Actinomyces qiguomingii sp. nov., isolated from the Pantholops hodgsonii.

Two previously undescribed, Gram-stain-positive, rod-shaped strains, 410T and 553, were isolated from faeces of the Tibetan antelope (Pantholops hodgsonii) from the Tibet-Qinghai Plateau, PR China. The optimum growth conditions of the two novel strains were 1 % (w/v) NaCl, 37 °C and pH 7. The end products from glucose fermentation included ethanol and lactic acid. Based on results of 16S rRNA gene sequence comparison and phylogenetic and phylogenomic analyses, strains 410T and 553 were classified into the genus Actinomyces, and were closely related to Actinomyces ruminicola (97.6 %), Actinomyces oricola (93.5 %) and Actinomyces dentalis (90.8 %). The genomic G+C content of strain 410T was 67.4 mol%. Digital DNA-DNA hybridization values between strain 410T and each of the closely related species were under 70 %. The respiratory quinones were MK-10 (68 %) and MK-9 (32 %). The main cellular fatty acids of the isolates were C16 : 0, followed by C18 : 1 ω9c. The major polar lipids were diphosphatidylglycerol and phosphatidylinositol-mannoside. The whole-cell sugars contained rhamnose, ribose and glucose. The diagnostic amino acids of cell-wall peptidoglycan included alanine, glutamic acid, lysine and ornithine. The results of biochemical, chemotaxonomic and genotypic analyses revealed that the two novel strains represent a novel species of genus Actinomyces, for which the name Actinomyces qiguomingii sp. nov. is proposed. The type strain is 410T (=CGMCC 1.16361T= DSM 106201T).

Enterovirus A Shows Unique Patterns of Codon Usage Bias in Conventional Versus Unconventional Clade.

Enterovirus A (EV-A) species cause hand, foot and mouth disease (HFMD), threatening the health of young children. Understanding the mutual codon usage pattern of the virus and its host(s) has fundamental and applied values. Here, through examining multiple codon usage parameters, we found that the codon usage bias among EV-A strains varies and is clade-specific. EVA76, EVA89, EVA90, EVA91 and EVA92, the unconventional clade of EV-A strains, show unique codon usage pattern relative to the two conventional clades, including EVA71, CVA16, CVA6 and CVA10, etc. Analyses of Effective Number of Codon (ENC), Correspondence Analysis (COA) and Parity Rule 2 (PR2), etc., revealed that the codon usage patterns of EV-A strains are shaped by mutation pressure and natural selection. Based on the neutrality analysis, we determined the dominant role of natural selection in the formation of the codon usage bias of EV-A. In addition, we have determined the codon usage compatibility of potential hosts for EV-A strains using codon adaptation index (CAI), relative codon deoptimization index (RCDI) and similarity index (SiD) analyses, and found that EV-A showed host-specific codon adaptation patterns in different clades. Finally, we confirmed that the unique codon usage pattern of the unconventional clade affected protein expression level in human cell lines. In conclusion, we identified novel characteristics of codon usage bias in distinct EV-A clades associated with their host range, transmission and pathogenicity.

The development and characterization of a stable Coxsackievirus A16 infectious clone with Nanoluc reporter gene.

Coxsackievirus A16 (CA16) belongs to the Human Enterovirus A species, which is a common pathogen causing hand, foot, and mouth disease in children. Currently, specific vaccines and drugs against CA16 are unavailable, and there is an unmet need to further understand the virus and invent effective treatment. Constructing a CA16 infectious clone with a reporter gene will greatly facilitate its virological studies. Here, we first reported the construction of a CA16 infectious clone (rCA16) whose progeny is highly replicative and virulent in suckling mice. On the basis of rCA16, we further inserted a NanoLuc (Nluc) reporter gene and made the rCA16-Nluc clone. We found that the Nluc gene in rCA16-Nluc is stable during continuous growing in Vero cells and thus allowed detection of a steady luciferase signal in rCA16-Nluc-infected Vero cells over 10 passages. Its application in antivirals characterization and high-throughput screening is exemplified by measuring IC50, CC50, and selection index of guanidine hydrochloride, ribavirin, chloroquine, and ammonium chloride against CA16. Finally, we showed that rCA16-Nluc based assay greatly simplified the CA16 neutralizing antibody tests. Thus, these two CA16 infectious clones will be robust tools for future enterovirus studies and antivirals development.

