英语：true of efforts The same is true of efforts to develop drugs to control the diseases spread by mosquitoes.PS:上一句：蚊子产生免疫力的速度与我们发明杀虫剂的速度一样快._作业帮
拍照搜题，秒出答案
英语：true of efforts The same is true of efforts to develop drugs to control the diseases spread by mosquitoes.PS:上一句：蚊子产生免疫力的速度与我们发明杀虫剂的速度一样快.
英语：true of efforts The same is true of efforts to develop drugs to control the diseases spread by mosquitoes.PS:上一句：蚊子产生免疫力的速度与我们发明杀虫剂的速度一样快.
true of efforts 真正的努力整句意思：The same is true of efforts to develop drugs to control the diseases spread by mosquitoes.也是同样的道理,努力开拓药物来控制疾病通过蚊子传播的.
与....(程度)一样
努力的事实，这句话的意思是“努力开发药品去控制蚊子传播的疾病也是一样的事”
你的问题是什么意思呢，楼主？是把英语翻译成中文吗？ True gold fears no fire ；True love can stand the test. 真金是不怕火的；真正的爱是禁受Associated material
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Pattern of activation of human antigen presenting cells by genotype GII.4 norovirus virus-like particles
Eleonora Ponterio†, Annacarmen Petrizzo†, Ilaria Di Bartolo, Franco Maria Buonaguro, Luigi Buonaguro and Franco Maria Ruggeri*
Corresponding author:
Franco M Ruggeri
† Equal contributors
Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanità, V.le Regina Elena, 299, 00161, Rome, Italy
Laboratory of Molecular Biology and Viral Oncology, Department of Experimental Oncology, Istituto Nazionale per lo Studio e la Cura dei Tumori “Fondazione Pascale” - IRCCS, Via Mariano Semmola 142, Naples, 80131, Italy
For all author emails, please .
Journal of Translational Medicine 2013, 11:127&
doi:10.76-11-127
The electronic version of this article is the complete one and can be found online at:
Received:7 February 2013
Accepted:20 May 2013
Published:24 May 2013
& 2013 Ponterio et al.; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background
Virus-like particles (VLPs) from an Italian GII.4 norovirus strain were used to investigate
activation and maturation of circulating antigen presenting cells (APCs) of human
Peripheral blood mononuclear cells (PBMCs) isolated from five healthy subjects were
pulsed ex vivo with VLPs, and stained with a set of monoclonal antibodies (MAbs) for phenotypic
analysis by flow cytometry. Cytokine release in cell supernatants was investigated
Norovirus VLPs induced activation and maturation of circulating APCs derived from
the five donors, as well as production of IL-6, IFN-γ and TNF-α cytokines.
Conclusions
The present results suggest that VLPs can activate antigen presenting cells for an
efficient induction of the adaptive immune response.
Keywords: N I PBMCs; VLPsBackground
Norovirus is the most important agent of gastroenteritis outbreaks worldwide, representing
a relevant problem of public health [,]. In Europe, a recent report of the EFSA authority recognized the increasing role
of norovirus in causing gastroenteritis outbreaks including foodborne [].
Norovirus (NoV) has a single stranded RNA genome, organized in three ORFs, coding
for non-structural proteins (ORF1), the major capsid protein (ORF2), and for a protein
(ORF3) of still unknown function. Based on nucleotide sequences, NoVs have been classified
into five genogroups (GI - GV), two of which (GI and GII) include at least 25 distinct
genotypes and the majority of strains responsible for human infections []. Genotype GII.4 is the one most frequently associated with gastroenteritis outbreaks
worldwide []. This genotype recurrently undergoes genetic changes, generating new variants [].
Norovirus infection occurs at all ages, suggesting that a long-lasting protective
immune response is not elicited, possibly related to a mostly strain-specific antibody
response []. However, mechanisms of immunity toward norovirus are poorly understood, and data
available on cross-protection following natural infection are contradictory [,].
Due to the absence of cell culture systems, most information on NoV has been obtained
through the expression of the viral capsid protein in eukaryotic systems, particularly
baculovirus. In insect cells, baculovirus recombinant capsid proteins self-assemble
into virus-like particles (VLP) [,], thus providing suitable particulate antigen for diagnostic assays, for viral antigen
characterization and for investigating norovirus-induced immune response [,]. X-ray crystallographic analysis of the recombinant capsid indicated that VLPs are
composed of 180 copies of monomeric protein, which is structurally divided into a
shell (S) and a protruding (P) domain, where most variable sequences are located.
