Optical and electrophysiological tools were used to map out the neural circuits within and between cortical layers in three different brain regions, and the results suggest regional specializations for sensory versus motor information processing. Rodents move their whiskers to locate and identify objects. Cortical areas involved in vibrissal somatosensation and sensorimotor integration include the vibrissal area of the primary motor cortex (vM1), primary somatosensory cortex , and secondary somatosensory cortex (S2). We mapped local excitatory pathways in each area across all cortical layers using glutamate uncaging and laser scanning photostimulation. We analyzed these maps to derive laminar connectivity matrices describing the average strengths of pathways between individual neurons in different layers and between entire cortical layers. In vM1, the strongest projection was L2/3→L5. In vS1, strong projections were L2/3→L5 and L4→L3. L6 input and output were weak in both areas. In S2, L2/3→L5 exceeded the strength of the ascending L4→L3 projection, and local input to L6 was prominent. The most conserved pathways were L2/3→L5, and the most variable were L4→L2/3 and pathways involving L6. Local excitatory circuits in different cortical areas are organized around a prominent descending pathway from L2/3→L5, suggesting that sensory cortices are elaborations on a basic motor cortex-like plan. The neocortex of the mammalian brain is divided into different regions that serve specific functions. These include sensory areas for vision, hearing, and touch, and motor areas for directing aspects of movement. However, the similarities and differences in local circuit organization between these areas are not well understood. The cortex is a layered structure numbered in an outside-in fashion, such that layer 1 is closest to the cortical surface and layer 6 is deepest. Each layer harbors distinct cell types. The precise circuit wiring within and between these layers allows for specific functions performed by particular cortical regions. To directly compare circuits from distinct cortical areas, we combined optical and electrophysiological tools to map connections between layers in different brain regions. We examined three regions of mouse neocortex that are involved in active whisker sensation: vibrissal motor cortex (vM1), primary somatosensory cortex (vS1), and secondary somatosensory cortex (S2). Our results demonstrate that excitatory connections from layer 2/3 to layer 5 are prominent in all three regions. In contrast, strong ascending pathways from middle layers (layer 4) to superficial ones (layer 3) and local inputs to layer 6 were prominent only in the two sensory cortical areas. These results indicate that cortical circuits employ regional specializations when processing motor versus sensory information. Moreover, our data suggest that sensory cortices are elaborations on a basic motor cortical plan involving layer 2/3 to layer 5 pathways. Sensation in the rodent vibrissal system relies on active whisking for interactions with the environment Ascending sensory and descending motor pathways interact at multiple levels including the brainstem In rodents, some of the cytoarchitectonic features of vM1, vS1, and S2 are area-specific, such as the presence of “barrels” in layer (L) 4 of vS1, and others are not, such as the presence of most cortical layers, including L1, L2/3, L5A, L5B, and L6 We identified vibrissal motor cortex (vM1), primary somatosensory (barrel) cortex (vS1), and secondary somatosensory cortex (S2) based on anatomical coordinates, cytoarchitectonic features, anatomical labeling experiments, and in the case of vM1, optical microstimulation mapping. vM1  was locaWe prepared coronal brain slices containing vM1, vS1, or S2  and usedTo calibrate LSPS, we recorded in cell-attached mode from excitatory neurons, while uncaging glutamate on a grid around the cell . The spaρcell is the neuronal density at the point of uncaging (neurons/µm3), Vexc is the volume of excited neurons (µm3), and SAP is number of APs fired per presynaptic neuron (AP/neuron). The average strength of a synaptic connection (qcon) is calculated from equation (1). The collection of qcon for different neuronal populations defined by laminar location is the basis of connectivity matrices. We first present the mapping data for each area in the more familiar form of average input maps. In subsequent sections we summarize connectivity in laminar connectivity matrices, which take into account the parameters in equation (1).An input map represents the aggregate functional synaptic connectivity between small clusters of presynaptic excitatory neurons at the stimulus locations and individual postsynaptic neurons. Pixels in input maps do not represent the strengths of unitary connections; rather, they measure average monosynaptic excitatory responses to a single uncaging event  and mapped the local sources of excitatory synaptic input with LSPS using a stimulus grid that spanned vM1 . We poolWe recorded from 80 excitatory neurons in vS1, using a different stimulus grid matched to the cortical thickness . In vS1,S2 abuts the lateral edge of vS1, where the barrel pattern terminates . The cytWe recorded input maps for 100 excitatory neurons in S2 . Similari, j) in the matrix  corresponds to the strength of a connection  from the jth presynaptic location  to the ith postsynaptic location . Distance is measured in normalized units along the radial directions . Because of the curvature of vM1 at the cortical flexure  to coordinates corresponding to an unfolded cortex , where h is the horizontal distance along the laminar contour and r is the distance along the radial axis. x-y coordinates  at a given presynaptic radial distance (r) into bins , describing connectivity between neurons at different locations along the radial axis , we binned the data and applied correction factors to derive the strength of input per presynaptic neuron per AP. We divided the connection strength in the raw connectivity matrix by the mean number of APs per uncaging event at the presynaptic region  we multiplied the neuron→neuron connections by the number of presynaptic and postsynaptic neurons per layer . Pixel values were 10–100 times lower than typical unitary EPSCs, reflecting both the generally low probability of connections between excitatory neurons in cortical circuits  The connectivity matrix description allows us to directly contrast local circuits in different cortical regions. The elements (pixels) in the neuron→neuron connectivity matrices, Wneuron , represeWlayer , the number of excited neurons needs to be estimated, based on the excitability (SAP), neuron density (ρcell), and excitation volume (Vexc) at the uncaging location (Equation 1). The accuracy of the estimate of qcon is limited by our measurement of ρcell and neuronal excitation , the forelimb region of somatic M1 A second main interlaminar hotspot in vS1 was the descending pathway(s) L2/3→L5, which was the predominant hotspot in the two motor areas. We noted that this pathway was present in all three cortical regions studied here and was similarly prominent in somatic M1 In three of the four areas, L6 neither received nor sent strong projections (but vS1 neurons in L6 received a weak projection from L4). L6 output is provided by an ascending connection to L4 in cat visual cortex One major difficulty in making a comparison of connectivity between two cortical areas is selecting the laminar position of pre- and postsynaptic neurons for the comparison. Is it better to compare identical relative laminar depths between cortical areas, not accounting for the decreased thickness of superficial layers, and increased thickness of deep layers, in motor areas? How shall we treat the presence or apparent absence of a distinct layer 4? We present a direct quantitative comparison of three major areas identified in our study, based on cytoarchitectonic laminar divisions  Figure . In vS1 radial to refer to the axis defined by the apical dendrites of pyramidal neurons; this axis is approximately normal to the cortical surface. Normalized radial distance is along the radial axis, bounded by the pia and the white matter, where pia  =  0 and the L6/white matter border  =  1. Vertical is synonymous with radial. Horizontal, or lateral, refers to planes normal to the radial axis, approximately parallel to layers, or laminae . Coronal brain slices (300 µm) were cut (Microm HM 650V), incubated 30 min at 37°C in oxygenated ACSF , and maintained in a holding chamber at 22°C for up to 5 h during recording. For vM1 slices, the brain was pitched upward ∼10° to optimize alignment with the radial axis of vM1, and slices ∼0.7–1.3 mm anterior to bregma were used; for vS1 and S2 slices, the brain was cut coronally, and slices ∼1–2 mm posterior to bregma were used , and NMDA receptor blockade with 5 µM 3-((R)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid . Patch pipettes were fabricated from borosilicate glass with filament (4–6 MΩ). Intracellular solution contained (in mM): 128 K-gluconate, 10 HEPES, 10 sodium phosphocreatine, 4 MgCl2, 4 Na2ATP, 3 sodium L-ascorbate, 1 EGTA, and 0.4 Na2GTP . To visualize dendritic arbors, 20 µM Alexa 594 or 488 (Molecular Probes) was added to the internal solution. In some cases, 2–3 mg/mL biocytin was included. Electrophysiological signals were amplified with an Axopatch 700B amplifier (Molecular Devices), filtered at 4 KHz, and digitized at 10 KHz. Data I/O were controlled by Ephus, a suite of custom Matlab-based (Mathworks) software tools available online (https://openwiki.janelia.orgRecordings were performed at room temperature (22°C) in ACSF. Neuronal excitability was reduced by increased divalent ions  and white matter Dataset S1Connectivity matrix values for neuron- and layer-based connectivity matrices. Matlab file containing the values for all neuron- and layer-based connectivity matrices of (0.01 MB TXT)Click here for additional data file.Figure S1Anatomical identification of vS1, vM1, and S2. (A) Injection of AAV-GFP into vM1 labeled axons projecting to vS1. Left, injection site in medial agranular cortex (vM1). Middle and right, axon termination zones in vS1. Arrow: L4. Str, striatum. cp, cerebral peduncle. VL, ventrolateral nucleus of thalamus. (B) Brainstem targets of vM1 were labeled following AAV-GFP . SpV, spinal trigeminal. 7, facial motor nucleus. (C) Injection of red Lumafluor beads into vM1 labeled somata in vS1. Left, brightfield image of injection site in vM1. Inset, fluorescence image of injection site. Middle, retrograde labeling of vM1-projecting somata in vS1 and S2. Regions of medial agranular cortex  are also labeled. Right, laminar distribution of vM1-projecting neurons in vS1 and S2. Note labeling in L2/3, L5A, and deep in cortical white matter (“L7”). Neurons are less densely labeled in L5B and L6; label is absent in L4. Labeling spreads across multiple barrels. POm, posterior nucleus of thalamus. Agm, medial agranular cortex. Cg, cingulate cortex. (D) Two additional examples of vS1 and S2 labeling following vM1 bead injection. (E) Injection of red Lumafluor beads into vS1 labeled somata in vM1. Left, fluorescent image of injection site in vS1. Right, retrograde labeling of vS1-projecting somata in vM1. VPM, ventroposteromedial nucleus of thalamus. (F) Injection of AAV-GFP into vS1 labeled S2 (left). Injection site is anterior to the plane of the slice. Note the lateral path of axons in white matter and layer 6. Simultaneous injection of red Lumafluor beads retrogradely labeled S2 (right).