Impact of Recombinant VSV-HIV Prime, DNA-Boost Vaccine Candidates on Immunogenicity and Viremia on SHIV-Infected Rhesus Macaques.

Currently, no effective vaccine to prevent human immunodeficiency virus (HIV) infection is available, and various platforms are being examined. The vesicular stomatitis virus (VSV) vaccine vehicle can induce robust humoral and cell-mediated immune responses, making it a suitable candidate for the development of an HIV vaccine. Here, we analyze the protective immunological impacts of recombinant VSV vaccine vectors that express chimeric HIV Envelope proteins (Env) in rhesus macaques. To improve the immunogenicity of these VSV-HIV Env vaccine candidates, we generated chimeric Envs containing the transmembrane and cytoplasmic tail of the simian immunodeficiency virus (SIV), which increases surface Env on the particle. Additionally, the Ebola virus glycoprotein was added to the VSV-HIV vaccine particles to divert tropism from CD4 T cells and enhance their replications both in vitro and in vivo. Animals were boosted with DNA constructs that encoded matching antigens. Vaccinated animals developed non-neutralizing antibody responses against both the HIV Env and the Ebola virus glycoprotein (EBOV GP) as well as systemic memory T-cell activation. However, these responses were not associated with observable protection against simian-HIV (SHIV) infection following repeated high-dose intra-rectal SHIV SF162p3 challenges.

Protocol to evaluate the inflammatory response in human macrophages induced by SARS-CoV-2 spike-pseudotyped VLPs.

The excessive release of pro-inflammatory cytokines in COVID-19 patients is deleterious to organs. The contribution of SARS-CoV-2 spike protein (S) to the inflammatory response is essential to understand its pathogenesis and virulence. Here, we present a protocol to produce and characterize HIV- and SARS-CoV-2-based virus-like particles and then evaluate the inflammatory cytokines' protein and mRNA levels produced in human macrophages by S of SARS-CoV-2 original strain and Delta variant. This protocol is applicable in evaluating S from different emerging variants. For complete details on the use and execution of this protocol, please refer to Ao et al. (2022).1.

Rhesus macaques show increased resistance to repeated SHIV intrarectal exposure following a heterologous regimen of rVSV vector vaccine expressing HIV antigen.

Despite the human immunodeficiency virus (HIV) pandemic continuing worldwide for 40 years, no vaccine to combat the disease has been licenced for use in at risk populations. Here, we describe a novel recombinant vesicular stomatitis virus (rVSV) vector vaccine expressing modified HIV envelope glycoproteins and Ebola virus glycoprotein. Three heterologous immunizations successfully prevented infection by a different clade SHIV in 60% of non-human primates (NHPs). No trend was observed between resistance and antibody interactions. Resistance to infection was associated with high proportions of central memory T-cell CD69 and CD154 marker upregulation, increased IL-2 production, and a reduced IFN-γ response, offering insight into correlates of protection.

Vpr R77Q is associated with long-term nonprogressive HIV infection and impaired induction of apoptosis.

The absence of immune defects that occurs in the syndrome of long-term nonprogressive (LTNP) HIV infection offers insights into the pathophysiology of HIV-induced immune disease. The (H[F/S]RIG)(2) domain of viral protein R (Vpr) induces apoptosis and may contribute to HIV-induced T cell depletion. We demonstrate a higher frequency of R77Q Vpr mutations in patients with LTNP than in patients with progressive disease. In addition, T cell infections using vesicular stomatitis virus G (VSV-G) pseudotyped HIV-1 Vpr R77Q result in less (P = 0.01) T cell death than infections using wild-type Vpr, despite similar levels of viral replication. Wild-type Vpr-associated events, including procaspase-8 and -3 cleavage, loss of mitochondrial transmembrane potential (deltapsi(m)), and DNA fragmentation factor activation are attenuated by R77Q Vpr. These data highlight the pathophysiologic role of Vpr in HIV-induced immune disease and suggest a novel mechanism of LTNP.

DNA vaccination protects mice against Zika virus-induced damage to the testes.