The latter interacts with both antibodies and cell receptors, including the histo-blood
group antigens (HBGAs) [,-]. High variability was described for HBGA affinity binding, depending on viral genotype
[]. Besides norovirus, the generation of VLPs has also been largely used to study the
immune response towards other fastidious or complex viruses [], and for production of vaccines against different human viruses, including HIV, papillomaviruses
and rotavirus [-].
Although human norovirus strains cannot cross the species barrier and infect mice,
injection with a low dose of human norovirus VLPs is effective at stimulating IgG
and IgA responses in these animals []. Preliminary phase I vaccination trials in humans using NoV VLPs have confirmed that
they are safe, and can effectively stimulate IgG and IgA responses also in man [-].
Besides antibody-mediated adaptive immunity, response to pathogenic viruses also relies
on the elicitation of cellular adaptive immunity [].
Peripheral blood mononuclear cells (PBMCs) consist mainly of monocytes, T-cells, B-cells,
smaller amounts of natural killer (NK) cells and dendritic cells (DCs) of both myeloid
and plasmacytoid origin. Dendritic cells are key determinants of v
they activate immune responses during viral infection and direct T-cells toward distinct
T-helper type responses [].
Multivariate and multiparametric analyses have been shown to predict the innate and
early adaptive immune response induced by a vaccine molecule in human monocyte-derived
dendritic cells (MDDCs) as well as PBMCs using an ex vivo experimental setting. In vitro stimulations performed on total PBMCs provide indication on the overall activation
response induced by the antigen, comparable to data generated on the MDDC subset of
cells []. This system biology approach involves high-throughput technologies such as global
gene expression profiling, multiplex analysis of cytokines and chemokines, and multiparameter
flow cytometry, combined with computational modeling [,-].
In the present study, recombinant VLPs have been produced by cloning and expressing
the ORF2 gene from a GII.4 norovirus strain detected in Italy in 2000 [], using the recombinant baculovirus expression system. The produced VLPs were used
to evaluate the immune response induced after ex vivo stimulation of human PBMCs. The results obtained show the ability of NoV VLPs to
induce activation and maturation of circulating antigen presenting cells derived from
five independent donors.
Production of Norovirus VLPs
Total RNA of a GII.4 norovirus strain (Hu/GII.4/00/IT; Grimsby-like) was extracted
from stools of an infected patient, identified during a gastroenteritis outbreak that
occurred in Italy in 2000 [], using the QIAmp Viral RNA Extraction kit (QIAgen Hilden, Germany). The cDNA was
obtained using primer (dT)20 and SuperScriptTM III reverse transcriptase (First-Strand
SuperScriptTM III Synthesis System, Life Technologies, Carlsbad, CA), following the
manufacturer’s instructions. The cDNA was used to amplify the entire ORF2, by PCR
using primers FWORF2 (5′-cgc cgg atc cat gaa gat ggc gtc gaa tga-3′), flanked by BamHI restriction enzyme site (underlined) and including the methionine codon (in bold)
and RWORF2 (5′-ctc gag taa tgc acg cct gcg ccc cgt tcc-3′), flanked by XhoI restriction enzyme site (underlined) and including the stop codon, indicated in
bold. The 1700 bp DNA fragment obtained was ligated into the pFastBac(TM)1 (Life Technologies)
that was introduced into E. coli DH10Bac (Life Technologies), yielding the DNA clone BacHu/GII.4/00/IT. Nucleotide
sequence was determined (Acc. No. KC462195). The bacmide was transfected into Sf9
insect cells to produce a high titer baculovirus. When a diffuse cytopathic effect
was observed, infected Sf9 monolayers were harvested, and VLPs were purified by ultracentrifugation
through a 30% (wt/vol) sucrose cushion, followed by a CsCl (1.362 g/cm3) density gradient []. Proper folding of the purified NoV VLPs was confirmed by electron microscopy (data
not shown). Absence of residual contaminating baculovirus was confirmed by both electron
microscopy and SDS-PAGE analysis.
Polystyrene microwell plates (NUNC, Rochester, NY) were coated with purified VLPs,
using a concentration of 0.01 μg/well. The optimal concentration of capture antigen
was established by chessboard titration using an anti-NoV positive mouse hyperimmune
serum (not shown). A second plate was coated with a baculovirus infected cell extract
expressing an unrelated protein, as negative control. After blocking by non fat milk,
the serum samples were added (1:100 of human sera and 1:1000 for hyperimmune mouse
serum). Binding of antibodies to the VLPs was detected using an anti-human GII.4 strain
(kindly provided by L Svensson, Sweden) or anti-mouse alkaline phosphatase-labeled
(AP) antibody. All assays were repeated three times. The cut-off value was determined
as the mean OD value of negative samples plus two times the standard deviation [].