(9.85 MB TIF)Click here for additional data file.Figure S2Uncaging MNI-glutamate evokes APs perisomatically. (A) Cell-attached recording in vM1 with high density  and low density  stimulus grids to examine regions of excitation for L5 vM1 neurons. At left, brightfield images with stimulus locations indicated. Middle, traces recorded at each map point. Right, maps quantified to show number and location of evoked APs. Stimulation in L2/3 did not evoke APs in the L5 neurons. (B) Two further examples of cell-attached recordings of L5 vM1 neurons stimulated throughout the width of cortex. (C) Cell-attached recording in L3 of vS1 with two example neurons (top and bottom), presented as above. Uncaging in L4 did not cause spiking of L3 neurons, but perisomatic stimulation did.(2.95 MB TIF)Click here for additional data file.Figure S3Profile of LSPS photoexcitability and neuron depth in coronal vS1 slices. (A) Photoexcitability of L3 neurons (blue) measured in loose-seal recording (excitation profiles) as in z, depth of soma (slice surface  =  0). Monoexponential fit is shown as a dashed line. (B) Photoexcitability of L5B neurons (red), plotted as above. Monoexponential fit is shown as a dashed line. (C) Photoexcitability of L3 and L5B neurons plotted on the same axes. Note that L5B is more excitable than L3. (D) Photoexcitability of all vS1 neurons (green) plotted with L3 and L5B data. Neurons deeper than 100 µm in the slice did not fire APs.(0.45 MB TIF)Click here for additional data file.Figure S4Neuronal density in vM1, vS1, and S2. (A) Coronal sections of mouse brain (50 µm) were stained for the neuronal marker NeuN and red fluorescent secondary antibody. Sections from three animals in vM1 (left), vS1 (middle), and S2 (right) shown. Scale bars, 200 µm. (B) Neuronal somata (red dots) were marked in Neurolucida, and coordinates were imported into Matlab for counting cell numbers as a function of radial distance. An example from vS1 is shown. Pia is at the top of the black rectangle; only neurons within the identified column were counted. (C) Neuron diameter was computed as a function of laminar depth (shown for vS1) and an Abercrombie correction factor [(thickness)/(slice thickness + object diameter)] was used to account for overcounting of neurons at the top and bottom of the section. (D) Neuronal density as a function of laminar position is plotted for vM1 (blue), vS1 (red), and S2 (green). Thick lines indicate the average of three sections. Thin lines indicate density for individual sections. Laminar boundaries for vS1 are indicated as horizontal lines.(2.03 MB TIF)Click here for additional data file.Figure S5Examples of vM1 input maps. (A) Bright-field image of vM1, with overlaid LSPS grid , aligned to pia medially and superiorly. (B) Example traces from three neurons' maps (boxed regions in C). Circles: somata. Dendritic responses omitted. (C) Examples of input maps for neurons at different laminar depths. Three examples are given (in a column) for each radial distance from the pia. Grouping corresponds to (0.55 MB TIF)Click here for additional data file.Figure S6Examples of vS1 input maps. (A) Bright-field image of vS1, with overlaid LSPS grid , aligned to pia superiorly and over neuron horizontally. (B) Example traces from three neurons' maps (boxed regions in C). Circles: somata. Dendritic responses omitted. (C) Examples of input maps for neurons at different laminar depths. Three examples are given (in a column) for each radial distance from the pia. Grouping corresponds to (0.55 MB TIF)Click here for additional data file.Figure S7Examples of S2 input maps. (A) Bright-field image of S2 at left  end of vS1, with overlaid LSPS grid , aligned to pia superiorly and over neuron horizontally. S2 maps are aligned such that the medial side (S1) is to the left, as indicated below the image. (B) Example traces from three neurons' maps (boxed regions in C). Circles: somata. Dendritic responses omitted. (C) Examples of input maps for neurons at different laminar depths. Three examples are given (in a column) for each radial distance from the pia. Grouping corresponds to (0.57 MB TIF)Click here for additional data file.Figure S8Comparison of molecular and anatomical definition of cortical lamination.  Sample in situ images of gene expression from coronal sections of vM1 in the Allen Brain Atlas. Etv1 and Wfs1 illustrated. (C) Brightfield image of vM1 with the axis along which measurements of relative laminar depth were taken indicated. Cortex is marked from 0 (pia) to 1 (white matter). White marks to the right of the ladder indicate cytoarchitectonic boundaries based on the video image. (D) Molecularly defined layers for vM1 plotted for comparison. Radial distance at which Etv1, Wfs1, Rorβ, Plexin D1, Enc1, Abat, and Fezf2 expression were measured based on images in the Allen Brain atlas. Boundaries indicate onset and offset of expression. There were two laminae for PlexinD1 and Enc1. Etv1 expression contained bands of high  and low (deep) expression. Thy1-ChR2 mouse (line 18) expressed in L5 neurons in motor cortex. Summary table of measurements for vM1 and vS1 are given below.(3.36 MB PDF)Click here for additional data file.Figure S9Analysis of horizontal and oblique pathways. (A) Each vM1 neuron's input map was projected onto a vector representing the horizontal profile of synaptic input. The vectors were sorted by postsynaptic position. (B) Same data as for (A), but grouped into distance bins and averaged. Equivalent to projection of the 3-D map data array onto the postsynaptic-horizontal plane. (C) Projection of the 3-D map data array onto the presynaptic-horizontal plane . (D–F) Same analyses as (A–C), for vS1 data set. (G–I) Same analyses as (A–C), for S2 data set. (J–L) Horizontal-only analysis. Only “home-layer”  data were used to generate each neuron's horizontal vector. For example, top vectors show L2 horizontal inputs to L2. Left: all neurons' vectors, sorted by postsynaptic position. Middle: vectors were grouped into distance-based bins and averaged. Black pixels: sites <100 µm from soma were excluded, and the top bin (corresponding to L1) was empty. Right: Mean weighted distance of individual and averaged horizontal data as a function of postsynaptic position. Values represent the mean, for each neuron, of the vector values times the pixel distances from the soma. Lines with error bars represent mean ± s.e.m.(0.66 MB TIF)Click here for additional data file.Figure S10Construction of neuron→neuron connectivity matrices. (A) Example of how neuron→neuron connectivity matrices are constructed; vS1 is used for this example. Given the individual input vectors to a given neuron (n neuron  averaged(0.45 MB TIF)Click here for additional data file.Figure S11Construction of neuron→neuron connectivity matrices in cortical layer bins. (A) Example of how neuron→neuron connectivity matrices are constructed; vS1 is used for this example. Process is identical to (0.39 MB TIF)Click here for additional data file.Figure S12Construction of layer→layer connectivity matrices in cortical layer bins. (A) Example of how layer→layer connectivity matrices are constructed from neuron→neuron connectivity matrices; vS1 is used for this example. The corrected neuron→neuron matrix of (0.23 MB TIF)Click here for additional data file.Figure S13Quantitative comparison of neuron→neuron connectivity derived from complementary methods. (A) Matrices of neuron→neuron connectivity based on pair recordings (0.43 MB TIF)Click here for additional data file.Figure S14Functional motor mapping using optical microstimulation. (A) Coronal brain slice through motor cortex, prepared from a Thy1-ChR2-YFP (line 18) mouse . (B) Functional motor map of whisker (red), forelimb (green), and tongue (blue), aligned to bregma (Br) and averaged across six animals. Distances are in mm from the midline or bregma. Colored lines delimit the area where movement could be evoked at threshold power in at least 50% of trials. The grid of the stimulation pattern is indicated with light blue dots .(2.18 MB TIF)Click here for additional data file.Figure S15Channelrhodopsin-based mapping of the extent of axonal innervations of local circuits in brain slice. (A) vM1 neurons were transfected with channelrhodopsin-2 by stereotactic injection of adeno-associated virus. vM1 slices were prepared as for LSPS circuit mapping and placed in normal artificial cerebrospinal fluid containing 2 mM Ca2+ and 1 mM Mg2+, as well as 5 µM CPP and 10 µM NBQX to block excitatory synaptic transmission. L2/3 pyramidal neurons were then recorded in cell-attached configuration while exciting the slice with 1 ms pulses of 1 mW 473 nm laser light. Grid spacing was selected to cover a large area  or a detailed area . Points where an action potential was evoked are indicated in red overlay; points without excitation are shown in gray. White circle indicates L2/3 soma. Left panels are large area maps overlaid on the slice image. The right image in (A) demonstrates the spatial resolution (∼50 µm) of the excitation on a medial extending branch of the axon.  Additional examples presented in the same format.(2.80 MB TIF)Click here for additional data file.Figure S16Input mapping of afferents to L5A neurons in vibrissal motor cortex using opposite faces of adjacent brain slices. (A) Experimental design. Two adjacent brain slices were prepared from the same animal. The posterior side of the anterior slice (PSAS) and anterior side of the posterior slice (ASPS) were used for recording (as previously described). (B) Representative input maps from neurons on PSAS (top) and ASPS (bottom) slices. (C) Group data for n  =  11 neurons on each side. Average input strength is quantified as the mean of the input vector for each given presynaptic depth bin, and presented ± s. e. m.(0.49 MB TIF)Click here for additional data file.Table S1Connectivity matrix values for neuron- and layer-based connectivity matrices. Excel file containing the values for all neuron- and layer-based connectivity matrices of (0.03 MB XLS)Click here for additional data file.Text S1Laminar analysis of excitatory local circuits in vibrissal motor and sensory cortical areas. Supplemental information to the article. Sections include: Supplemental Methods ; converting input maps to connectivity matrices; methods for optical microstimulation), Supplemental Discussion , and References.(0.32 MB RTF)Click here for additional data file.


