Higher click here frequency and avidity responses were observed to human IgG1 DNA when compared to Ag DNA (p=0.0047) (Fig. 4D). High-avidity CTL responses should result in effective anti-tumor responses. The TRP2/HepB human IgG1 DNA vaccine was screened for prevention of lung metastases and inhibition of growth of established subcutaneous lesions. The B16F10 cells expressing IFN-α (B16F10 IFN-α) have a moderate growth rate of 4 wk, which is more representative of human cancer and were thus chosen for preliminary in vivo studies. Forty days post final immunization and forty nine days after tumor cell injection TRP2/HepB human IgG1 DNA

immunized mice exhibited peptide and tumor-specific immune responses (data not shown). The tumor area was selleck products quantified and expressed as percentage of total lung area. TRP2/HepB human IgG1 DNA immunized mice demonstrated a significant reduction in tumor burden compared to untreated control mice (p=0.0098) (Fig. 4E). When the hair was permitted to grow back after last immunization, mice immunized with TRP2/HepB human IgG1 DNA were observed to have growth of white hair at the site of immunization, which was not apparent in control mice. TRP2/HepB human IgG1 DNA was

evaluated for its ability to prevent the growth of the aggressive parental B16F10 tumor line in a therapeutic model. Figure 4f shows that immunization with TRP2/HepB human IgG1 DNA significantly (p=0.019) delays growth of the aggressive B16F10 melanoma compared to a control human IgG1 DNA vaccine. This suggests that delivering epitope-based DNA vaccines in the context of an inert carrier (i.e. Ab) has advantages. We have previously

shown that Ab protein vaccines can target Ag presenting cells through the high affinity FcγR1 receptors. Ab–DNA vaccination was therefore compared to protein vaccination and also to vaccination in Fcγ knockout mice. DNA vaccination gene gun can stimulate naïve T-cell responses by direct transfection of DC allowing direct presentation CTL epitope. Alternatively, transfection of non-professional APC and secretion of protein leading to cross presentation can occur. In contrast, generation of an immune response from protein immunization can only occur by cross presentation. TRP2 human IgG1 DNA vaccine was compared to Buspirone HCl an identical protein vaccine. TRP2 human IgG1 DNA immunized mice generate superior frequency and avidity epitope-specific responses (p=0.0028) (Fig. 5A). The results indicate that DNA vaccine is superior to protein possibly by allowing both direct and cross-presentation of CTL epitopes. A suggested mechanism for the cross presentation of epitopes from human IgG1 DNA is the binding and uptake of protein by the FcγR1. To examine if the Fc region was important mice were immunized with TRP2/HepB human IgG1 DNA constructs lacking the Fc region. Mice immunized with the vaccine lacking the Fc region demonstrate a significantly reduced response specific (p=0.

1c,d, respectively). A 70% reduction in the number of LAG-3+ cells was observed both in the CD4 and the CD8 subsets at a 10 ng/ml antibody concentration. The half-maximum effective concentration was found at the ng/ml level [1 ± 0·4 ng/ml for CD4+ T cells and 0·7 ± 0·4 ng/ml for CD8+ T cells, mean ± standard deviation (s.d.) of five experiments]. The observed effect is not due to competition

between the chimeric A9H12 mAb and the 17B4-FITC mAb used to reveal LAG-3, as the binding of 17B4-FITC is not inhibited by a threefold excess of the chimeric A9H12 mAb (not shown). A putative internalization of the membrane LAG-3 induced by the chimeric A9H12 was excluded because the disappearance of activated T cells was also observed with an anti-CD25 antibody (not shown). CDC and ADCC are probably the dominant mode of action of this antibody, as no agonist