Zika virus (ZIKV) is an emerging pathogen causally associated with serious sequelae in fetuses, inducing fetal microcephaly and other neurodevelopment defects. ZIKV is primarily transmitted by mosquitoes, but can persist in human semen and sperm, and sexual transmission has been documented. Moreover, exposure of type-I interferon knockout mice to ZIKV results in severe damage to the testes, epididymis and sperm. Candidate ZIKV vaccines have shown protective efficacy in preclinical studies carried out in animal models, and several vaccines have entered clinical trials. Here, we report that administration of a synthetic DNA vaccine encoding ZIKV pre-membrane and envelope (prME) completely protects mice against ZIKV-associated damage to the testes and sperm and prevents viral persistence in the testes following challenge with a contemporary strain of ZIKV. These data suggest that DNA vaccination merits further investigation as a potential means to reduce ZIKV persistence in the male reproductive tract.

Analysis of HIV-1 Vpr determinants responsible for cell growth arrest in Saccharomyces cerevisiae.

BACKGROUND: The HIV-1 genome encodes a well-conserved accessory gene product, Vpr, that serves multiple functions in the retroviral life cycle, including the enhancement of viral replication in nondividing macrophages, the induction of G2 cell-cycle arrest, and the modulation of HIV-1-induced apoptosis. We previously reported the genetic selection of a panel of di-tryptophan (W)-containing peptides capable of interacting with HIV-1 Vpr and inhibiting its cytostatic activity in Saccharomyces cerevisiae (Yao, X.-J., J. Lemay, N. Rougeau, M. Clement, S. Kurtz, P. Belhumeur, and E. A. Cohen, J. Biol. Chem. v. 277, p. 48816-48826, 2002). In this study, we performed a mutagenic analysis of Vpr to identify sequence and/or structural determinants implicated in the interaction with di-W-containing peptides and assessed the effect of mutations on Vpr-induced cytostatic activity in S. cerevisiae. RESULTS: Our data clearly shows that integrity of N-terminal alpha-helix I (17-33) and alpha-helix III (53-83) is crucial for Vpr interaction with di-W-containing peptides as well as for the protein-induced cytostatic effect in budding yeast. Interestingly, several Vpr mutants, mainly in the N- and C-terminal domains, which were previously reported to be defective for cell-cycle arrest or apoptosis in human cells, still displayed a cytostatic activity in S. cerevisiae and remained sensitive to the inhibitory effect of di-W-containing peptides. CONCLUSIONS: Vpr-induced growth arrest in budding yeast can be effectively inhibited by GST-fused di-W peptide through a specific interaction of di-W peptide with Vpr functional domain, which includes alpha-helix I (17-33) and alpha-helix III (53-83). Furthermore, the mechanism(s) underlying Vpr-induced cytostatic effect in budding yeast are likely to be distinct from those implicated in cell-cycle alteration and apoptosis in human cells.

[Relationship of MPO and NQO1 gene polymorphisms with susceptibility to acute leukemia].

The aim of this study was to investigate the relationship of the gene polymorphisms of myeloperoxidase (MPO) and NAD (P) H: quinone oxidoreductase 1 (NQO1) with the susceptibility to acute leukemia (AL) in Chinese Gansu population. A 1:1 paired case-control study of 150 patients with acute leukemia and 150 cancer-free inpatients as a control was conducted to detect the polymorphisms of MPO and NQO1 by LDR techniques. The results showed that the MPO-463A genotype frequency in patient group was lower than that in control group, and there was significant difference of MPO (G-463A) genotype between patient group and control group (χ(2) = 11.828, P < 0.05, OR = 0.368, 95%CI = 0.205 - 0.610). The NQO1-609T genotype frequency in patient group was higher than that in control group, and there was significant difference of NQO1 (C-609T) genotype between patient group and control group (χ(2) = 17.931, P < 0.05, OR = 1.428, 95%CI = 1.237 - 3.339). The combined gene analysis showed that the AML risk in patients carrying the wild genotypes of MPO and NQO1 was dropped to 33.6%. It is concluded that the MPO and NQO1 gene polymorphisms are associated with susceptibility to AL. The AL risk may decrease in patients carrying MPO (G-463A) mutant gene (GA/AA), while the AL risk may increase in patients carrying NQO1 (C-609T) mutant gene (TC/TT). The combined effect of MPO and NQO1 wild genotypes may further decrease AL risk.

[Relationship between polymorphisms of myeloperoxidase gene and susceptibility of acute leukemia in Chinese Gansu population].