From Monovalent to Multivalent Vaccines, the Exploration for Potential Preventive Strategies Against Hand, Foot, and Mouth Disease (HFMD).

Hand, foot, and mouth disease (HFMD) recently emerged as a global public threat. The licensure of inactivated enterovirus A71 (EV-A71) vaccine was the first step in using a vaccine to control HFMD. New challenges arise from changes in the pathogen spectrum while vaccines directed against other common serotypes are in the preclinical stage. The mission of a broad-spectrum prevention strategy clearly favors multivalent vaccines. The development of multivalent vaccines was attempted via the simple combination of potent monovalent vaccines or the construction of chimeric vaccines comprised of epitopes derived from different virus serotypes. The present review summarizes recent advances in HFMD vaccine development and discusses the next steps toward a safe and effective HFMD vaccine that is capable of establishing a cross-protective antibody response.

Amphiphilic methoxy poly(ethylene glycol)-b-poly(ε-caprolactone)-b-poly(2-dimethylaminoethyl methacrylate) cationic copolymer nanoparticles as a vector for gene and drug delivery.

We studied methoxy poly(ethylene glycol)-b-poly(ε-caprolactone)-b-poly(2-dimethylaminoethyl methacrylate) (mPEG-b-PCL-b-PDMAEMA) nanoparticles as the codelivery vector of hydrophobic drug and pDNA by employing dynamic light scattering (DLS), ζ potential, transmission electron microscopy (TEM), gel retardation assay, and confocal microscopy, and subsequently its in vitro cytotoxicity and transfection efficiency were tested. mPEG-b-PCL-b-PDMAEMA nanoparticles (NPs) with or without paclitaxel are both able to complex with pDNA completely when N/P ratio is equal to or above 3, and the combinatorial deliveries of paclitaxel and pDNA have equivalent transfection efficiency compared to blank NPs/pDNA complexes when N/P ratio is equal to or above 15, which indicates that the payload of hydrophobic drug does not influence pDNA condensation and transfection efficiency. Importantly, the in vitro cell experiment results confirm that the introduction of hydrophobic segment between mPEG and PDMAEMA segments can largely improve the gene transfection efficiency, which is about 15 times that of mPEG-b-PDMAEMA. NPs/pDNA complexes can be efficiently internalized into 293T cells after transfection for 2 h. The drug release rate of paclitaxel-loaded NPs in pH 5.0 release medium is higher than that in pH 7.2 release medium. These results suggest that mPEG-b-PCL-b-PDMAEMA NPs may be a promising vector to deliver anticancer drugs and pDNA simultaneously for achieving the synergistic/combined effect on cancer therapies.

Efficacy, stability, and biosafety of sifuvirtide gel as a microbicide candidate against HIV-1.

Sifuvirtide is a proven effective HIV-1 entry inhibitor and its safety profile has been established for systemic administration. The present study evaluated the potential of sifuvirtide formulated in a universal gel for topical use as a microbicide candidate for preventing sexual transmission of HIV. Our data showed that sifuvirtide formulated in HEC gel is effective against HIV-1 B, C subtypes, CRF07_BC and CRF01_AE, the latter two recombinants represents the most prevalent strains in China. In addition, we demonstrated that sifuvirtide in gel is stable for at least 8 weeks even at 40°C, and did not cause the disruption of integrity of mucosal epithelial surface, or the up-regulation of inflammatory cytokines both in vitro or in vivo. These results suggest that sifuvirtide gel is an effective, safe and stable product, and should be further tested as a vaginal or rectal microbicide in pre-clinical model or clinical trial for preventing HIV sexual transmission.

Minocycline down-regulates topical mucosal inflammation during the application of microbicide candidates.

An effective anti-human immunodeficiency virus-1 (HIV-1) microbicide should exert its action in the absence of causing aberrant activation of topical immunity that will increase the risk of HIV acquisition. In the present study, we demonstrated that the vaginal application of cellulose sulfate (CS) gel induced topical mucosal inflammatory responses; the addition of minocycline to CS gel could significantly attenuate the inflammation in a mice model. The combined gel of CS plus minocycline not only reduced the production of inflammatory cytokines in cervicovaginal lavages (CVLs), also down-regulated the activation of CD4+ T cells and the recruitment of other immune cells including HIV target cells into vaginal tissues. Furthermore, an In vitro HIV-1 pseudovirus infection inhibition assay showed that the combined gel decreased the infection efficacy of different subtypes of HIV-1 pseudoviruses compared with that of CS gel alone. These results implicate that minocycline could be integrated into microbicide formulation to suppress the aberrant activation of topical mucosal immunity and enhance the safety profile during the application of microbicides.