or antagonist effect could be evidenced in mixed lymphocyte reactions Trichostatin A (data not shown). The chimeric A9H12 mAb cross-reacted with baboon LAG-3 because it bound to similar percentages of activated PBMC to that found for human cells, and did not bind to resting baboon PBMC (Fig. 1e). According to a two-compartment model, after an intravenous bolus administration of 1 mg/kg of chimeric A9H12 (n = 2), the elimination half-life was 86·1 ± 31·3 h (Fig. 2a). Three other animals received 0·1 mg/kg of chimeric A9H12. In that case, the elimination half-life was calculated as 23·8 ± 6·8 h (Fig. 2a). In order to evaluate whether chimeric A9H12 can deplete LAG-3+ target cells in vivo, Lumacaftor nmr inguinal lymph nodes were biopsied before, and on days 1 and 4 after treatment. The percentage of LAG-3+ cells was then evaluated by flow cytometry. We observed a reduction of both CD4+ and CD4–LAG3+CD3+ T lymphocytes after chimeric A9H12 administration (Fig. 2b). CD4–CD3+ T lymphocytes represent mainly CD8+ T cells, but can also contain a few NK T cells. This was not due to immunological masking,

as Sorafenib the detecting fluorescent anti-LAG-3 antibody used did not compete with chimeric A9H12. As expected, administration of chimeric A9H12 induced no modification of lymphocyte count in the peripheral blood. To test the efficacy of chimeric A9H12 in vivo, we established a DTH model in baboons after sensitization with BCG vaccine. That sensitized animals were indeed immunized was controlled after 1 month with an IFN-γ ELISPOT assay on PBMC. Of eight baboons vaccinated with BCG, all but one became immunized. Unsensitized animals presented a frequency of 1/61 845 ± 1/13 329 PBMC responding in vitro to tuberculin-PPD, and this rose to a frequency of 1/7 842 ± 1/1578 in sensitized animals. Two immunized baboons used as controls were challenged with tuberculin IDR three consecutive times over 5 months and demonstrated consistent and reproducible erythema after each IDR (Table 1).

Thus, CD4+ T cells have not been widely exploited in ACT as well as the properties (i.e. homing potential, functionality, and survival) that CD4+ T cells might require for successful applications in ACT are much less known than in the case for CD8+ CTL. A large, still not definitive, amount of literature underline how IL-2, IL-7 and IL-15 play non-redundant

roles in shaping the representation of memory cells 19–23. IL-2 controls T-cell clonal expansion and contraction, and promotes lymphocyte differentiation. IL-2 and IL-15 can also support memory cell division and have been used in combination with Ag-driven stimulation, for the expansion of CTL 24–29. IL-7 regulates peripheral T-cell homeostasis, and contributes to the generation and selleck inhibitor long-term survival of both CD4+ and CD8+ memory T lymphocytes in vivo30, 31. In some cases IL-7 amplifies Ag-driven T-cell responses 32–36, favors the transition of effector to memory cells 31, 37–39, and sustains a slow, homeostatic-like, Ag-independent memory T-cell proliferation 24, 30, 40. Furthermore, its administration

at the time of Ag withdrawal supports Buparlisib chemical structure memory CD8+ T-cell generation 41, and enhances vaccine-mediated immunity when provided in adjuvant settings 42, 43. Based on our previous results showing that tumours only allow a limited expansion of effector CD4+ T cells, while hinder both natural and vaccine-induced memory-like cell responses 10, 15, we attempted the ex vivo expansion of tumour-specific CD4+ T cells to be used in ACT, using common-γ-chain receptor cytokines. We report the ability of IL-7, rather than IL-2 in expanding tumour-sensitized T cells in short-term cultures, capable of sustaining anti-tumour protection in ACT settings. We and others previously characterized Ag-specific CD4+ T-cell responses by fluorescent 5-FU manufacturer MHC class II/peptide multimer and Ag-specific intracellular cytokine staining in 16.2β mice 10, 44, which express a Tg TCR-β-chain specific

for the Leishmania receptor for Activated C Kinase (LACK, derived from Leishmania Major) Ag coupled to a polyclonal α-chain TCR repertoire. This allows the identification of both naive (∼0.5% of CD4+ cells) and memory polyclonal LACK-specific CD4+ T cells. By using this model, we found that TS/A tumours expressing LACK as an intracellular tumour-associated Ag (TS/A-LACK tumour cells) promote the expansion of short-lived LACK-specific effector-like CD4+ T cells, while hinder the accumulation of both natural- and vaccine-induced central memory-like T cells 10, 15. As IL-7 is known to support memory CD4+ T-cell expansion following Ag withdrawal 41, we asked whether this cytokine could be used in short-term in vitro cultures for the expansion of tumour-sensitized CD4+ T cells useful in ACT settings. In agreement with our previous findings, CD4+CD44high T cells able to bind I-Ad/LACK fluorescent multimers (Fig. 1A) and to secrete IL-2 and/or IFN-γ upon LACK-specific stimulation (Fig.