PBMC donor subjects
Five healthy volunteers (4 females and 1 male), with a mean age of 30 years (range
25– 47) were enrolled into the study. Peripheral blood was collected from each subject
in 2011, under informed consent, and processed at the National Cancer Institute of
Naples, as approved by the Institutional Review Board.
PBMCs preparation
Fresh human PBMCs were isolated by Ficoll-Hypaque density gradient centrifugation,
and plated in 6-well plates at a concentration of approximately 1 × 107 cells/well in a maximum volume of 3 ml/well. Isolated PBMCs were incubated for 24
hours (short-term culture) or for 6 days (medium-term culture) in RPMI 1640 medium
(Life Technologies, Carlsbad, CA).
Cell culture medium
PBMCs culture medium consisted of RPMI 1640 medium (Life Technologies) supplemented
with 2mM L-glutamine (Sigma), 10% fetal calf serum (Life Technologies), and 2% penicillin/streptomycin
(5,000 IU, and 5 mg per ml, respectively. MP Biomedicals, Segrate, Italy). Recombinant
interleukin-2 (rIL-2; R&D Systems, Minneapolis, MN) was added at a concentration of
75 U/ml for medium-term culture (6 days).
PBMCs stimulation with VLPs
PBMCs were pulsed with purified NoV VLPs (10 μg/ml) for 24 hours or 6 days. In the
latter case, VLPs where added to PBMCs at day 0 and 3. Residual endotoxin activity,
due to lipopolysaccharide (LPS) possibly present in NoV VLP preparation, was blocked
by pre-incubation with polymyxin B sulfate (SIGMA) at a concentration of 10 μg/ml.
The absence of interference with activation due to polymyxin B sulfate was verified
as previously described []. In parallel, cells were pulsed with 8 μg/ml of LPS, PBS was
added to unstimulated PBMCs. At the end of incubation, PBMCs were harvested, washed
with PBS without calcium and magnesium, and stained for phenotypic analysis by flow
cytometry. Cell supernatants were collected to quantify cytokine production, by ELISA.
Flow cytometry
Short-term culture of PBMCs were incubated for 30 min at 4°C with human monoclonal
antibodies specific for CD3, CD40, CD80, CD83, CD86, HLA-DR, CD123, CD11c and CD14
(BD Pharmingen, San Diego, CA), washed and analyzed with a FACScalibur flow cytometer
(BD Pharmingen).
Mononuclear cells were gated by their specific forward (FSC) and side (SSC) scatters,
excluding dead cells and debris. Among selected mononuclear cells, peripheral blood
dendritic cells (PBDCs) were identified as cells positive for HLA-DR and negative
for CD3 and CD14. Within HLA-DR+CD3-CD14- PBDCs, myeloid dendritic cells (mDCs) were subsequently defined as CD11c-positive
and CD123-negative cells, whereas plasmacytoid dendritic cells (pDCs) were defined
as CD123-positive and CD11c-negative cells. Alternatively, within selected mononuclear
cells monocytes were identified as HLA-DR+CD3-CD14+ cells.
Data analysis was carried out with the WinMDI2.8 Software. A paired t test was performed,
all p-values were two-tailed and considered significant if less than 0.05.
Cytokine analysis
At the time of cell harvesting, supernatants were collected and analyzed. Cytokine
production was assessed using the Instant ELISA system (Bender Medsystems) for quantitative
detection of human cytokines, according to the manufacturer’s instructions. Data acquisition
was performed using a Sirio-S ELISA reader. A paired t test was performed, all p-values
were two-tailed and considered significant if less than 0.05.
GII.4 NoV VLPs induce a maturation phenotype in PBMCs
Human sera were collected from five healthy volunteers, and were assayed against purified
NoV VLPs, by ELISA (Figure&). Sera were positive for antibodies against GII.4 NoV at dilutions between 1:100
to 1:3200, suggesting that the five subjects had experienced a previous infection
with a GII.4 NoV strain or another norovirus genotype cross-reacting antigenically
with GII.4.
Immunoreactivity of human GII.4 NoV VLPs with five human volunteers serum samples,
human positive serum and negative serum using ELISA. The cut-off value was determined as the mean OD value of negative samples plus two
times the standard deviation [].