Recurrent outbreaks of H5, H7 and H9 avian influenza viruses in domestic poultry accompanied by their occasional transmission to humans have highlighted the public health threat posed by these viruses. Newer vaccine approaches for pandemic preparedness against these viruses are needed, given the limitations of vaccines currently approved for H5N1 viruses in terms of their production timelines and the ability to induce protective immune responses in the absence of adjuvants. In this study, we evaluated the feasibility of an adenovirus (AdV)-based multivalent vaccine approach for pandemic preparedness against H5, H7 and H9 avian influenza viruses in a mouse model. Replication-defective AdV vectors expressing hemagglutinin (HA) from different subtypes and nucleoprotein (NP) from one subtype induced high levels of humoral and cellular immune responses and conferred protection against virus replication following challenge with H5, H7 and H9 avian influenza virus subtypes. Inclusion of HA from the 2009 H1N1 pandemic virus in the vaccine formulation further broadened the vaccine coverage. Significantly high levels of HA stalk-specific antibodies were observed following immunization with the multivalent vaccine. Inclusion of NP into the multivalent HA vaccine formulation resulted in the induction of CD8 T cell responses. These results suggest that a multivalent vaccine strategy may provide reasonable protection in the event of a pandemic caused by H5, H7, or H9 avian influenza virus before a strain-matched vaccine can be produced. Occasional reports of human infection with both low and highly pathogenic avian influenza A viruses of H5, H7, and H9 subtypes underscore the public health threat and pandemic potential posed by these avian influenza subtypes Vaccination remains the most effective and economically prudent strategy to combat the threat posed by avian influenza viruses with pandemic potential. For pandemic preparedness purposes, vaccines against H7, H9 and multiple clades of H5N1 viruses have been developed and clinically evaluated, and in the case of H5N1 vaccines, stockpiled by national and international human health agencies. The use of an adjuvant is necessary to heighten and broaden neutralizing antibody responses elicited by H5N1 inactivated split or subunit vaccines to achieve cross-reactivity across H5N1 clades and subclades In the present study, the feasibility of an adenovirus (AdV) -based multivalent vaccination approach against multiple avian influenza viruses of the H5, H7, and H9 subtypes was evaluated in a mouse model. Replication-incompetent AdV vectors expressing HA derived from H5, H7, and H9 avian influenza subtypes were generated and evaluated for their ability to induce protective immunity against heterologous viruses when used alone or in combination as a multivalent vaccine formulation. The influenza virus nucleoprotein (NP) has been shown to be a target of virus-specific CD8+ T cells that contribute to virus clearance following infection The Purdue University Biosafety Committee and Animal Care and Use Committee approved the protocol for all animal studies described in this manuscript and conducted at Purdue University, under the auspices of the Institutional Animal Care and Use Committee (IACUC) #A3231-01 which is supported by the American Association for Laboratory Animal Science (AALAS). All animals were humanely euthanized by an overdose of anesthetic (ketamine/xylazine). The 293 and MDCK cell lines were obtained commercially from American Type Culture Collection (ATCC), and the 293Cre cell line was obtained from Merck & Co., Inc., Whitehouse Station, NJ. The BHH2C cell line which was created in the PI’s laboratory using a combination of commercially available cell lines, MDBK and 293. The use of all human cell lines and the construction of BHH2C hybrid cell line were with permission from the Purdue University Institutional Review Boards (IRB) formed in accordance with federal regulations. A Research Exemption was obtained for the use of commercially available human cell lines. The IRBs are a unit of the Human Research Protection Program (HRPP) which is housed within the Office of Research Administration (ORA).MDCK (Madin-Darby canine kidney), 293 [human embryonic kidney cells expressing human AdV serotype 5 (HAdV5) E1 gene products], 293Cre [293 cell line that constitutively expresses Cre recombinase ], and BHH2C (bovine-human hybrid clone 2C) -pathogenic strains. All vaccine constructs were purified by cesium chloride density-gradient centrifugation and titrated by plaque assays on the BHH2C cell line as described HAdV vector-based monovalent vaccine constructs, HAd-H1HA (H1HA), HAd-H7HA (H7HA), and HAd-H9HA (H9HA), were generated after placing the whole length coding region of the HA gene of H1, H7, or H9 subtype influenza virus, A/California/04/2009 (H1N1), A/Netherlands/219/2003 (H7N7), or A/chicken/Hong Kong/G9/1997(H9N2), respectively. The gene was under the control of the human cytomegalovirus (HCMV) immediate early promoter and the bovine growth hormone (BGH) polyadenylation signal (PolyA) and was inserted in the early (E) 1 region of HAdV5 genome (containing deletions in E1 and E3 regions) using a Cre-Lox recombination system in a 293Cre cell line 8 plaque-forming units (PFU) of each of mono or bivalent AdV vector alone: H1HA; 1203HA; 05HA; H7HA; H9HA; H5b; H7H9b or used in these combinations: H5b+H7H9b; H5b+NP; H7H9b+NP; H5b+H7H9b+NP; and H5b+H7H9b+H1HA (combination formulation contained 108 PFU of each AdV vector) twice at four-week intervals. In this study we used a 10-fold higher vaccine dose than previously used to induce protective immune responses in mice 50) of influenza viruses, Egypt/08, TK/VA . Briefly, thawed lung tissues were homogenized in 1 ml of sterile phosphate-buffered saline (PBS), and then 10-fold serially diluted lung homogenates were used to infect MDCK cells seeded in 96-well plates. After incubation for 72 hours (h) at 37°C, the HA activity of the culture supernatants was determined by hemagglutination of turkey red blood cells (TRBC). The limit of virus detection was 0.5 log10 50% tissue culture infectious dose (TCID50) per ml.All animal studies were conducted following guidelines and approval from Institutional Biosafety Committee and Institutional Animal Care and Use Committee at Purdue University. Experiments involving influenza viruses were conducted under Biosafety Level 2+ containment. To assess the breadth of immune response, groups of six-to-eight week-old female BALB/c mice  obtained from Harlan Sprague Dawley Inc., Indianapolis were inoculated intramuscularly (i.m.) with 10Vibrio cholerae  at 37°C for 16 h and later heat inactivated at 56°C for 30 minutes. The presence of hemagglutination inhibition (HI) antibody was determined using four hemagglutination units of each of influenza viruses and 0.5% TRBC. The HI titer was defined as the reciprocal of the highest dilution of serum which completely prevented the agglutination of TRBC.Sera from all mice were treated with a receptor-destroying enzyme (RDE) from 50 of each influenza virus. Serial two-fold dilutions of RDE-treated and heat-inactivated serum samples were mixed with 100 TCID50 of an influenza virus and incubated at room temperature for 1 h. The virus antibody mixture was then added to the monolayer of MDCK cells, and the plates were incubated for 72 h at 37°C. After incubation, the HA activity of the supernatant was assessed by HA assay with 0.5% TRBC. The virus neutralization titer was defined as the reciprocal of the highest dilution of serum which showed complete absence of TRBC agglutination. The assay was done in triplicate. We did not check the induction of antibodies by vaccine constructs against the A/Indonesia/5/2005 (H5N1)-PR8/CDC-RG virus strain as it was not available for distribution by the CDC at the request of the Government of Indonesia.The microneutralization assay was performed using MDCK cells and 100 TCIDThe protocol was similar as described previously -mouse–IgG  at a dilution of 1:2000 was added to the wells followed by 1 h incubation at room temperature. The plates were developed with a TMB substrate . Optical density measurements were taken at 405 nm using an EMax microplate reader  after stopping the reaction with 1 M sulfuric acid. The cut-off value was set as the mean plus 3× standard deviation of the negative control.Streptavidin-coated 96-well plates  were incubated with 2 μg/ml of either P1 or P2 peptide in PBS overnight at 4°C. After blocking with PBS containing 1% bovine serum albumin (BSA), two-fold serially diluted serum samples (starting at 1:50 dilution) were added and incubated at room temperature for 3 h. The horseradish peroxidase-conjugated goat anti5 or 1×106 cells/well) isolated from inoculated mice were cultured in the presence of HA518 (IYSTVASSL) or NP147 (TYQRTRALV) peptides  in RPMI medium , supplemented with 10% reconstituted fetal bovine serum for 60 h and developed according to an ELISpot protocol 96-well flat-bottom polyvinyl chloride micro-titer plates  were coated overnight at 4°C with an anti-mouse IFN-γ antibody . Splenocytes   and an anti-CD8 antibody conjugated with allophycocyanin (APC)  as described previously P<0.05.The Kruskall-Wallis test was used for calculation of significance. The significance was set at Cre recombinase–mediated site-specific recombination technique was used to insert the full-length coding regions of the HA genes of the A/California/04/2009 (H1N1), A/Netherlands/219/2003 (H7N7), or A/Chicken/Hong Kong/G9/1997(H9N2) viruses into the E1 region of the HAdV genome under the control of the HCMV promoter and the BGH PolyA to create HAd-H1HA (H1HA), HAd-H7HA (H7HA) and HAd-H9HA (H9HA) monovalent vaccine constructs, respectively  [Cell-mediated immune (CMI) responses induced against influenza has been shown to aid in the clearance of influenza virus infection P<0.005) .50 of Egypt/08, TK/VA, or G1/99 influenza virus. The protection efficacy was determined by assessing virus titers in the lungs collected three days after challenge. Mean lung virus titers of at least 4 log10 TCID50/ml were detected in mice that received ΔE1E3 and were challenged with any of the three viruses. In contrast, mice immunized with monovalent vaccines  had no detectable virus in the lungs following challenge with a heterologous virus from the same subtype, but no cross-protection was observed in mouse groups challenged with a heterologous influenza subtype  developed substantial levels of virus neutralization antibodies against pH1N1 in H1HA immunized animals or pH1N1, Egypt/08, TK/VA, or G1/99 in H5b+H7H9b+H1HA immunized animals . FurtherP≤0.010) and NP147 (P≤0.005) epitope-specific CD8+T cells were observed in all the vaccine groups but not in the groups that received ΔE1E3 (P≤0.005) increased numbers of both HA518-specific and NP147-specific interferon-γ-secreting cells were detected in spleen cells of all vaccine groups compared to the group receiving only ΔE1E3  in mediating protection against pandemic virus infection was clearly demonstrated during the 2009 pH1N1pandemic Earlier we had demonstrated the feasibility of the AdV vector-based vaccine approach for pandemic preparedness against H5N1 influenza viruses Consistent with previous findings Although antibody titers against the homologous subtype induced by monovalent vaccines were higher, the bivalent vaccine further broadened vaccine efficacy by inducing humoral responses against both homologous and antigenically distinct virus strain of the same subtype. Furthermore, when used together as a multivalent vaccine composition, protective immune responses against all the three avian influenza subtypes were induced. Although, the antibody titers  in the combination vaccine group were two-fold lower compared to the bivalent vaccines administered separately, the difference was not statistically significant (P>0.05). Further inclusion of an AdV vector-based H1N1 vaccine into the multivalent vaccine composition resulted in an even broader multi-subtype vaccine that also elicited protective responses against the 2009 pandemic H1N1 virus.Unlike the variable head region of HA, the stalk region is relatively conserved across influenza A viruses. Several recent studies have demonstrated the importance of HA stalk-specific antibodies in providing heterosubtypic protection. The cellular immune responses induced against NP play an important role in the virus clearance from the lungs, and, therefore, are critical for recovery from influenza virus infections. Since NP is highly conserved across influenza A virus subtypes, the CMI responses generated against it are usually broadly cross-protective Due to high prevalence of AdV infections in humans, there is variable levels of pre-existing AdV immunity in the majority of human population and it is believed that it can adversely impact the vaccine efficacy of AdV vector-based vaccines 8 VP when given i.n. resulted in approximately 80% seroconversion (at least four-fold rise in HI titers) in humans without any major safety concerns 10 VP of an AdV vector expressing the M. tuberculosis antigens Ag85A, Ag85B, and TB10.4 resulted in a more than 50-fold increase in the frequencies of TB-specific CD8+ T cells in humans primed with the BCG vaccine 18 purified VP are projected to be produced from a single 10,000 L bioreactor within 7–8 weeks 11 VP are used per individual as a one- or two-dose regiment suggesting that AdV vector-based vaccines can be produced at large scale for meeting the pandemic influenza vaccine requirement.Eventually the HA and/or NP gene cassettes of H5, H7, and H9 avian influenza subtypes will be expressed in a single AdV vector to reduce the vaccine dose and avoid the complexity of vaccine production, testing and formulation. It has been demonstrated that an AdV-based influenza vaccine dose of 5×10In summary, our findings demonstrate the feasibility of a multivalent vaccine approach for pandemic preparedness against multiple avian influenza virus subtypes. Such a multicomponent vaccine could provide a first line of defense following the emergence of a pandemic threat and/or could be used to prime the population in the inter-pandemic period to induce some level of immunity that would be boosted following infection with the pandemic strain.