The patient had undetectable levels of IgG, IgA and IgM and normal numbers of circulating lymphocytes (10 686 cells per µl) with remarkable eosinophilia (4030 cells per µl). The rest of his initial immune work-up is summarized

in Table 1. Genetic work-up revealed a compound heterozygous RAG2 defect (G95V+E480X). The patient was commenced on CsA treatment; however, his cutaneous symptoms did not improve despite maintaining a high CsA trough level (100–150 ng/ml). Therefore, methylprednisone (2 mg/kg/day) was added and slow resolution of his cutaneous symptoms was observed. The patient was kept on both CsA and methylprednisone treatments until a successful HLA-matched cord blood transplantation was performed https://www.selleckchem.com/products/bmn-673.html at the age of 6 months. In both patients, transplantations were successful and they have been currently followed for 2 years (patient 1) and 1 year (patient 2), with complete recovery of their symptoms and full reconstitution of their immune system. TCR repertoire.  Examination of TCR-Vβ at presentation

revealed peripheral expansion of oligoclonal T cells with dominant specific receptors. In patient 1, the dominant clone was TCR-Vβ 20, while in patient 2, TCR-Vβ 17 and TCR-Vβ 7·2 were dominant (Fig. 1a,b). Clonal patterns were also seen in the examined TCR-Vγ repertoire in both patients (Fig. 2a,b). These results suggest abnormal thymocyte selection and peripheral 5-Fluoracil expansion, as expected in

Omenn patients. Patient 1 showed a significant clinical improvement during CsA therapy; therefore, a follow-up analysis of his TCR repertoire see more was not indicated. However, in order to show that the patient did not have any expanded peripheral T cells, prior to the HSCT procedure, analysis of his TCR-Vγ repertoire was performed. The analysis revealed complete lymphopenia and no TCR expansion (Fig. 2c). In contrast, patient 2 did not respond completely to the initial treatment with CsA and remained symptomatic, therefore a follow-up analysis of his TCR repertoire was performed (Fig. 1c–e). Surprisingly, while the expression of the dominant TCR-Vβ 17 clone was reduced, the TCR-Vβ 7·2 clone did not respond to CsA therapy. Moreover, a few other TCRs, such as TCR-Vβ 14 and TCR-Vβ 5·1, started to appear (Fig. 1c). Only the addition of methylprednisone treatment resulted in suppression of these clones (Fig. 1d). However, even before the transplant, the patient still suffered mild skin symptoms, which were probably attributed to the presence of the TCR-Vβ 14 clone (Fig. 1e). Changes in the relevant TCRs during the treatment are presented in Fig. 3. During that time the patient was clinically stable apart from his skin symptoms and had no overt infection or other reason to explain clonal expansion. Trec quantification.

Caby et al. examined plasma samples from healthy donors and successfully identified vesicles of 50–90 nm in diameter that have the molecular and biophysical properties of exosomes.[70] Besides blood, exosomes have also been detected in various bodily fluids such as urine, cerebrospinal fluid, saliva, breast learn more milk, semen, amniotic fluid, malignant ascites, bronchoalveolar lavage fluid and synovial fluid.[71] The presence of urinary exosomes was verified when small vesicles (<100 nm in diameter) orientated ‘cytoplasmic-side inward’

were observed in normal urine with functions in urinary secretion of aquaporin-2 and other membrane-associated proteins[72] (see Fig. 2). The proteomic analysis of urinary exosomes identified proteins

characteristically restricted in expression to renal epithelia of the glomerular podocytes, the proximal tubule, the thick ascending limb of Henle, the distal convoluted tubule and the collecting duct. Proteins from the transitional epithelium of the urinary bladder were also identified, suggesting urinary exosomes may be derived from cells throughout the renal tract.[72-74] Thus, analysis of urinary exosomes provides an attractive non-invasive means of acquiring information about the pathophysiological state of their renal cells of origin. CD24, a small but extensively glycosylated protein linked to the cell surface by means of a glycosyl-phosphatidylinositol anchor, has been reported to be a marker for urinary exosomes.[75] It was previously thought that the main physiological role for urinary see more exosomes is the disposal of senescent Farnesyltransferase proteins from cells, which may be a more efficient way of protein elimination than proteasomal and lysosomal degradation,[76] similar to the process by which maturing