This study was aimed to explore the relationship between gene polymorphisms of myeloperoxidase (MPO) and the susceptibility of acute leukemia in Chinese Gansu population. G463A mutation of mpo gene was analyzed by polymerase chain reaction-ligase detection reaction (PCR-LDR) in 100 normal individuals (control group) and 100 patients with acute leukemia (AL group). The results showed that the a allele genotype and ga/aa genotype of mpo gene occurred more frequently in control group (28% and 54%) than those in AL group (19% and 31%) (p < 0.05). The AL risk for controls was decreased by 0.383-fold, compared with the individuals with gg genotype (95%CI = 0.215 - 0.682, p < 0.01). By further stratified analysis, the ga/aa genotype of mpo gene occurred more frequently in control group (54%) than those in AML group (28.2%) (p < 0.01). AML risk (95%CI = 0.157 - 0.546, p < 0.01) in the controls was decreased by 0.346-fold compared with the individual with gg genotype, however, the acute lymphoblastic leukemia (ALL) showed no significant difference from control group in the incidence of the allele a genotype and ga/aa genotype of mpo gene. It is concluded that mpo gene polymorphism is associated with susceptibility of acute myeloid leukemia in Chinese Gansu population. The risk of AML decreases in the persons carrying a allele, but mpo gene polymorphism is not associated with susceptibility of acute lymphoblastic leukemia.

Intracellular transport of human immunodeficiency virus type 1 genomic RNA and viral production are dependent on dynein motor function and late endosome positioning.

Our earlier work indicated that the human immunodeficiency virus type 1 (HIV-1) genomic RNA (vRNA) is trafficked to the microtubule-organizing center (MTOC) when heterogeneous nuclear ribonucleoprotein A2/B1 is depleted from cells. Also, Rab7-interacting lysosomal protein promoted dynein motor complex, late endosome and vRNA clustering at the MTOC suggesting that the dynein motor and late endosomes were involved in vRNA trafficking. To investigate the role of the dynein motor in vRNA trafficking, dynein motor function was disrupted by small interference RNA-mediated depletion of the dynein heavy chain or by p50/dynamitin overexpression. These treatments led to a marked relocalization of vRNA and viral structural protein Gag to the cell periphery with late endosomes and a severalfold increase in HIV-1 production. In contrast, rerouting vRNA to the MTOC reduced virus production. vRNA localization depended on Gag membrane association as shown using both myristoylation and Gag nucleocapsid domain proviral mutants. Furthermore, the cytoplasmic localization of vRNA and Gag was not attributable to intracellular or internalized endocytosed virus particles. Our results demonstrate that dynein motor function is important for regulating Gag and vRNA egress on endosomal membranes in the cytoplasm to directly impact on viral production.

[Observation on myeloid origin of neovascular endothelial cells and infiltration of bone marrow-originated inflammatory cells in a murine tumor model].

BACKGROUND & OBJECTIVE: Some studies indicate that endothelial progenitor cells (EPCs) originated from the bone marrow participate in neoplastic angiogenesis, and that bone marrow origin of inflammatory cells potentially contribute to neoplastic invasion, angiogenesis and metastasis. This study was to observe the origin of neovascular endothelial cells and infiltration of bone marrow-originated inflammatory cells in a murine tumor model. METHODS: Healthy C57BL/6 mice were irradiated with 60Co at 8 Gy. Bone marrow cells of green fluorescent protein (GFP) transgenic C57BL/6 mice (donators) were transplanted intravenously into C57BL/6 mice (recipients) via the tail vein 24 h after irradiation. Lewis lung tumor cells were inoculated subcutaneously into recipient mice 2 weeks after transplantation. The xenograft tumors were removed until their diameters reached approximately 1- 2 cm. Subsequently, tumor vessels and inflammatory cells were observed under fluorescent microscopy and detected using immunohistochemistry (IHC). RESULTS: Unsuccessive green fluorescence emitted by neoplastic vascular endothelial cells and inflammatory cells was observed, most of which appeared positive IHC staining. A large number of macrophages were observed inside or adjacent to the necrotic areas of the tumor. A few lymphatic cells were mainly dispersed inside tumor stroma and tumor cells. CONCLUSIONS: Partial endothelial cells of neoplastic neovessels originate from the bone marrow. The murine tumor model could be used as a specific and direct approach to observe bone marrow-originated cells in neoplasms.

Hypophosphorylation of poly(A) polymerase and increased polyadenylation activity are associated with human immunodeficiency virus type 1 Vpr expression.

