccine were capable of neutralizing only 1 of 5 viruses, however, sera primed with the polyvalent DNA vaccines were able to neutralize at least 2 of 5 viruses.</p><p><b>CONCLUSION</b>Polyvalent DNA prime plus protein boost is an effective immunization strategy to broaden the neutralization breadth and further research should be performed on the basis of this pilot study.</p>

Expression and purification of HIV-1 subtype C Gp120, and its antibodies preparation / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To prepare HIV-1 subtype C Gp120 protein and to produce its polyclonal antibodies.</p><p><b>METHODS</b>A C-terminal fragment of gp120 gene was amplified by PCR from a plasmid expressing full-length HIV-1 subtype C gp160 gene. The length of the subtype C gp120 fragment was 612 nt and it encodes 204 amino acid residues. The resulting DNA construct was cloned into a prokaryotic expression vector (pET-30a) and recombinant pET-30a-gp120 was expressed in Escherichia coli BL21 (DE3) as an insoluble protein. The vector also contained a six-histidine (His6) tag at the C-terminus for convenient purification. To produce subtype C Gp120-specific polyclonal antibodies, New-Zealand rabbit was immunized with the purified Gp120 protein. Serum samples were tested by enzyme-linked immunosorbent assays (ELISA) to determine the level of antibodies. And Western blotting was used to further verify whether the polyclonal antibodies could specifically recognize subtype C Gp160 protein expressed in mammalian cells.</p><p><b>RESULTS</b>HIV-1 subtype C Gp120 protein was successfully acquired and the titer of its polyclonal antibodies was 1:204 800. The polyclonal antibodies efficiently recognized Subtype C Gp160 protein expressed in COS-1 cells.</p><p><b>CONCLUSION</b>HIV-1 subtype C Gp120 fusion protein with high purity was obtained and its corresponding polyclonal antibodies with high titer were produced.</p>