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Antiretroviral therapy (ART) significantly reduced the CD8+ cell noncytotoxic anti-HIV response in 12 HIV-1-infected subjects (P < 0.0001). In separate experiments, CD8+ cells from long-term survivors were cocultured with HIV-infected CD4+ cells using varying concentrations of anti-HIV drugs. The antiviral function of CD8+ cells from 4 of the 14 LTSs was reduced with exposure to 10 μM of nevirapine (P < 0.05). The antiviral activity of CD8+ cells from 2 LTSs was inhibited by 5 μM of zidovudine. These studies indicate that nevirapine and probably zidovudine can inhibit the anti-HIV activity of CD8+ cells and thus could influence the effectiveness of antiretroviral therapy. [ABSTRACT FROM AUTHOR]

Abstract: Objective: To develop an effective HIV vaccine strategy that can induce cross-reactive neutralizing antibody. Methods: Codon-optimized gp140 and gp145 env genes derived from HIV-1cn54, a CRF07 B′/C recombinant strain, were constructed as DNA and recombinant Tiantan vaccinia (rTV) vaccines. The effect of heterologous immunization with gp140 and gp145 was tested in mice and guinea pigs. T cell responses were detected using the IFN-γ ELISPOT assay. A panel of primary isolates of clade B′ and B′/C HIV-1 and TZM-bl cells was used to determine the neutralizing activity of immunized sera. Results: The neutralizing antibodies (NAbs) induced by the heterologous immunogen immunization neutralized all HIV-1 B′ and B′/C primary isolates in the guinea pig model. Gp145 and gp140 heterologous prime-boost induced the best neutralizing antibody response with a broad neutralizing spectrum and the highest titer of 1:270 at 6 weeks after the last inoculation. However, the T cell response to HIV-1 peptides was significantly weaker than the gp145+gp145 homologous prime-boost. Conclusions: This heterologous prime-boost immunization strategy could be used to design immunogen-generating broad neutralizing antibodies against genetic variance pathogens. [Copyright &y& Elsevier]

Abstract: Electroporation, a non-virus-mediated gene transfection method, has traditionally had poor outcomes with low gene transfection efficiency and poor cellular viability, particularly in primary human lymphocytes. Herein we have optimized the electroporation conditions for primary CD8+ cells resulting in a maximum rate of 81.3%, and a mean transfection efficiency of 59.6%. After removal of dead cells, the viability of transfected primary CD8+ cells was greater than 90%, similar to untransfected controls. Using this procedure, primary human CD8+ cells transfected with an interferon α8 plasmid produced fluids that inhibited HIV-1 replication by >95%. This transfection protocol is useful for transfection of other primary blood cells, such as CD4+ T cells, and for studying the function of genes in primary human blood cells instead of cell lines. The transfection procedure also has potential application in gene therapy clinical trials to treat diseases utilizing transfected primary human cells. [Copyright &y& Elsevier]

Abstract: An effective vaccine strategy for HIV-1 will probably require the induction and maintenance of both humoral and cellular immunity at mucosal and systemic sites. We tested a new prime-boost approach of intranasal priming with 3×106 PFU of replicative recombinant Tiantan vaccinia virus (rTTV) and intramuscular boosting with 100μg DNA plasmid expressing HIV-1 Gag in BALB/c mice along with other strategies. Our data demonstrated that intranasal priming with replicative recombinant Tiantan vaccinia and intramuscular boosting with DNA vaccine raised the highest vaginal IgA and systemic T-cell responses, and modest lung IgA and sera IgG responses among all vaccination regimens; each vaccination regimen generated its own imprint of the most preferential T-cell receptor usage of Vβ. These results demonstrate that the combination of intranasal priming with replicative recombinant Tiantan vaccinia and intramuscular boosting with DNA vaccine is a preferable regimen for induction of both T-cell and humoral immune responses at mucosal and systemic sites. [Copyright &y& Elsevier]

Abstract: Equine Infectious Anemia Virus (EIAV), like other lentiviruses, has a transmembrane glycoprotein with an unusually long cytoplasmic tail (CT). Viral envelope (Env) proteins having CT truncations just downstream the putative membrane-spanning domain (PMSD) are assumed to exist among all wild-type budded virions, and also in some cell-adapted strains. To determine whether CT-truncated Env proteins can cause particularly deleterious effects on the Env expressing cells and/or their neighboring cells, plasmids encoding codon-optimized env gene including full-length (pE863) or CT-truncated (pE686* and pE676*) were transiently transfected into 293T cells, respectively. Data from intracellular protein expression and cell death assays revealed that CT-truncated Env, compared to full-length Env, not only induced comparable apoptosis, but also caused much more intensive mitochondria-mediated necrosis that could simultaneously induce significant decrease of intracellular protein expression in the Env expressing cells. Moreover, results from flow cytometric analysis showed that mitochondrial depolarization preceded the caspase activation in cells no matter which env construct was delivered, and indicated that both full-length and CT-truncated Env proteins share a common intrinsic mitochondrial pathway to induce apoptosis. Our results partially elucidate the mechanisms underlying cell death resulting from EIAV pathogenesis. [Copyright &y& Elsevier]