This manuscript describes the phenotypic and genotypic characteristics of two selected EV71 strains (EV71:TLLm and EV71:TLLmv), which have been adapted to replicate in mouse-derived NIH/3T3 cells, in contrast to the original parental virus which is only able to replicate in primate cell lines. The EV71:TLLm strain exhibited productive infection in all primate and rodent cell lines tested, while EV71:TLLmv exhibited greater preference for mouse cell lines. EV71:TLLmv displayed higher degree of adaptation and temperature adaptability in NIH/3T3 cells than in Vero cells, suggesting much higher fitness in NIH/3T3 cells. In comparison with the parental EV71:BS strain, the adapted strains accumulated multiple adaptive mutations in the genome resulting in amino acid substitutions, most notably in the capsid-encoding region (P1) and viral RNA-dependent RNA polymerase (3D). Two mutations, E167D and L169F, were mapped to the VP1 canyon that binds the SCARB2 receptor on host cells. Another two mutations, S135T and K140I, were located in the VP2 neutralization epitope spanning amino acids 136–150. This is the first report of human EV71 with the ability to productively infect rodent cell lines in vitro.Since its identification in 1969, Enterovirus 71 (EV71) has been causing periodic outbreaks of infection in children worldwide and most prominently in the Asia-Pacific Region. Understanding the pathogenesis of Enterovirus 71 (EV71) is hampered by the virus’s inability to infect small animals and replicate in their derived  P1, P2 and P3. P1 encodes four viral structural proteins 1A-1D ; P2 and P3 encode seven non-structural proteins 2A-2C and 3A-3D Enterovirus 71 (EV71) is a small non-enveloped virus approximately 30 nm in diameter. The viral capsid exhibits icosahedral symmetry and is comprised of 60 identical units (protomers), with each consisting of four viral structural proteins VP1–VP4. The capsid surrounds a core of a single-stranded positive-sense RNA genome of 7,450 nucleotides (nt) long. The genome contains a single open reading frame which encodes a polyprotein of 2193 amino acids (aa) and is flanked by a long 5′ untranslated region (UTR) of 745 nt and a shorter 3′ UTR of 85 nt with a poly-A tract of variable length at its 3′ terminus. The polyprotein is divided into three regions, Enterovirus in 1974 EV71 causes an array of clinical diseases including hand, foot and mouth disease (HFMD), aseptic meningitis, encephalitis and poliomyelitis-like paralysis mainly in infants and young children Similar to other human enteroviruses, EV71 is unable to infect animals other than humans, although rhesus and cynomolgous monkeys can be experimentally infected quasispeciesRNA viruses, by virtue of their error-prone replication and high mutation rates 9 CCID50/ml, while virus titer from COS-7 was 106 CCID50/ml  within 48 hours post-infection (hpi)  M, and vCID50/ml . EV71:BSCID50/ml  D, 1G, aCID50/ml  and Hep-CID50/ml  indicatiAll the rodent cell lines tested were determined to be non-permissive to EV71:BS infection. Lytic CPE of cells was absent following infection , virus tEV71:TLLm was derived following serial passage of EV71:BS in NIH/3T3 mouse cell line for a minimum of 60 cycles. All primate and rodent cell lines tested, with the exception of NRK cells, were permissible to productive infection by EV71:TLLm. Full CPE was observed in RD, Vero, and COS-7 , as wellThe EV71:TLLmv virus strain was derived from further passage of EV71:TLLm in NIH/3T3 cells for another 40 cycles. EV71:TLLmv caused lytic CPE in fewer number of cell lines - RD, Vero, NIH/3T3, Neuro-2A, and TCMK cells , and fulOn the other hand, the primate cell lines HeLa, Hep-2, and COS-7 were observed to be non-permissible to EV71:TLLmv infection, as shown by the absence of CPE , immeasuRRR), calculated by taking the ratio of virus titer values assayed in NIH/3T3 to the titer values assayed in Vero, was used as a surrogate measure of the degree of virus adaptation to NIH/3T3 cells. The parental EV71:BS virus displayed highly negative RRR values for RD, Vero, and COS-7 . Alignment of the full genome sequences of EV71:TLLm (GenBank Accession No. KF514879) against EV71:BS (GenBank Accession No. KF514878) revealed 60 nucleotide mutations, 21 of which resulted in amino acid substitutions . On the Amino acid changes were also observed within the P2 and P3 regions, most notably in the RNA-dependent RNA polymerase (3D). EV71:TLLm acquired four amino acid changes, mostly in the palm and thumb domains of the enzyme . EV71:TLVero and NIH/3T3 cells transfected with viral RNA exhibited full CPE at 7 days post-transfection (dpt) (data not shown). Viral antigens were detected in NIH/3T3 cells transfected with viral RNA of EV71:BS , but notin vitro cultured rodent cell lines. The mouse adherent fibroblast cell line NIH/3T3, which was derived from the NIH/Swiss mouse embryo Sequential passage of the human EV71 isolate (EV71:BS) generated virus strains that gained the ability to infect Whereas the clinical isolate EV71:BS infects only primate cell lines, EV71:TLLm productively infects both primate and rodent cell lines. It is worth noting that although EV71:TLLm virus has successfully achieved the ability to infect rodent cells, the degree of adaptation to NIH/3T3 cells is less pronounced. The virus titer determined using Vero cells is much higher than that determined in NIH/3T3 cells, as indicated by negative values in relative replication rates (RRR) assay . In addiAlthough it is not possible to pinpoint which amino acid substitutions are adaptive to the mouse NIH/3T3 host cell, and this subject is not covered in this manuscript, viral whole genome sequencing may shed light to potential adaptive mechanisms. Most of the amino acid substitutions identified reside in the P1 (capsid) and RNA polymerase (3D) proteins , 3, suggMapping of the capsid protein mutations indicate that majority of the identified amino acid substitutions in the viral P1 region reside in exposed regions of the protein , specifiTo explore the possible role of the P1 region mutations in binding the virus receptor for host cell entry, EV71:BS viral RNA was transfected into murine cells. Direct introduction of EV71:BS RNA into the mouse cell cytoplasm results to productive infection in NIH/3T3 cells, as suggested by the observation of virus-induced CPE and measurable virus titers in the culture supernatant as assayed in Vero cells . Re-inocSeveral amino acid mutations were also observed in the P2 and P3 regions, which encode virus proteins that are crucial for virus replication and hijacking the host cell protein translation machinery in vitro cultured rodent cells. This may be due to the high genetic barrier associated with major changes in phenotype and host range To our knowledge, this is the first report of EV71 strains originally derived from human clinical sample that have successfully gained the ability to productively infect several rodent cell lines following successive passages in a mouse cell line. The relatively high mutation rates during RNA replication results in variant genomes that serve as genetic reservoirs of phenotypic traits for future adaptive potential, and the consequential replication as a quasispecies distribution 6 CCID50 per mouse) via intragastric and intraperitoneal routes . A proportion of the infected pups succumbed to acute illness leading to death within a few days post-infection (unpublished data). This preliminary result seems promising, and further studies are required to ascertain the usefulness of these virus strains in establishing mouse models of EV71 infection, pathogenesis and disease.The mouse cell line (NIH/3T3)-adapted virus strains presented in this study may provide an alternative opportunity in establishing mouse models of EV71 infections in immune-competent mice. Preliminary findings of animal studies demonstrated that BALB/c mice aged 14 days old could be acutely infected with EV71:TLLm . Studies were performed using various mammalian cell lines; human adenocarcinoma cell lines HeLa (CCL-2) and HEp-2 (CCL-23), and rhabdomyosarcoma RD (CCL-136); African green monkey kidney Vero (CCL-81), and Vervet monkey kidney fibroblast COS-7 (CRL-1651); mouse neuroblastoma Neuro2A (CCL-131), embryonic fibroblast NIH/3T3 (CRL-1658), and kidney epithelial TCMK (CCL-139); hamster ovarian epithelial-like CHO-K1 (CCL-61), and normal rat kidney epithelial NRK (CRL-6509).The human EV71 BS strain (EV71:BS) was previously isolated from the brainstem of a deceased patient infected with EV71. The virus was passaged in Vero cells for four cycles prior to storage at −80°C until further use. The mouse cell (NIH/3T3)-adapted EV71:TLLm strain was derived from the EV71:BS strain via continuous serial passage (>60 cycles) in mouse NIH/3T3 cells. The EV71:TLLm strain was further passaged (40 cycles) in NIH/3T3 cells to generate the mouse cell-adapted virulent strain (EV71:TLLmv).v/v) of fetal bovine serum  and 0.22% (w/v) sodium bicarbonate  and incubated at 37°C and 5% CO2, unless otherwise stated. All infected cells were incubated in maintenance medium (DMEM supplemented with 1% FBS and 0.22% NaHCO3).All cell lines were grown in Dulbecco’s Modified Eagle’s Medium  supplemented with 10% (5 cells per well) were seeded in tissue culture-treated six-well plates  overnight, infected with 500 μl of virus suspension (MOI 1), and incubated at 30°C, 37°C, or 39°C for 2 hours. Cells were washed twice in sterile Phosphate-Buffered Saline  solution before addition of fresh maintenance medium . Infected cells were observed daily for appearance of distinct lytic cytopathic effects (CPE).Cells . Plates were subjected to three cycles of freezing and thawing, and lysates were harvested and cleared by vigorous vortexing followed by centrifugation at 1,500×g for 10 minutes. Cleared supernatants were stored in cryovials  at −80°C until further use.For temperature adaptation assays, inoculated Vero and NIH/3T3 cells were incubated at 30°C, 37°C, and 39°C and observed daily for appearance of CPE. Respective culture supernatants were harvested at 48 hpi and stored in cryovials at −80°C until further use.Various mammalian cell lines, i.e. RD, HeLa, and HEp-2 (human), Vero and COS-7 (monkey), NIH/3T3, Neuro-2A, and TCMK (mouse), CHO-K1 (hamster), and NRK cells (rat), were infected with either parental EV71:BS or derived NIH/3T3-adapted EV71 strains at MOI (multiplicity of infection) of 1 and incubated at 37°C for 10 days. Cultures were observed daily for appearance of CPE.4cells per well) and Vero cells (4×103 cells per well) were seeded overnight in a 96-well plate. Frozen virus thawed to room temperature were diluted (10−1) in sterile 1% aqueous sodium deoxycholate , and vigorously mixed for 15 minutes to disaggregate virus. Disaggregated virus was subjected to ten-fold serial dilution in maintenance medium, and 100 μl diluted virus from 10−3 dilution onwards was added onto each well of cells. Plates were incubated at 37°C and observed daily under inverted light microscopy for the appearance of distinct CPE. Virus titer was reported as 50% cell culture-infectious doses per volume (CCID50/ml).Virus supernatants were subjected to endpoint titration and assayed in both NIH/3T3 and Vero cells. The virus titer was enumerated using the Reed and Muench method relative replication rates (RRR) in NIH/3T3 and Vero cells was calculated using the following formula:A is the virus titer assayed in NIH/3T3 cells, and B is the virus titer assayed in Vero cells.To assess the degree of adaptation of EV71:TLLm and EV71:TLLmv in NIH/3T3 cells, virus supernatants harvested from previously infected primate and rodent cell lines were subjected to virus titer assay using both NIH/3T3 and Vero cells. The titer ratio used to measure w/v) Evan’s blue counter stain.For infected cells that did not exhibit significant CPE, immunofluorescence (IF) staining was performed to verify infection. Cells were trypsinized at 72 hpi, washed twice in sterile PBS, and coated onto Teflon slides . Slides were air-dried inside the biosafety cabinet and UV-treated for 15 minutes to inactivate live virus prior to fixation in cold acetone at 4°C for 10 minutes. Slides were probed with pan-Enterovirus antibody  and subsequently with FITC-conjugated mouse IgG  mixed with 0.01% (4 cells per well) seeded overnight in 24-well plates were transfected with viral RNA extracted from 4×106 CCID50 virus using Lipofectamine 2000  following the manufacturer’s protocol. RNA from EV71:BS, EV71:TLLm, and EV71:TLLmv was extracted using Viral RNA kit  and incubated with Lipofectamine 2000 on cells for 6 hours at 37°C. Transfected cells were observed daily for appearance of CPE. At 7 dpi, supernatant was harvested from infected cells and passaged onto freshly seeded Vero and NIH/3T3 cells. Cells were observed daily for appearance of CPE, and at 7 dpi, cells were trypsinized and processed for immunofluorescence viral antigen detection.Vero and NIH/3T3 cells (3×10http://expasy.org/spdbv/) and the SWISS-MODEL server Viral RNA of EV71:BS, EV71:TLLm, and EV71:TLLmv strains was extracted using Viral RNA kit  and reverse-transcribed using Superscript II . The cDNA obtained was amplified with GoTaq Green  and degenerate EV71 primers (primers’ sequences are available upon request). The amplicon was purified using PCR clean up kit  and cloned into pZero2 vector . Clones were selected from Kanamycin plates, inoculated into LB broth (50 μg/ml Kanamycin) and allowed to grow overnight at 37°C for plasmid extraction with QiaSpin Miniprep kit . Plasmids were subsequently sequenced by the BigDye terminator method  using the same primers. The 500-bp fragment sequences obtained were aligned using BioEdit v7.0.9.0 Figure S1Virus fitness assessment of EV71:BS, EV71:TLLm, and EV71:TLLmv in NIH/3T3 and Vero cells at 30°C. Overnight seeded (A) NIH/3T3 and (B) Vero cells infected with EV71:BS , EV71:TLLm , or EV71:TLLmv  were incubated at 30°C and observed under the light microscope with phase-contrast at 24 hpi (a–c), 48 hpi (d–f), and 72 hpi (g–i). Images taken are representative of two independent experiments.