The HIV-1 encoded accessory protein, viral protein R (Vpr) is responsible for several biological effects in HIV-1-infected cells including nuclear transport of the preintegration complex, activation of long terminal repeat (LTR)-mediated transcription, and the induction of cell-cycle arrest and apoptosis. Vpr's ability to arrest cells at the G2 phase of the cell cycle is due to the inactivation of p34(cdc2) cyclin B complex, resulting in hypophosphorylation of substrates involved in cell-cycle progression from G2 to mitosis (M). Poly(A) polymerase (PAP), the enzyme responsible for poly(A) addition to primary transcripts, contains multiple consensus phosphorylation sites for p34(cdc2) cyclin B kinase that regulates its catalytic activity. We investigated the effects of Vpr on the activity of PAP in Jurkat cells using a superinfection system. Superinfection of cells using Vpr+ vesicular stomatitis virus G protein (VSV-G)-pseudotyped virus caused a complete dephosphorylation of PAP. Cotransfection studies in 293T cells and Xenopus oocyte RNA injection experiments mirrored these effects. Vpr's dramatic effect on PAP dephosphorylation was reflected in enhanced polyadenylation activity in PAP activity assays. HIV-1 Vpr appears to enhance processes that are coupled to transcription such as polyadenylation and could ultimately prove to optimize HIV-1 replication and contribute to HIV-1 pathogenesis. (C)2002 Elsevier Science.

Genetic selection of peptide inhibitors of human immunodeficiency virus type 1 Vpr.

Human immunodeficiency virus 1 (HIV-1) encodes a gene product, Vpr, that facilitates the nuclear uptake of the viral pre-integration complex in non-dividing cells and causes infected cells to arrest in the G(2) phase of the cell cycle. Vpr was also shown to cause mitochondrial dysfunction in human cells and budding yeasts, an effect that was proposed to lead to growth arrest and cell killing in budding yeasts and apoptosis in human cells. In this study, we used a genetic selection in Saccharomyces cerevisiae to identify hexameric peptides that suppress the growth arrest phenotype mediated by Vpr. Fifteen selected glutathione S-transferase (GST)-fused peptides were found to overcome to different extents Vpr-mediated growth arrest. Amino acid analysis of the inhibitory peptide sequences revealed the conservation of a di-tryptophan (diW) motif. DiW-containing GST-peptides interacted with Vpr in GST pull-down assays, and their level of interaction correlated with their ability to overcome Vpr-mediated growth arrest. Importantly, Vpr-binding GST-peptides were also found to alleviate Vpr-mediated apoptosis and G(2) arrest in HIV-1-producing CD4(+) T cell lines. Furthermore, they co-localized with Vpr and interfered with its nuclear translocation. Overall, this study defines a class of diW-containing peptides that inhibit HIV-1 Vpr biological activities most likely by interacting with Vpr and interfering with critical protein interactions.

[Arsenic trioxide inhibits P-glycoprotein expression in multidrug-resistant human leukemia K562/ADM cell line that overexpresses mdr-1 gene and enhances their chemotherapeutic sensitivity].

OBJECTIVE: To investigate the effects of arsenic trioxide (As(2)O(3)) on the apoptosis and P-glyco-protein (P-gp) expression of multidrug-resistant human leukemia K562/ADM cells, and the combined effects of As(2)O(3) with conventional chemotherapeutic agents. METHODS: Multidrug-resistant human leukemia cell line K562/ADM that overexpresses mdr-1 gene was used as the target cells. The cell proliferating activity was assessed with a MTT assay. Cell morphology was examined by light microscopy, confocal microscopy and electron-microscopy. P-gp expression, cell-cycle status were determined by flow cytometry. RESULTS: K562/ADM cells were highly resistant to adriamycin, and cross-resistant to daunorubicin and etoposide. As(2)O(3) at concentrations of 0.5 to 20 micromol/L inhibited the proliferation of K562/ADM cells, and K562/ADM cells were more sensitive to As(2)O(3) than their parent K562 cells did. As(2)O(3) induced marked apoptosis of K562/ADM cells showed by typical apoptotic morphological changes and the appearance of high sub-G(1) cell population. As(2)O(3) significantly inhibited the P-gp expression in K562/ADM cells, and exerted a synergistic effect on the enhancement of the cell sensitivity to adriamycin, daunorubicin and etoposide. CONCLUSION: As(2)O(3) induces growth-inhibition and apoptosis of multidrug-resistant K562/ADM cells, and augments synergistically the sensitivity of the cells to conventional chemotherapeutic agents via down-regulation of P-gp expression.