Abstract: HIV-1 pandemic posed an unprecedent challenge to the global health and it is believed that an effective vaccine will be the final solution against HIV-1. HIV-1 envelope is the primary immunogen in developing neutralization antibody based HIV vaccine. To define the suitable Env derived immunogen, we systemically compared the immunogenicity of gp140 and gp145 in a DNA vaccination alone and a prime-boost modalities. Two DNA vaccines and two recombinant Tiantan vaccinia vaccines (rTTV) were constructed for vaccination of female Balb/c mice. Elispot assay was used to read out the T cell immunity and ELISA assay was used to quantify antibody immunity. PLL (poly-l-leucine)-ELISA assay was used in linear antibody epitope mapping. Mice primed with gp145 tended to elicit more Env-specific T cells responses than those primed with gp140, significant difference was observed in DNA immunization alone. The ultimate T cell responses in prime-boost regimen tend to be determined mainly by the priming efficacy. Linear antibody epitope mapping displayed that sera raised by gp145 priming were vigorously reactive to more peptides than that by gp140. Our data demonstrated HIV-1 Thailand B-derived gp145 may raise higher T-cell responses and broader linear peptide-specific antibody responses than gp140 does. However, it remains to be determined that how these observations are relevant to the neutralization of antibody activities. [Copyright &y& Elsevier]

Helical milling (HM) is a developing hole-making technology in aircraft assembly. Nevertheless, fundamental problems, such as tool deflection and chip evacuation, when helical milling small-diameter or deep holes still exist. To solve these problems and improve the machining quality of Ti-6Al-4V holes, an ultrasonic transversal vibration-assisted helical milling (UTVHM) method is developed. The motion paths of the peripheral cutting edges are established, and the tool-workpiece contact behaviour is analysed to explore how transversal vibration affects the material removal of UTVHM. The hole surface strengthening mechanisms in the UTVHM were revealed and explored. Moreover, the verification experiments were conducted, and the results suggested that the transverse force and thrust force in the UTVHM reduced up to 38.1% and 13.9%, respectively, because of the intermittent cutting mode realised in the UTVHM compared to HM. Significantly improved surface integrity, excellent chip evacuation conditions, and narrow hole geometric tolerances were obtained using UTVHM. A maximum depth of 9.7 µm for the plastic deformation layer was generated in the UTVHM, which was 6.5 times that of HM. The maximum increases in surface microhardness and residual compressive stresses in the UTVHM were 184.7% and 150.2%, respectively. Electron backscattered diffraction (EBSD) analysis showed that a higher plastic strain and smaller grain size occurred in the UTVHM. This study enhances the understanding of the surface and subsurface formation and material removal mechanisms of UTVHM, providing a theoretical basis and practical method for machining holes with high precision and excellent surface integrity in aircraft structures. [Display omitted] • A novel hole-making method of ultrasonic transversal vibration-assisted helical milling (UTVHM) is proposed. • UTVHM exhibits superior cutting performance compared to conventional helical milling. • Lower cutting force and higher hole precision are obtained in UTVHM. • Strengthening effect for the machined surface is realized in UTVHM. [ABSTRACT FROM AUTHOR]

In twist drilling, severe extrusion is unavoidable at the chisel edge zone and results in high thrust force and poor surface integrity. Traditional axial vibration-assisted twist drilling only modifies the material removed from the continuous extrusion to the interrupted extrusion for the chisel edge centre, and must satisfy the chip-breaking conditions to ensure that the cutting edge is periodically separated from the workpiece. Hence, transversal vibration, has been introduced in twist drilling to overcome the severe extrusion behaviour of the chisel edges of the twist drills, referred to as ultrasonic transversal vibration-assisted drilling (UTVD). Theoretical models revealed that the material removal behaviour of the chisel edge centre changed from extrusion in CD to cutting in UTVD. Experiments were performed on UTVD of Ti-6Al-4V to confirm the feasibility with this novel method. The results indicated that transverse vibration significantly affected the thrust force and torque, and the maximum decline was 29.4% and 31.5%, respectively, compared with CD. Generated chips verified the material removal behaviour of the chisel edge centre zone changed from extrusion in CD to cutting in UTVD. Therefore, the hole quality and surface integrity were improved significantly by UTVD. Deeper plastic deformation (∼2.5 times), higher surface microhardness and compressive residual stress were realised using UTVD. This study enhanced the understanding of material removal and formation mechanisms of the surface and subsurface in UTVD, thereby providing a novel method and theoretical basis for the high-performance twist drilling of difficult-to-cut alloys. [ABSTRACT FROM AUTHOR]