Establishing a Th17 based mouse model for preclinical assessment of the toxicity of candidate microbicides.

BACKGROUND: To effectively block the invasion of human immunodeficiency virus (HIV)-1 on mucosal surface, vaginal anti-HIV-1 microbicides should avoid inflammatory responses and disruption of mucosa integrity because these will facilitate transepithelial viral penetration and replication. However, existing models fail to predict and evaluate vaginal mucosal toxicity induced by microbicides, and most importantly, they are unable to identify subtle or subclinical inflammatory reactions. This study was designed to develop a cost-effective in vivo model to evaluate microbicide safety in a preclinical study which can recapitulate the mucosal topical reaction. METHODS: A murine model was employed with nonoxynol-9 (N-9) as the topical stimulant within the vagina. Different concentrations of N-9 (1%, 3%, and 4%) were topically applied to the vagina for five consecutive days. A panel of inflammatory cytokines including interleukine-2 (IL-2), IL-4, IL-6, IL-17A, interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and immuno-regulatory IL-10 were assayed in vaginal lavage. Cytokines were quantified by using cytometric bead array (CBA) and reverse transcript (RT) real-time PCR. Histopathological evaluation of vaginal tissues was conducted on hematoxylin-eosin stained slides and scored with a semi-quantitative system according to the severity of epithelial disruption, leucocyte infiltration, edema, and vascular injection. The association between the cytokines and histopathological scores was assessed by linear regression analysis. RESULTS: All three concentrations of N-9 induced inflammatory cytokine production. The 4% N-9 application resulted in a consistent production of cytokines in a time-dependent manner. The cytokines reached peak expression on day three with the exception of IL-4 which reached its peak on day one. Histopathological examination of 4% N-9 treated cervicovaginal tissues on day three showed intensive damage in four mice (sores: 10 - 13) and moderate damage in one mouse (score: 8), which were significantly associated with both inflammatory cytokines IL-17A and IL-6 and anti-inflammatory cytokines IL-4 and IL-10. Interestingly, IL-17A showed significant positive association with inflammatory cytokine TNF-α (r = 0.739; P < 0.05), anti-inflammatory cytokines IL-10 (r = 0.804; P < 0.01) and IL-4 (r = 0.668; P < 0.05). CONCLUSIONS: Our data demonstrate that a panel of cytokines (IL-17A, IL-6, IL-4 and IL-10) could be used as surrogate biomarkers to predict the histopathological damage. Th17 may play a central role in orchestrating inflammatory cytokine responses. This Th17 based mouse model is cost-effective and suitable to assess the toxicity of candidate microbicides in preclinical studies.

The use of PEGylated poly [2-(N,N-dimethylamino) ethyl methacrylate] as a mucosal DNA delivery vector and the activation of innate immunity and improvement of HIV-1-specific immune responses.

To minimize the cytotoxicity of poly (2-(dimethylamino) ethyl methacrylate) (PDMAEMA) as a gene delivery vector, we synthesized PEGylated PDMAEMA by atom transfer radical polymerization (ATRP). Here we report its effects on transfection efficiency in vitro delivered with a GFP expression plasmid and immunogenicity in vivo after complexed with a HIV gag gene DNA vaccine. mPEG(113)-b-PDMAEMA(94) was efficient in condensing DNA and formed polyplexes with an average diameter of about 150 nm. The in vitro transfection experiments demonstrated that PEGylation dramatically decreased the cytotoxicity at the N/P ratios above 30, although the transfection efficiency in vitro was reduced. Interestingly, mice in vivo vaccination study clearly showed that PEGylated PDMAEMA used as DNA delivery vector significantly improved the prime effect of DNA vaccine through intranasal administration. Importantly, PEGylated PDMAEMA was further proved its ability to induce cytokines production by murine macrophages. Overall, mPEG-b-PDMAEMA can be used as an efficient DNA vaccine vector which enhances adaptive immune responses by activating innate immunity.

Mucosal priming with PEI/DNA complex and systemic boosting with recombinant TianTan vaccinia stimulate vigorous mucosal and systemic immune responses.

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 10 microg DNA plasmid in the PEI/DNA complexes and boosting with 10(7)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.