(TIF)Click here for additional data file.Figure S2Virus fitness assessment of EV71:BS, EV71:TLLm, and EV71:TLLmv in NIH/3T3 and Vero cells at 37°C. Overnight seeded (A) NIH/3T3 and (B) Vero cells infected with EV71:BS , EV71:TLLm , or EV71:TLLmv  were incubated at 37°C and observed under the light microscope with phase-contrast at 24 hpi (a–c), 48 hpi (d–f), and 72 hpi (g–i). Images taken are representative of two independent experiments.(TIF)Click here for additional data file.Figure S3Virus fitness assessment of EV71:BS, EV71:TLLm, and EV71:TLLmv in NIH/3T3 and Vero cells at 39°C. Overnight seeded (A) NIH/3T3 and (B) Vero cells infected with EV71:BS , EV71:TLLm , or EV71:TLLmv  were incubated at 39°C and observed under the light microscope with phase-contrast at 24 hpi (a–c), 48 hpi (d–f), and 72 hpi (g–i). Images taken are representative of two independent experiments.(TIF)Click here for additional data file.Figure S4Transfection of murine cell lines NIH/3T3, Neuro-2A, and TCMK with EV71:BS viral RNA for evidence of virus replication. Overnight seeded NIH/3T3, Neuro-2A, and TCMK cells were either infected with 1000 CCID50 of EV71:BS virus  or transfected with equivalent amounts of viral RNA . and harvested at 48 hpi for viral antigen detection. Virus in the supernatants were harvested at 7 dpi and passaged onto fresh Vero  and NIH/3T3 cells . Cells were harvested and stained for viral antigens at 48 hpi.(TIF)Click here for additional data file.Figure S5Localization in VP1 and VP2 of adaptive mutations in the genomes of EV71:TLLm and EV71:TLLmv. Adaptive mutations observed in the VP1  and VP2  regions of EV71:TLLm  and EV71:TLLmv  were modelled using DeepView/SwissPDBviewer v3.7 and the 3D structure of EV71 capsid P1 region (PDB ID 4AED). The mutations were observed to be mostly localized to the surface-exposed loops of the protein. The B–C loop is shown in red; D–E loop in yellow; E–F loop in orange; and G–H loop in pink.(TIF)Click here for additional data file.Figure S6Titer ratio in NIH/3T3 cells relative to titer in Vero cells of virus supernatant harvested from cells either transfected with EV71:BS viral RNA or infected with live virus. Supernatants from NIH/3T3, Neuro-2A, Vero, and TCMK either transfected with viral RNA or infected with live virus were harvested and subjected to virus enumeration by Reed-and Muench method. The ratio of the log(titer) determined in NIH/3T3 cells relative to the titer determined in Vero cells is shown. 3T3-TRANS: RNA transfected NIH/3T3 cells; 3T3-INF: virus infected NIH/3T3 cells. Asterisks indicate Student’s t-test with p-value <0.05.(TIF)Click here for additional data file.


Plasmodium falciparum employs antigenic variation to evade the human immune response by switching the expression of different variant surface antigens encoded by the var gene family. Epigenetic mechanisms including histone modifications and sub-nuclear compartmentalization contribute to transcriptional regulation in the malaria parasite, in particular to control antigenic variation. Another mechanism of epigenetic control is the exchange of canonical histones with alternative variants to generate functionally specialized chromatin domains. Here we demonstrate that the alternative histone PfH2A.Z is associated with the epigenetic regulation of var genes. In many eukaryotic organisms the histone variant H2A.Z mediates an open chromatin structure at promoters and facilitates diverse levels of regulation, including transcriptional activation. Throughout the asexual, intraerythrocytic lifecycle of P. falciparum we found that the P. falciparum ortholog of H2A.Z (PfH2A.Z) colocalizes with histone modifications that are characteristic of transcriptionally-permissive euchromatin, but not with markers of heterochromatin. Consistent with this finding, antibodies to PfH2A.Z co-precipitate the permissive modification H3K4me3. By chromatin-immunoprecipitation we show that PfH2A.Z is enriched in nucleosomes around the transcription start site (TSS) in both transcriptionally active and silent stage-specific genes. In var genes, however, PfH2A.Z is enriched at the TSS only during active transcription in ring stage parasites. Thus, in contrast to other genes, temporal var gene regulation involves histone variant exchange at promoter nucleosomes. Sir2 histone deacetylases are important for var gene silencing and their yeast ortholog antagonises H2A.Z function in subtelomeric yeast genes. In immature P. falciparum parasites lacking Sir2A or Sir2B high var transcription levels correlate with enrichment of PfH2A.Z at the TSS. As Sir2A knock out parasites mature the var genes are silenced, but PfH2A.Z remains enriched at the TSS of var genes; in contrast, PfH2A.Z is lost from the TSS of de-repressed var genes in mature Sir2B knock out parasites. This result indicates that PfH2A.Z occupancy at the active var promoter is antagonized by PfSir2A during the intraerythrocytic life cycle. We conclude that PfH2A.Z contributes to the nucleosome architecture at promoters and is regulated dynamically in active var genes. Plasmodium falciparum is a protist parasite that causes malaria and kills more than 800,000 people per year. The parasite escapes from the human immune response by antigenic variation through switching between expression of different var genes. These encode different variant antigens that are expressed on the surface of the infected erythrocyte and mediate pathogenic adhesion of the infected erythrocytes to host receptors. Understanding how this process is regulated may lead to the identification of factors that are essential for immune evasion and that could represent novel drug targets. Here, we have identified the parasite's histone variant PfH2A.Z as a novel contributor to the transcriptional regulation of antigenic variation. PfH2A.Z is enriched in the promoter of many genes, but enrichment correlates with gene expression only in var genes. Furthermore we show that PfH2A.Z enrichment in var promoters is antagonised by the var gene silencing factor PfSir2A. These findings further extend our knowledge of the complex mechanisms regulating gene expression in P. falciparum. Plasmodium falciparum causes the majority of malaria-induced morbidity and mortality, resulting in approximately 860,000 deaths each year Plasmodium parasites have a complicated life cycle, during which they differentiate into several morphologically distinct asexual and sexual stages in the human host and the mosquito vector.P. falciparum erythrocyte membrane protein 1 (PfEMP1) variant antigens, which are expressed on the IE surface and are encoded by approximately 60 genes of the var multigene family var gene is expressed at a time P. falciparum and chronic infection Disease occurs during the repeated cycles of invasion and asexual replication of the parasite inside human erythrocytes. A central mechanism of malaria pathogenesis is the ability of the infected erythrocytes (IE) to sequester at vascular sites by cytoadherence to host receptors. Through this process the parasite avoids clearance by splenic macrophages and contributes to severe malaria complications such as cerebral and placental malaria P. falciparum employs stringent regulatory mechanisms to achieve stage-specific gene expression P. falciparum, emphasizing the significance of epigenetic control in the parasite During its asexual, intaerythrocytic life cycle, Plasmodium. The Plasmodium genome encodes a broad set of common chromatin remodelling and modifying factors, including many that are novel and that may be employed in unique epigenetic mechanisms Epigenetic gene regulation confers a heritable state of gene expression and is typically mediated by changes in chromatin structure without a change in DNA sequence. The fundamental units of chromatin are nucleosomes and are formed by ∼146 bp of DNA wrapping around an octamer of histones. The canonical nucleosome components are two H2A/H2B dimers and an H3/H4 tetramer. In most eukaruyotes an additional subunit, H1, links nucleosomes, but this subunit is missing in Plasmodium species. Chromatin can exist as compact, silent heterochromatin and as open, transcriptionally competent euchromatin. These different physical states are essentially determined by the composition and distribution of nucleosomes, post-translational modification of histones, presence of chromatin associated trans-factors and by the covalent modification of DNA, although this latter mechanism has not yet been shown in P. falciparum chromatin has unique features that differ markedly from other eukaryotes. P. falciparum intergenic regions, including promoters, display a global nucleosome depletion P. falciparum promoters does not correlate inversely with steady-state mRNA levels P. falciparum exists in an unusually euchromatic state conferred by the euchromatin marks trimethylated lysine 4 of histone 3 (H3K4me3) and acetylated lysine 9 of histone 3 (H3K9ac) P. falciparum heterochromatin, defined by the histone post-translational modification H3K9me3 and its cognate trans-factor heterochromatin protein 1 (HP1), is restricted to subtelomeric and several chromosome internal domains in P. falciparum that contain gene families including the majority of var genes The organisation of var genes present both in subtelomeric and central chromosomal positions form clusters at the nuclear periphery var gene appears to require it to leave the heterochromatic cluster and relocate to a specific perinuclear expression site var genes is controlled by epigenetic mechanisms var gene must acquire the histone marks H3K4me3 and H3K9ac in its promoter var promoter is the only cis element required for monoallelic exclusive transcription var gene intron is important for var gene silencing cis sequence elements contribute to the rate of var gene switching var gene regulation was proven by the upregulation of numerous var genes and the loss of monoallelic var gene expression that occurred when either of two Sir2 histone deacetylase genes were disrupted The P. falciparum gene regulation is apparent, the role of alternative histones has not yet been investigated. H2A.Z is an H2A variant, which is essential for viability in most organisms apart from yeast and has been highly conserved through evolution Toxoplasma gondii, H2A.Z enrichment at promoters (and enhancers in humans) correlates with gene activity Alternative histones can replace canonical histones through ATP-dependent deposition to create structurally and functionally specialized chromatin domains P. falciparum. Using immunofluoresecence analysis (IFA), co-immunoprecipitation (Co-IP) and chromatin immunoprecipitation (ChIP) in conjunction with quantitative real time PCR (qPCR), we show that PfH2A.Z is enriched in the promoter of a set of developmentally regulated genes in the euchromatin compartment. In these genes promoter occupancy of PfH2A.Z does not correlate with transcription levels, suggesting that presence of PfH2A.Z is important for providing a transcriptionally competent chromatin structure at the promoter but does not directly relate to promoter activity. In var genes by contrast, PfH2A.Z promoter occupancy is strongly associated with transcriptional activity, as PfH2AZ is periodically enriched in the active var gene and is depleted in silent var genes, which is consistent with our IFA evidence that H2A.Z is absent from the subtelomeric heterochromatin compartment. This balance is distorted in cells in which the histone deacetylase Sir2A has been disrupted. Thus, these data suggest that var gene silencing requires expulsion of PfH2A.Z from the var promoter and that this involves histone deacetylation.In this study, we provide the first characterization of the alternative histone H2A.Z in P. falciparum H2A.Z variant (PfH2A.Z) is encoded by the gene PFC0920w S. cerevisiae H2A.Z variant Htz1 and 67.3% identity with the human and mouse H2A.Z proteins  followed by quantitative PCR in ring stage parasites, trophozoites and schizonts. H2A.Z is enriched in nucleosomes surrounding the transcription start site (TSS) in other organisms, therefore at least two quantitative (q) PCR reactions were performed for each gene to amplify regions near the predicted TSS upstream of the start codon (ups) as well as in the open reading frame (orf).Our results show that PfH2A.Z is enriched in all three stages in the promoter regions of candidate genes that are differentially regulated throughout the life cycle , S3A. InTo determine whether the level of PfH2A.Z promoter occupancy in the examined genes correlated with gene expression, mRNA levels were quantified by q-RT-PCR . The upsTo further investigate the relationship between PfH2A.Z and euchromatic and heterochromatic histone marks, ChIP was performed in parallel for PfH2A.Z as well as H3K4me3, H3K9ac and H3K9me3. PfH2A.Z and the two euchromatic marks, H3K4me3, H3K9ac all showed significant enrichment in the ups region when compared to the orf in ring and schizont stage parasites, in contrast the levels of the heterochromatic mark H3K9me3 were the same in ups and orf . This isTogether, these results demonstrate that PfH2A.Z is enriched near the TSS in genes, independently of their transcriptional activity, and that PfH2A.Z enrichment near the TSS correlates with enrichment of the euchromatin marks H3K4me3 and H3K9ac.P. falciparum histone deacetylases Sir2A and Sir2B silence subtelomeric and central var genes S. cerevisiae H2A.Z antagonises subtelomeric gene silencing by Sir2 var genes. We harvested chromatin and RNA from a parasite culture that had been selected for the expression of a single var gene encoding VAR2CSA by panning on chondroitin sulphate A (CSA). To map the position of PfH2A.Z along the var2csa gene, we designed seven qPCR reactions spanning this var locus. The TSS of var2csa has previously been mapped to −1475 bp upstream of the start codon in the FCR3 parasite line The var2csa transcription peaks , and var2csa expressing parasites (var2csa ON) at ring stage. Transcription levels were verified by q-RT-PCR , but not the TSS of inactive var2csa (var2csa OFF) nor the TSS of the other two silent var genes PF08_0141 (var41) and PFL0020w (var20). In contrast to the upstream region, all three var genes were moderately enriched in PfH2A.Z in the introns  var genes in ring stages of both 3D7Δsir2 parasite lines  and p = 0.0152 (Δsir2B))  (var gene expression is down-regulated in mature 3D7Δsir2 parasites (data not shown) Although ups/orf ratios were generally quite low in ring stages, PfH2A.Z enrichment at Δsir2B)) . In schiΔsir2B)) . This re 0.0159) , althougvar genes in 3D7Δsir2A schizonts we analysed by ChIP the relationship between enrichment of PfH2A.Z and H3K9me3 or H3K4me3, respectively , orthologues of which mediate the post-replicative H2A.Z incorporation into nucleosomes in yeast and humans T. brucei and T. gondiiP. falciparum conforms to this pattern, with an enrichment of PfH2A.Z, but not H2A, in the upstream regions of genes , as described previously 5′-GGGATCCGGATGGAAGTTCCAGGAAAAGT and PfH2A.ZEcoRIRev: 5′-GAATTCTTATTGAGCTGTTGGGGGAAGTG. The PCR product was cloned into pGEX-5X-3 . PfH2A.ZGST fusion protein was expressed in BL21 cells in Luria Broth and induced with 1mM IPTG. Bacteria pellets were resuspended in Bugsbuster reagent and benzonase (Novagen) and the soluble proteins were purified on GST-Bind Resin (Novagen). Antibodies were generated in rabbits by the Walter and Eliza Hall Institute Monoclonal Antibody Facility .The full length PfH2A.Z (PFC0920w) sequence was amplified from 3D7 cDNA using oligonucleotides PfH2A.ZBamHIFor: Abcam Ab1791), rabbit anti-H2A (Millipore 07-146), rabbit anti-H2B (Abcam Ab1790), non-immune rabbit IgG (Abcam Ab 46540) and pre-immune rabbit serum. Primary antibodies used for IFA co-localization and WB were rabbit anti-H3K9me3 (Abcam Ab8898), rabbit anti-H3K4me3 (Millipore 04-745) and rabbit anti-H3K9ac (Millipore 06-942). Secondary antibodies for IFA were goat anti-rabbit AlexaFluor488 or chicken anti-rabbit AlexaFluor594 (Molecular Probes). Secondary antibodies for WB were goat anti-rabbit HRP (Invitrogen).Primary antibodies employed in ChIP assays in this study were rabbit anti-PfH2A.Z, rabbit anti-H3 (7 IE were separated by SDS-PAGE on 10% Bis-Tris gels (Invitrogen) and analysed by Western Blotting as described previously Parasites were harvested in 8 hour intervals at 5–10% parasitemia. Lysates were generated by saponin lysis of cultures and extraction of the resulting parasite pellets with 2 x SDS PAGE loading buffer. Equivalents of 5×10Molecular Probes) as a secondary antibody. PfH2A.Z was subsequently detected with affinity purified rabbit anti-PfH2A.Z (10 ng/µl) directly labelled using the Zenon Rabbit IgG labelling kit (Invitrogen) according to the manufacturers instructions. IE were mounted onto slides using ProLong antifade (Invitrogen), left overnight to cure and analysed with an Olympus FV1000 Confocal Laser Scanning Microscope and the FluoView software.IFA was performed on paraformaldehyde/glutaraldehyde-fixed cultures as described previously Electron Microscopy Sciences, Fort Washington, PA). Ultrathin (90–100 nm) sections were cut using a Leica Ultracut R microtome, labeled with rabbit anti-PfH2A.Z and goat-anti-rabbit IgG conjugated to 12 nm colloidal gold (Jackson ImmunoResearch Laboratories). Sections were poststained with uranyl acetate and lead citrate and observed using a Philips CM120 BioTwin Transmission Electron Microscope.Parasites were fixed in 1% glutaraldehyde for 1 h at 4°C, dehydrated in increasing ethanol concentrations, then embedded in LR Gold resin (Roche)) followed by dounce homogenization (Pestle B). 0.25% NP40 was added to the parasite suspension prior to homogenization. Nuclei were pelleted by centrifugation at 21,000×g for 10 min at 4°C and resuspended in SDS lysis buffer . Chromatin was sheared into 200–1000 bp fragments by sonication for 2×8 min at 30 sec intervals using a Bioruptor UCD-200 (Diagenode) and diluted 1∶10 in ChIP dilution buffer .Chromatin was isolated at three time points during the intra-erythrocytic developmental cycle (IDC) from early ring (6–14 hpi), trophozoite (24–32 hpi) and schizont (36–44 hpi) stage parasites. Parasite cultures were cross-linked with 1% paraformaldehyde for 10 min at 37°C and the reaction subsequently quenched with 125 mM glycine. After one wash with PBS parasites were released from IE by saponin lysis, and nuclei were isolated by incubation for 30 min on ice in lysis buffer . For each IP, 1×109 ring stage parasites, 2.5×108 trophozoites or 1×108 schizonts were used. Optimal antibody dilutions were determined empirically and ranged between 1∶50 and 1∶200.Immunoprecipitation was performed with the EZ ChIP Kit (Applied Biosystems) (Applied Biosystems 7900HT) with SYBR PCR master mix (Applied Biosystems). Optimal PCR conditions were determined for each primer pair using serial dilutions of gDNA. PCR was performed in duplicates and melting curves were analysed after each run to confirm the specificity of the amplification. ChIP recoveries were normalized for input signals (ΔCt) and corrected for values obtained with non-immune control antibodies (ΔΔCt). The site specific enrichment of histones was calculated as the 2−ΔΔCt value. To correct for differences in nucleosome density, enrichment of PfH2A.Z and H2A was expressed as a ratio over H3. As in the course of the study different H3 antibody lots were used to precipitate biological replicates, the H3 recoveries and therefore ratios varied considerably between experiments. However, the pattern of histone variant enrichment across genes was reproducible between experiments (Primers amplifying upstream regions near the TSS (ups) and in the open reading frame (orf) were designed using the Primer Express software (systems) . For generiments , 4 & S4.For statistical analysis of PfH2A.Z enrichment in 3D7ΔSir2A and 3D7 parasites, the ratio of PfH2A.Z enrichment near the TSS and in the open reading frame (ups/orf ratio) was calculated. Paired t-test was performed using the GraphPad Prism software (Version 4).9 IE and extraction with salt-free buffers 8 IE were incubated with 10 µl of antiserum overnight at 4°C and precipitated with 30 µl protein G agarose (Millipore). After extensive washing proteins were eluted with 2 x SDS PAGE loading buffer and analysed by SDS-PAGE and Western Blotting.Mononucleosomes were prepared from freshly isolated nuclei by MNase (NEB) digestion with 20 KU MNase per 1×10Invitrogen). RNA was purified as described previously Invitrogen). Quantitative RT-PCR was performed as described previously −ΔΔCt analysis (Applied Biosystems user bulletin 2).Total RNA was harvested in parallel to the chromatin preparation by lysis of pelleted IEs in 20 pellet volumes of TRIzol  as well as loop 1 for H2A.Z/H2A.Z self interaction and loop 2 for nucleosome/DNA interaction (green). The region underlined in yellow encompasses the C-terminal docking domain which mediates interaction with the H3/H4 dimer. Lysine residues printed in red in PfH2A.Z were shown to be acetylated (0.98 MB TIF)Click here for additional data file.Figure S2Nuclear localization of PfH2A.ZGFP. Confocal microscopy of PfH2A.ZGFP parasites at the (A) merozoite, (B) ring, (C) trophozoite and (D) schizont stage. Blue: DNA stained with DAPI. Green: PfH2A.ZGFP. Overlays in the lower panels in each picture demonstrate the nuclear localization of PfH2A.ZGFP.(1.27 MB TIF)Click here for additional data file.Figure S3var genes. (B) Enrichment of H2A.Z/H3 in var genes var2csa, var20 and var41. Amplified regions are depicted in the gene models under each graph. Error bars represent standard deviation from two technical replicates.Biological replicate of ChIP analysis of PfH2A.Z occupancy in genes. ChIP was performed in ring , trophozoite (mid gray bars) and schizont stage (black bars) parasites with antibodies against PfH2A.Z and H3 and non-immune control antibodies. Real time qPCR was performed targeting sequences near the TSS and in the open reading frame of genes with different expression profiles . Enrichment was calculated and the data are presented as ratio over H3 to correct for differences in nucleosome density in the inter- and intra-genic regions. The left scale corresponds to ring data, the right scale to trophozoite and schizont data. (A) Enrichment of PfH2A.Z/H3 in non-(0.40 MB TIF)Click here for additional data file.Figure S4−ΔΔCt analysis. (A) Stage specific expression of ring, schizont, and constitutively expressed genes was verified. Sporozoite specific genes were not expressed in any stage. (B) Var2csa was expressed at high levels in ring stages but not in trophozoites and schizonts. Transcripts of two other var genes (var20 and var41) were undetectable.Verification of stage specific transcription profiles. Quantitative real time PCR was performed on cDNA prepared from ring (light grey), trophozoite (mid grey) and schizont (dark grey) stage parasites. The levels of each sequence in cDNA was determined relative to its levels in a constant quantity of 3D7 strain gDNA and the amount of cDNA normalised using the housekeeping gene arginyl-tRNA synthetase by 2(0.26 MB TIF)Click here for additional data file.Figure S5ChIP analysis of PfH2A.Z and histone modifications in upstream regions and open reading frames in (A) ring stage and (B) schizont stage 3D7 parasites. ChIP enrichment (over pre-immune serum) in the upstream region (ups) and open reading frame (orf) is shown for each antibody. PfH2A.Z (light grey), H3K4me3 (dark grey) and H3K9ac (white) concomitantly show a significant increase in the ups region in comparison to the orf. In contrast, H3K9me3 (black) is not significantly enriched. Shown is the median boxed with 25th and 75th percentile and minimum/maximum values as whiskers. A non-parametric Mann-Whitney test was performed and significant differences are indicated. 