In addition, Ficoll-Hypaque isolated PBMCs were prepared from the volunteers’ blood,
and incubated with VLPs, or with LPS or PBS as positive and negative controls. After
24 hours stimulation, the expression of surface maturation/activation markers of immune
cells, such as CD80, HLADR, CD83, CD86 and CD40 was evaluated by flow cytometry analysis.
VLP stimulation induced a trend of increased expression of all evaluated activation/maturation
markers in circulating monocytes, as well as in myeloid dendritic cells (mDCs) (Figure&).
Expression of surface maturation/activation markers, indicated as mean fluorescence
intensity (MFI), induced by NoV VLPs and LPS on PBMCs from five healthy subjects. * P & 0.05; ** P & 0.01; *** P & 0.001; c = control (un-stimulated PBMCs); CD14+ = CD14+ CD11c + = CD11c+ mDCs.
In particular, an increased expression of CD80, CD40 and HLADR activation markers
was induced in the CD14+ monocyte cell population (p & 0.05), whereas in CD11c+ mDC cells the CD40, CD86 and HLADR activation markers resulted significantly up-regulated
(p & 0.05) (Figure&). Only a weak activation signal was observed for CD123+ plasmacytoid dendritic cells (pDCs) (data not shown). No increase of CD86 was detected
in either CD14+ monocytes or CD11c+ mDC cells upon stimulation with LPS, neither was HLADR induced in CD14+ cells, but
the reasons for this remain unclear.
Cytokine production by NoV VLP-loaded PBMCs
The level of interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and IL-6
was determined in the supernatant of PBMCs loaded with NoV-VLPs ex vivo, after 24 hours or 6 days of incubation, except for IL-2 whose level was only determined
at 24 hours. The results reported in Figure& show that NoV-VLPs induced significant production of IL-6 (p & 0.05). In particular,
IL-6 level at 24 hours was significantly higher in VLP-stimulated PBMCs than in unstimulated
PBMCs (p & 0.001), and the production of IL-6 persisted for the 6 days of induction
(p & 0.05). No IFN-γ production was observed at 24 hours, and the cytokine level only
increased after 6 days incubation for three of the five subjects (i.e. EP, LG, PP)
(Figure&). On the other hand, TNF-α induction was only detected for some of the donors at
either 24 (i.e. AP, PP) or 6 days (i.e. EP, PP) (Figure&), whereas no IL-2 production was induced in any subject by NoV-VLPs at 24 hours (data
not shown). The overall results indicate that NoV-VLPs induced activation of PBMCs,
which is coupled with the production of IL-6, IFN-γ and TNF-α.
Cytokine analysis in supernatants of PBMCs from five healthy subjects, induced by
NoV VLPs and LPS. Ranges of cytokine concentration detectable in the experimental conditions were:
IL-6 (3–200 pg/ml), TNF-α (7–500 pg/ml), IFN-γ (1.6 - 100 pg/ml). Higher concentrations
were deduced by analysis of diluted samples.
Discussion
The nature of immunity to norovirus is a key determinant if considering the prospect
of future prevention of disease by possible vaccines. To date, little is known about
the protective immune response elicited by norovirus infection, that is largely due
to the lack of cell culture systems for noroviruses pathogenic to humans.
In the absence of in vitro replicating viruses, VLPs represent a useful tool for investigating virus-ligand
interactions and the anti-viral immune response, since they strictly resemble infectious
viruses both antigenically and morphologically [,,]. VLPs might also be suitable for vaccination against NoV infection [], similar to vaccines already in use for other viruses such as papillomavirus [].
In the present study, we used a baculovirus expression system to generate VLPs from
a NoV GII.4 strain identified during a major gastroenteritis outbreak in Italy in
2000 []. As previously reported by others [-], also the VLPs described in this study induced a strong immune response in Balb/c
mice, eliciting elevated antibody levels (data not shown). The VLPs prepared in this
study persisted during stressful CsCl centrifugation and proved antigenically stable
at 4°C. They were thus suitable for investigating the cell-mediated immune response
to GII.4 norovirus, which was done using peripheral blood cells from five asymptomatic
adult volunteers. Although these subjects had not been affected by gastroenteritis
in the 4 weeks before testing, they all resulted positive for NoV-antibodies in a
NoV-VLP based ELISA test, indicating a previous infection with this or a similar norovirus
genotype. This observation is not surprising since GII.4 is a pandemic genotype, and
is in line with previous findings that between 90-100% healthy adult humans are seropositive
for norovirus [,,].