Ultrasonic assisted milling (UAM) has been demonstrated excellent cutting performance for nickel-based superalloy machining. However, the machining quality improvement achieved by traditional UAM is limited at high cutting speed. Rotary ultrasonic elliptical milling (RUEM) is an unconventional cutting method recently proposed for high quality and efficient machining of difficult-to-cut alloy, breaking the cutting speed limit for UAM. In order to verify the feasibility of this method in thin-walled curved surface machining, the ball end milling cutter was adopted in feasibility experiments of RUEM on Inconel 718 with the evaluation of cutting force, surface integrity and tool wear. The results indicate that compared with conventional milling (CM) without ultrasonic vibration assistance, the cutting force reduction reaches 31.33% in RUEM. The material strength enhancement was obtained in RUEM with the disturbed strengthening phases of Inconel 718. RUEM achieved − 728.65 MPa of surface compressive residual stress and 140 µm depth of compressive residual stress layer beneath the machined surface simultaneously, while CM provided tensile residual stress field near the machined surface. Moreover, the tool flank wear status was significantly improved in RUEM. These results demonstrate that various machinability benefits of Inconel 718 are obtained by RUEM, which is a promising method for nickel-based superalloy thin-walled structure finish machining. [ABSTRACT FROM AUTHOR]

Abstract: Interest is increasing regarding replicating poxvirus as HIV vaccine vector. In China, the Tiantan Vaccinia Virus (TV) has been used most extensively in the battle of eradicating smallpox. Recently, TV was developing as vaccine vector to fight against infectious diseases such as human immunodeficiency virus (HIV). However, replicating vaccinia virus sometimes may pose serious post-vaccination complications, especially in immunosuppressed individuals. To develop a safer and more effective TV-based vector, we constructed C12L (vIL-18 binding protein) and A53R (vTNF receptor homolog) gene-deleted mutants which are based on parental TV and VTKgpe (TV expressing HIV gagpol and env gene), respectively. The pathogenicity and immunogenicity were also evaluated. Deleting these two immunomodulatory genes lessened the virulence of the parental virus in both mice and rabbit models. Notably, C12L deletion mutant attenuated the skin virulence of parental virus by as high as approximate 2 logs. Furthermore, VTKgpe with A53R and C12L gene deletion retains the high immunogenicity of the parental virus to elicit strong humoral and cellular responses to the HIV target genes despite the remarkable attenuation. These data suggest that deletion of the cytokine viroceptor gene is feasible to obtain a safer and replication-competent TV vector for vaccination and immunotherapy. [Copyright &y& Elsevier]

Abstract: An effective vaccine strategy for HIV-1 will probably requires the induction and maintenance of both humoral and cellular immunity. We tested a new prime–boost approach of intranasal priming with 10μg DNA plasmid in the PEI/DNA complexes and boosting with 107 PFU of replicative recombinant TianTan vaccinia virus (rTTV) expressing HIV-1 Gag in BALB/c mice. Intranasal priming with PEI/DNA complexes elicited strikingly stronger HIV-specific T-cell (p =0.0358) and IgA immune responses at mucosal sites of lung (p =0.0445) and vaginal tract (p =0.0469) than intranasal priming with naked DNA, though both are followed by the same rTTV boosting. Furthermore, an intramuscular boosting with rTTV could profoundly enhance both T-cell and antibody immune responses raised by intranasal priming. These results demonstrate that the combination of intranasal priming with PEI/DNA complexes and systemic boosting with rTTV is a preferable regimen for induction of both T-cell and humoral immune responses. [Copyright &y& Elsevier]

Depletion of T regulatory cells (Tregs) in the tumor microenvironment is a promising cancer immunotherapy strategy. Current approaches for depleting Tregs are limited by lack of specificity and concurrent depletion of anti-tumor effector T cells. The transcription factor forkhead box p3 (Foxp3) plays a central role in the development and function of Tregs and is an ideal target in Tregs, but Foxp3 is an intracellular, undruggable protein to date. We have generated a T cell receptor mimic antibody, "Foxp3-#32," recognizing a Foxp3-derived epitope in the context of HLA-A*02:01. The mAb Foxp3-#32 selectively recognizes CD4 + CD25 + CD127low and Foxp3 + Tregs also expressing HLA-A*02:01 and depletes these cells via antibody-mediated cellular cytotoxicity. Foxp3-#32 mAb depleted Tregs in xenografts of PBMCs from a healthy donor and ascites fluid from a cancer patient. A TCRm mAb targeting intracellular Foxp3 epitope represents an approach to deplete Tregs. [ABSTRACT FROM AUTHOR]