12 genes were analysed (N = 12) in two technical replicates of one experiment.(0.24 MB TIF)Click here for additional data file.Figure S6var genes. The var genes cluster together in a group with low PfH2A.Z enrichment and low euchromatic histone marks (H3K9ac and H3K4me3) but high levels of the heterochromatin mark H3K9me3. Data are compiled from two technical replicates of one ChIP experiment.Correlation between PfH2A.Z and histone modifications in the upstream region of genes in (A) ring and (B) schizont stage 3D7 parasites. Enrichment of PfH2A.Z (X-axis) positively correlates with H3K4me3 (p<0.0001) and H3K9ac (p<0.0001) and negatively correlates with H3K9me3 (p<0.0001) at both stages. P-value and spearman correlation coefficient (r) are indicated. A total of 36 genes were analysed, including 20 (0.40 MB TIF)Click here for additional data file.Figure S7var gene dominantly transcribed in ICAM-selected 3D7 parasites. (A) qPCR analysis of ICAM selected parasites identifies PFL0020w as the most abundantly transcribed var gene. (B) Northern Blot analysis confirms PFL0020w as the dominant transcript in ICAM selected parasites. A PFL0020w specific probe labels a band of about 10 kb in ICAM selected rings stage parasites (3D7-ICAM), but not in unselected parasites (3D7). A probe representing the conserved exon 2 hybridizes to multiple transcripts in 3D7, but only one dominant transcript in 3D7-ICAM which corresponds to PFL0020w. (C) Model of the PFL0020w gene. The predicted transcription start site (TSS) at approximately −400 bp upstream of the start codon is represented by the arrow. Coordinates of loci amplified by qPCR are indicated below the graph. (D) ChIP analysis of PfH2A.Z distribution along the PFL0020w gene in 3D7-ICAM at ring stage (light grey bars), schizont stage (mid grey bars) or in unselected 3D7 ring stage parasites (dark grey bars). PfH2A.Z is enriched near the TSS at −400 bp at ring stage of 3D7-ICAM but not unselected parasites, and not in 3D7-ICAM schizonts. PfH2A.Z is also enriched in the intron of PFL0020w in both ICAM selected and unselected lines and slightly enriched in the sequence amplified by qPCR from DBL5 which is directly adjacent to the intron. Error bars represent two biological replicates.PfH2A.Z is enriched in the upstream region of a (0.89 MB TIF)Click here for additional data file.Figure S8var genes in 3D7ΔSir2A, 3D7ΔSir2B and 3D7 parasites at ring stage. Multiple var genes are expressed at high levels in 3D7ΔSir2A parasites (red bars) and 3D7ΔSir2B parasites (blue bars). Only marginal var gene expression is detected in 3D7 (black bars). Levels of each var gene sequence in cDNA were normalised using the gene arginyl-tRNA synthetase and expressed relative to the level of the same var gene sequence detected in a constant amount of 3D7 strain gDNA using 2−ΔΔCt analysis. Var groups according to Transcription profiles of (0.39 MB TIF)Click here for additional data file.Figure S9var genes in 3D7ΔSir2A trophozoites. Enrichment of PfH2A.Z (X-axis) positively correlates with H3K4me3 (upper panel) and negatively correlates with H3K9me3 (lower panel). P-value and spearman correlation coefficient (r) are indicated. 11 var genes were analysed in one experiment.Correlation between PfH2A.Z and histone modifications in the upstream region of (0.09 MB TIF)Click here for additional data file.Table S1Gene accession numbers and oligonucleotide sequences used for qPCR.(0.08 MB PDF)Click here for additional data file.Video S1PfH2A.Z shows a polarized distribution in the nucleus. 3D reconstruction using serial Z-stacks of confocal images was performed. The animation shows a 360° rotation. The blue area depicts DAPI-stained DNA, the green area depicts PfH2A.Z labelled with rabbit anti-PfH2A.Z antiserum. PfH2A.Z labelling is concentrated towards one side of the nucleus.(0.04 MB MP4)Click here for additional data file.


In addition to 100% survival, there were no signs of weight loss and disease in 7 out of 8 groups of high dose vaccinated mice. Lower doses of vaccine showed a reduction of protection in a dose-dependent manner. However, even the lowest dose of vaccine provided significant levels of protection against the divergent influenza strains, especially considering the stringency of the challenge virus. In addition, we found that all doses of H5-Con vaccine were capable of providing complete protection against mortality when challenged with lethal doses of all 3 H5N1 influenza strains. This data demonstrates that centralized H1-Con, H3-Con and H5-Con genes can be effectively used to completely protect mice against many diverse strains of influenza. Therefore, we believe that these Ad-vectored centralized genes could be easily translated into new human vaccines.With the exception of the live attenuated influenza vaccine there have been no substantial changes in influenza vaccine strategies since the 1940’s. Here we report an alternative vaccine approach that uses Adenovirus-vectored centralized hemagglutinin (HA) genes as vaccine antigens. Consensus H1-Con, H3-Con and H5-Con HA genes were computationally derived. Mice were immunized with Ad vaccines expressing the centralized genes individually. Groups of mice were vaccinated with 1 X 10 Influenza A virus infections impose a significant burden on our healthcare systems. The influenza pandemic of 1918–1919 infected ~25% of the world’s population. Studies have estimated that the mortality rate of this pandemic was greater than 2.5% . In the A universal influenza vaccine has been the ultimate goal for vaccine researchers. Several strategies have been proposed to expand the breadth of immunity against divergent strains of influenza. The use of conserved genes such as the nucleoprotein (NP) and matrix (M) have shown the potential to induce cross-reactive T cell immunity against divergent influenza strains –8. Studiin vitro and in vivo analyses indicate that the Ad-vectored centralized vaccines can provide high levels of protection against a wide array of divergent influenza strains. We feel that combining the robust gene delivery of the Ad vaccine system with the use of centralized genes may provide a foundation of immunity that is capable of providing high levels of cross-protective immunity against many, if not all, human influenza strains.In this study we have applied the centralized gene vaccine approach to three relevant human influenza virus HA genes, H1, H3, and H5. The centralized genes were incorporated into replication-defective Adenovirus (Ad) viral vector systems and tested to protect against a wide panel of divergent H1, H3 and H5 influenza isolates. We found that Centralized H3-Con and H5-Con HA genes were created using human influenza virus sequences created as previously described for the H1-Con gene . BrieflyAll aligned sequences were then inspected manually to correct for apparent mistakes. Positions containing gaps or ambiguously aligned positions were removed from the datasets. The amino acid consensus sequences were generated from this alignment by using the most common amino acid at each position. The 566, 566 and 568 amino acid consensus sequences were named H1-Con, H3-Con, and H5-Con, respectively. The polybasic connecting peptide region was not included in the construction of the centralized H5-Con gene. The HA genes were codon-optimized for mammalian expression and synthesized by Genscript. Inc.The centralized genes created by this approach retain critical functional domains such as the secretory signal, polybasic connecting peptide, cleavage and fusion sites, transmembrane domains and cytoplasmic tail. The ability of the consensus genes to hemagglutinate cRBCs indicating that they posses the receptor binding domains and transmembrane domains of a functional influenza HA gene is shown . All 3 cin vitro. The first generation RD (E1/E3 deleted) Ad5 vectors were constructed using the Ad-Easy system in 293A cells as described in [In order to provide optimal boosting we designed the Adenovirus (Ad) vaccine vectors based on two distinct Ad types 4 and 5 . We have previously described the use of Ad4 vaccine vectors as influenza vaccines. However, these vectors were replication-competent (RC) E3 deleted vectors . In thisribed in . Ultimat7 virus particles (vp)/mouse, 5 X 107 vp/mouse and 1 X 1010 vp/mouse.Groups of 5 mice were anesthetized i.p. with ketamine (140 mg/kg)/xylazine (5.55 mg/kg). Using various doses of the Ad4 vaccines the mice were primed with the consensus HA genes. The mice were boosted 4 weeks after priming with the Ad5 HA-con expressing vaccines . The Ad in vivo studies were mouse-adapted through serial lung passage as previously described [50).Influenza virus H1N1 A/PR/8/34 (PR/34) was obtained from ATCC (VR95). Influenza viruses BEI NR-3170 H1N1 A/Fort Monmouth/1/47 (FM/47), BEI-2759 H1N1 A/WS/33 (WS/33), BEI-NR13663 H1N1 A/California/07/09 (CA/09), BEI NR-3604 H3N1 A/Texas/1/77 (TX/77), BEI NR-3502 H3N2 A/Mississippi/1/85 (MS/85), BEI NR-3483 H3N2 A/Aichi/2/68 (Aichi/68), and BEI NR-12283 H3N2 A/Brisbane/10/2007 (Bris/07) were obtained from the Biodefense and Emerging Infections Research Resources Repository. H5N1 A/BH Goose/Qinghai/A/05 (Goose/05), H5N1 A/Vietnam/1203/04 (Viet/04), and H5N1 A/Japanese White Eye/Hong Kong/1038/2006 (White Eye/06) were received from the WHO GISRS network. The H3 and H5 influenza viruses used in the escribed . All of Female BALB/c mice (6–8 weeks old) were purchased from Charles River Laboratories  and housed in the Mayo Clinic Animal Facility under the Association for Assessment and Accreditation of Laboratory Animal Care (AALAC) guidelines with animal use protocols approved by the corresponding the Mayo Clinic Institutional Animal Care and Use Committee (IACUC protocol No. A64612). All animal experiments were carried out according to the provisions of the Animal Welfare Act, PHS Animal Welfare Policy, the principles of the NIH Guide for the Care and Use of Laboratory Animals, and the policies and procedures of Mayo Clinic.Serum was collected using Becton Dickinson microtainer tubes with serum separator and heat-inactivated at 56°C for 1h. Starting at a dilution of 1:5 sera were diluted two-fold in 50 μl of DPBS in a 96-well, nonsterile, nontissue culture–treated, round bottom microtiter plate. Four HAU of influenza virus in 50 ul was added to the diluted sera and incubated at room temperature (RT) for 1 hr. After incubation, 50 μl of a 1% chicken RBC solution was added and incubated at RT for 1 hr. The HI titer was determined to be the highest serum dilution to inhibit hemagglutination.50 of influenza A/PR/8/34, A/FM/1/47 virus, A/WS/33 virus, A/TX/1/77, A/Vietnam/04, A/BH Goose/Qinghai/A/05, A/Japanese White Eye/Hong Kong/1038/2006 virus. Mice were challenged with 10MLD50 of A/Mississippi/1/85. The mice were placed on their backs and 10 μl of influenza virus was pipetted into each nare in a total volume of 20 μl. The mice were then weighed and monitored daily for signs of disease. Mice were humanely euthanized if their body weight dropped ≥25% of baseline weights.Four weeks after the boosting immunization the mice were anesthetized i.p. with ketamine (140 mg/kg)/xylazine (5.55 mg/kg). The mice were weighed for baseline measurements and challenged intranasally with 100MLD10 vp was capable of inducing a protective HI titer against the CA/09 virus  or greater strongly correlates with a protective immune response when evaluating inactivated influenza vaccines in adults , 26. The09 virus . The H3-09 virus . The H5-09 virus .10 vp) was capable of providing complete protection from morbidity where there was no weight loss or signs of disease and 100% survival against lethal A/PR/8/34, A/FM/1/47 and A/WS/33 influenza virus challenges. Mice vaccinated with a lower dose of 5 X 107 vp had 100%, 100% and 75% survival against lethal A/PR/8/34, A/FM/1/47 and A/WS/33 influenza virus challenges, respectively. The lowest dose of vaccine, 1 X 107 vp, provided 80%, 60% and 80% survival against lethal A/PR/8/34, A/FM/1/47 and A/WS/33 influenza virus challenges, respectively . This would be logical as the various influenza strains would be less divergent from the vaccine gene. On the other hand, the H1-Con vaccine appears to induce greater levels of immunity as compared to the H3-Con vaccine. While both the H1 and H3 serotypes have greater levels of genetic diversity between strains, as compared to the H5 serotype, there is more genetic divergence in the H1 serotype than the H3 serotype, ~21% and ~14%, respectively. A more exhaustive analysis using more mouse-adapted influenza viruses may better elucidate the correlation between genetic diversity and the efficacy of centralized vaccine genes. However, only these two H3 mouse-adapted influenza viruses were available for this study.In general, mice vaccinated at the highest dose of vaccine do not show any signs of disease. This may be due primarily to antibody titers that are high enough to completely neutralize the virus prior to infection. However, the mice vaccinated with lower doses of vaccine do show signs of disease and weight loss when challenged with lethal influenza viruses. These data indicate that the primary infection is not circumvented solely due to sterilizing antibody responses. Anti-influenza T cell immunity as well as changes in the cytokine responses due to vaccination may play a significant role in the protective effects observed at the lower doses. T cell depletion and cytokine profile studies may provide important details into this mechanism of protection.7 vp/mouse equates to ~1.5 X 1011 vp/human. This is a dose of Ad that is commonly used in human clinical trials and found to be safe and well tolerated [While the lower doses were less effective, it should be pointed out that the challenge viruses consisted of stringent doses that had 100-fold greater lethality than has ever been observed in humans . Therefore the protection we observed at the low dose vaccine would likely be much more effective against nonlethal endemic and pandemic strains. Therefore these doses are very translatable to humans and indicate just how effective these vaccines can be against extremely pathogenic influenza viruses. Based on body weight, the intermediate vaccine dose of 5 X 10olerated –32. Agaiolerated . Our datS1 FigA hemagglutination assay was performed by serially diluting the cells in DPBS in a U bottom assay plate. Fifty microliters of a 1.0% solution chicken red blood cells was added and incubated at room-temperature for 1 hour. A control virus expressing the eGFP-Lucifease expression gene was included as a control for the detection of possible Ad-specific agglutination.(TIFF)Click here for additional data file.S2 Fig(TIFF)Click here for additional data file.S3 Fig(TIFF)Click here for additional data file.


Here we use meta-analysis of 46 published studies to investigate the response of plant N and P to elevated CO2 alone and in combination with nutrient (N and P) additions across temperate vs. tropical biomes. Elevated CO2 reduces plant N concentrations more than plant P concentrations in total biomass pools, resulting in a significant decline in vegetation N/P. However, elevated CO2 treatments in combination with N additions increase plant P concentrations, whereas P additions have no statistical effect on plant N concentrations under CO2 enrichment. These results point to compensatory but asymmetrical interactions between N, P and CO2; that changes in N rapidly alter the availability of P, but not the converse, in response to increased CO2. Our finding implies widespread N limitation with increasing atmospheric CO2 concentrations alone. We also suggest that increased anthropogenic N deposition inputs could enhance plant N and P in a progressively CO2-enriched biosphere.Rising levels of atmospheric CO More recently, Yuan and Chen2 on average decreases plant N/P across an array of sites; however, this study did not examine tropical plant N/P responses, despite tropical systems having different nutrient conditions than many temperate ones2 in combination with other N, P or N plus P additions. Questions remain over whether N or P will become progressively more limiting under elevated CO2, and how such limitations will be affected by changes in P and N inputs across temperate vs. tropical ecosystems. This is critical given that C, N and P are among the most anthropogenically altered biogeochemical cycles on EarthThe stoichiometry of plant N and P concentrations has provided insight in patterns of N versus P limitation across terrestrial ecosystems672 are acting in combination with other global changes, particularly changes in anthropogenic N deposition and P inputs through cropland fertilization, which can also affect patterns of nutrient cycling and limitation on land. Nitrogen inputs can increase plant productivity and lead to over-enrichment with N18It is increasingly important to recognize that plant responses to CO2, precipitation, warming and N deposition result in additive effects on plant N/P ratios. However, they did not study the effects of elevated CO2 with P additions or with N and P additions combination. Evidence for widespread nutrient co-limitation suggests that the cycling of one nutrient can influence the availability of another2-fixationYuan and Chen2 alone and in combination with N, P and N + P additions to test the hypotheses that (1) elevated CO2 decreases plant N and P concentrations;(2) nutrient inputs prevent declines in plant nutrient contents of the fertilized nutrient under elevated CO2; and (3) inputs of N or P alters plant nutrient contents of the other nutrient under elevated CO2 due to compensatory interactions among C, N and P cycles.Here, we analyze plant N/P in response to elevated CO2 treatment effects on plant N/P  compared to 27 belowground observations and 4 whole-plant concentrations reported. Of the compiled studies, there were 34 observations for elevated CO2 with N alone, 22 observations for elevated CO2 with added P alone, and 15 observations for elevated CO2 with added N and P in combination (see We collected 133 observations from 46 separate studies to examine CO N/P see . The maj N/P see . Of thestion see .2 (~12%), regardless of the climatic zones, the kind of CO2 delivery, plant functional group or plant tissue examined tropics. CO2-incuced declines in plant P concentrations were observed for non-woody plants and aboveground tissues, but not for woody plants and belowground tissues. Hence, elevated CO2 significantly decreased plant N/P by 11% compared to control conditions, but this response varied with climatic zones (2 decreased plant N/P to a greater extent in (sub-)tropics (23%) than in temperate regions (6%). CO2 enrichment caused significant declines in plant N/P for chambers (14%) but not FACE experiments, for woody species (13%) but not non-woody species, and for legumes (11%) but not for non-legumes.Plant N concentrations decreased significantly with elevated COexamined . Across trations . Rather,ic zones . Overall2 with N fertilization, elevated CO2 alone (without N fertilization) significantly decreased plant N concentration (3%) and increased plant P concentration (16%), thus resulting in a decrease of 20% in plant N/P  and in plant P concentration (16%) (2 enrichment and N fertilization were not related to the amount of N added (see In the dataset of elevated CO N/P see . Howeveron (16%) . The resdded see .2 without P fertilization tended to negatively affect plant N concentrations, P concentrations and N/P (see 2 plus P treatments consistently decreased plant N concentrations (7%), increased plant P concentrations (24%) and decreased plant N/P ratios (26%) (2 with P fertilization were not related to the amount of P added (see Elevated CO N/P see . Howeveros (26%) . The resdded see .2 with N and P fertilizations, N and P fertilizations did not significantly affect plant N concentration, P concentration and N/P responses to elevated CO2   of plant N concentrations to elevated CO < 0.01) . There w N added , while nNP added ,d.2 enrichment singly. This result was clear and systematic in the case of plant N concentrations and generally confirmed results of previous research1011282 have been less-examined. Our results showed that P exhibited a more complex CO2 response than N across a broad range of terrestrial ecosystems, which resulted in some differences in plan N/P. For example, plant P was increased under elevated CO2 in (sub-)tropics rather than temperate regions, which imply that relatively high soil N availability in (sub-)tropics could help plants acquire P with high energy (C) inputs2 enrichment, P in belowground tissues did not change with elevated CO2. This suggests that plants may be allocating more P to roots under elevated CO2, or actively mining soil P pools, perhaps via increased investment in mycorrhizal fungi and fine roots302, whereas P concentrations of herbaceous vegetation declined significantly, revealing strong functional-group dependencies in plant P responses. The relative stability in legume N/P under elevated CO2 indicate that N2-fixing species has the ability to balance N and P through direct access to N from atmosphere and investment in phosphatase and mycorrhizae to acquire P26This cross-system analysis supports expectations for our first hypothesis – that plant N and P contents decline systematically under CO2, which were in line with those reported for temperate ecosystems by Yuan and Chen2 was less than unity (P < 0.01) (2 concentrationsOur results showed net declines in plant N/P under elevated CO < 0.01) . This me2 enrichment, which can explained by factors that differentially affect plant metabolism of N vs. P112 may increase the efficiency of photosynthesis or metabolically down-regulate photosynthetic enzymes, thus causing increasing photosynthetic N use efficiency with decreasing N supplies342 would disproportionally increase P vs. N demands in ecosystems362 could increase plant growth rates, which requires P-rich ribosomal RNA (rRNA)382.Generally, the decline in plant N/P we observed points to more substantial N vs. P limitation with CO2  under ambient CO2 and elevated CO2. We also limited our data to studies where means, standard deviations of the mean, and number of replicates were reported or could be calculated. For studies that measured N and P concentrations at multiple time-points, only the final value was used to maintain the statistically independence between individual observations. DataThief−1. All N/P are presented here on a mass basis.We searched ISI Web of Science, using the terms “elevated CO2 concentration were classified as Free-Air Carbon dioxide Enrichment (FACE) and chambers; plants were categorized based on functional type  and plant tissues examined .We categorized the data by climatic zones, experiment types, plant characteristics, and presence any kind of nutrient fertilization involving N and P. The climatic zones included arctic, temperate and (sub-)tropical zones. Research facilities used to increase CO2 and nutrient (N and P) fertilization, we also collected data on the level N and/or P additions where available, including elevated CO2 with N fertilization (from 1 g N m−2 yr−1 to 20 g N m−2 yr−1) (CO2 + N), elevated CO2 with P fertilization (from 1.5 g P m−2 yr−1 to 16 g P m−2 yr−1) (CO2 + P), and elevated CO2 with N and P fertilizations (CO2 + NP). For each of the three datasets, we made two comparisons: elevated CO2 without nutrient fertilization vs. control (ambient CO2 without nutrient fertilization) and elevated CO2 with nutrient fertilization vs. control.To examine interactions between elevated CO2 without nutrient fertilization)P < 0.05)The treatment effects were examined by calculating response ratios (RR) from each individual study. RR is used as an indicator of effect size, and is calculated as the experimental mean divided by the control mean .