reticulocytes shed obsolete membrane proteins and remodel their plasma membrane through the exosomal pathway.[52] However, increasing evidence is suggesting that urinary exosomes play a role beyond exocytic cell waste elimination.[75, 77] Another possible role of exosomes in the urinary tract is to regulate the co-functioning between different parts of the nephron, through secretion and reuptake of their contents such as mRNAs and miRNAs that can affect the function of the recipient cell[73] (Fig. 1). Functional transfer of molecules such as aquaporin-2 between different renal cells has been described[78] and could mediate coordinate adaptation of nephron function. The role of circulating exosomes in physiological messaging remains poorly defined, but pathophysiological roles have been increasingly explored. Endothelial dysfunction is thought to be the key event in the pathogenesis of atherosclerosis. Endothelial dysfunction is a systemic inflammatory process associated with increased adhesion molecule expression, loss of anti-thrombotic factors, increase in vasoconstrictor products and platelet activation.

After overnight

α-CD3 stimulation, both TSC1KO CD4+ and CD8+ T cells upregulated CD25 and CD69 in a heterogeneous manner. A portion of TSC1KO T cells showed decreased CD25 and CD69 upregulation as compared with WT T cells (Fig. 2F), suggesting impaired early activation of T cells in the absence of TSC1. α-CD3 stimulation resulted in expansion of WT CD4+ T cells in vitro. Such expansion appeared blunted in the absence of TSC1 (Fig. 2G). However, TSC1KO CD4+ as well as CD8+ T cells underwent similar or even more divisions than WT T cells during the same time of α-CD3 stimulation (Fig. 2H). Although a decrease in CD4+ T-cell expansion was observed, elevated levels of IL-2 were detected in the supernatants of TSC1KO CD4+ T cells compared with that of WT CD4+ T cells after 48 or 72 h of stimulation with α-CD3 (Fig. 2I), suggesting increased IL-2 production by TSC1KO T cells on GSK1120212 cost JAK inhibitor a per cell basis. These results indicate that TSC1 deficiency results in constitutive activation of mTORC1 in thymocytes and peripheral T cells, and has complex effects on T-cell activation manifested by decreased early activation and blunted expansion, but increased

IL-2 production and slightly enhanced proliferation. The decreases in both CD4+ and CD8+ peripheral T-cell compartments in TSC1-deficient mice, and the blunted expansion concordant with normal or enhanced proliferation of TSC1KO T cells in vitro led us to hypothesize that TSC1 might control T-cell survival. Indeed, an increased proportion of freshly isolated TSC1KO CD4+ and CD8+ T cells stained positive for 7-AAD ex vivo (Fig. 3A). The increase in cell death of TSC1KO T cells was not associated with the upregulation of Fas or FasL (Fig. 3B). The vast majority of cell death within the T-cell subsets is confined to the CD44hiCD62Llow population in both WT and TSC1KO T cells, and the death occurring in this particular subset is noticeably pronounced NADPH-cytochrome-c2 reductase in TSC1KO T cells (Fig. 3C). The amount of cell death seen in TSC1KO T cells was intensified after α-CD3

stimulation (Fig. 3D). Collectively, these observations demonstrate that the absence of TSC1 in T cells renders them less fit for survival in the periphery, particularly during T-cell activating conditions. The mitochondrion plays a central role in apoptosis 22. In HSCs, TSC1-deficiency results in increased mitochondrial content and the production of harmful ROS 18. To our surprise, TSC1KO T cells exhibited decreased mitochondrial content compared with WT T cells based on MitoTracker Green staining (Fig. 4A). Also, the ratio of mitochondrial DNA to nuclear DNA using the 12S rRNA gene and 18S rRNA as mitochondrial and nuclear DNA markers, respectively, was significantly decreased in TSC1KO T cells (Fig. 4B).