The innate and early adaptive immune response induced by GII.4 NoV-VLPs was evaluated
on ex vivo stimulated PBMCs from the volunteers by means of activation/maturation phenotype
analysis and cytokine production analysis. NoV-VLPs induced an increased expression
of activation markers and co-stimulatory molecules in circulating APCs, particularly
in CD14+ monocyte and CD11+ mDC cell populations. In particular, PBMC stimulation resulted in increased expression
of surface activation/maturation markers (i.e. CD80, CD86, CD40 and HLA-DR), whose
role in the initiation of the immune response is well defined. Both CD80 and CD86
are able to prime T cells, providing a co-stimulatory signal necessary for T cell
activation and survival [,]. In addition, CD40 expression on antigen-presenting cells is known to be enhanced
by CD40L on activated T cells, in a loop of induction of the immune response []. Finally, the HLA-DR molecule, which represents a ligand for the T-cell receptor
(TCR), is up-regulated as well in response to antigenic stimuli []. The overall results provide interesting clue on the responsiveness of ex vivo loaded circulating APCs.
Moreover, PBMC stimulation resulted in increased production of specific cytokines,
such as TNF-α, IL-6, whose level persisted for the 6 days of stimulation, and IFN-γ,
whose level increased only after prolonged stimulation.
Several studies have been performed to identify the cytokine profile induced by NoV.
In particular, an investigation on the T-cell response, induced in humans after a
challenge with a GII.2 norovirus strain, identified a predominant Th1 CD4-dependent
cell response, characterized by significant IFN-γ secretion []. Moreover, experimental infection of gnotobiotic piglets with a GII.4 human NoV strain
was also reported to induce both antibodies and Th1/Th2 cytokine responses, locally
and systemically []. These authors detected persistently higher Th1-specific cytokines (low transient
IFN-γ and high IL-12) in infected pig sera, but also Th2-specific IL-4 and IL-6 cytokines,
although to a lower level. Notably, a delayed IFN-α response was also evident [].
Several authors suggest that response to norovirus infection mainly involves IFN-γ
secretion by CD4 Th1 T cells [,,,].
Since the production of cytokines was not investigated in isolated cell subpopulations
in the present study, no conclusion can be drawn about a specific pattern of Th1 versus
Th2 response upon stimulation with GII.4 VLPs. However, our present observations suggest
that despite an initial status characterized by IL-6 production, a prolonged stimulation
may induce viable T cells to produce IFN-γ, implying that NoV-VLPs might activate
autologous T cells.
The low reactivity shown by ELISA testing of some donors’ sera may be due to the antigenic
differences between the strain(s) which had infected the five subjects in the past
and the virus genotype corresponding to the VLPs used. The observation that IFN-γ
became detectable only at later time points after in vitro stimulation might either indicate a switch in cytokine production or a low rate of
secretion of this cytokine that would require a longer time to reach a detectable
concentration in the culture supernatant.
Conclusions
The present study on norovirus VLPs supports earlier findings, confirming that NoV
VLP administration can specifically activate human PBMCs ex vivo.
Further studies are needed to clarify the reported lack of a long-lasting protective
immune response following natural infection in man.
Competing interests
The authors declare that they have no competing interests of either financial or non-financial
nature regarding the work described in the present manuscript and its publication.
Authors’ contributions
EP drafted the first version of the manuscript, designed experiments, analyzed and
interpreted data, conducted serological testing, and participated to production of
VLP and experiments for PBMC activation. AP analyzed PBMC data, participated to experiments
for PBMC activation and contributed to a final draft. IDB participated to production
of VLP and contributed to final draft preparation. LB and FMB designed experiments,
and participated to final draft preparation. FMR supervised the activities, and reviewed
the manuscript. All authors read and approved the final manuscript, and agreed with
the conclusions of the work. None of the authors has any conflict of interest with
the work being presented.
Authors’ information
E Ponterio BSc, PhD, post- I Di Bartolo BSc, PhD FM Ruggeri BSc, PhD
Chief of Unit, Viral Zoonoses Unit, Department of Veterinary Public Health and Food
Safety, Istituto Superiore di Sanità, Rome, Italy. L Buonaguro MD, C
FM Buonaguro MD, Chief of Unit, and A Petrizzo BSc, PhD, post-doc, Laboratory of Molecular
Biology and Viral Oncology, Department of Experimental Oncology, Istituto Nazionale
per lo Studio e la Cura dei Tumori “Fondazione Pascale” - IRCCS, Naples, Italy.
Acknowledgements
This study was partially supported by grants from the Ministry of Health, Italy: “Creazione
di una rete di laboratori virologici di riferimento (Noronet-Italia) per il controllo
delle epidemie di gastroenterite da Norovirus in Italia” CCM 2009.
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