[9] Stimulation indices (SI) were calculated

as proliferative response in the presence of antigen divided by response in the absence of antigen. Brains and spinal cords were fixed in 5% formalin saline and processed for routine histology. Sections, 5 μm thick, were cut and stained with haematoxylin & eosin to evaluate inflammatory infiltrates or Luxol fast blue/cresyl fast violet (LFB/CFV) to assess the degree of demyelination. Data were analysed using Graphpad prism and expressed as mean ± standard error of the mean (SEM). The EAE clinical scores were assessed by Mann–Whitney U-test and day of onset and disease incidence were analysed by Kaplan–Meier using sigmastat software (SPSS Inc., Chicago, IL). Group EAE score represents the maximum neurological deficit in all animals within the group and mean EAE score represents the maximum neurological deficit developed by mice, which exhibited EAE, as

previously described EPZ-6438 supplier and the mean day of onset of signs.[3, 16] P-values < 0·05 were considered significant. To identify the immunodominant B-cell epitopes C57BL/6 WT (MOG+/+) and MOG-deficient (MOG−/−) mice, which will lack any immune tolerance and deficits in their immune repertoire to MOG, were immunized with rmMOG corresponding to MOG sequence 1–116. On day 20, plasma was collected and examined using ELISA to identify responses to 23 mer overlapping peptides (Table S3). No differences were observed between the responses of MOG+/+ and MOG−/− mice to rmMOG on day 20 (Fig. 1). Similarly, antibody responses to peptides in both Celecoxib selleck WT and MOG−/− knockout mice were restricted to sequences below residues 82 and dominant responses to epitopes within residues MOG45–67 and MOG50–72 (Fig. 1a).

Similar to responses to MOG35–55 (see ref. [9]) antibody responses to the 23 mer peptide MOG35–57, encompassing the encephalitogenic peptide MOG35–55, were not dominant. As expected, no responses were found in peptides above residues 116 (Fig. 1a). To examine antibody responses in more detail, C57BL/6 WT (MOG+/+) and MOG-deficient (MOG−/−) mice (n = 5) were immunized with a pool of 15 mer peptides and recall responses on day 20 to individual peptides were examined using ELISA. We identified immunodominant epitopes with residues MOG113–127 and MOG148–162 (Fig. 1b) in C57BL/6 WT (MOG+/+) and MOG-deficient (MOG−/−) mice. No responses were observed to any other peptide or in mice immunized with complete Freund’s adjuvant only. No differences were observed between responses in C57BL/6 WT (MOG+/+) and MOG-deficient (MOG−/−) mice (Fig. 1). Next, to identify the immunogenic T-cell epitopes within mouse MOG, mice were immunized with the overlapping peptide spanning the mouse MOG sequences. On day 10 responses were examined using a thymine incorporation assay as described previously.[9] This study revealed that while a T-cell response to MOG36–50 (SI = 3·90) was detectable (Fig. 2) a stronger response to peptide MOG183–197 (SI = 5·2) was also induced.

1). Responder PBMC were incubated with sotrastaurin 0, 25, 50, 100 or 250 ng/ml 60 min before the stimulator cells were added. A dose-dependent effect of the study drug on alloresponsiveness was observed: the mean proliferative response decreased LEE011 order in the presence of 25, 50 100 and 250 ng/ml sotrastaurin from 37250

to 21617, 18487, 9500 and 3191 cpm, respectively (all P < 0·0001; mean percentage of inhibition 40, 49, 74 and 92, respectively, Fig. 1). For each experiment the IC50 was calculated. The median IC50 for sotrastaurin was 90 nM (45 ng/ml) (molecular mass 499 acetate). To study the effect of sotrastaurin on the IL-2-driven STAT-5 activation by Tregs, whole blood samples of three healthy volunteers were incubated with and without 100 ng/ml sotrastaurin in the presence of IL-2. In the absence of this cytokine STAT-5 was not phosphorylated in Tregs (all <4% pSTAT-5). After stimulation Talazoparib supplier with IL-2, 47·5% (median) of Tregs phosphorylated STAT-5, which was similar in the presence of sotrastaurin (median

50·5%, Fig. 2). To study the effect of sotrastaurin on the function of CD4+CD25high Treg, PBMC and CD25low populations, co-culture experiments were performed in blood bank donor samples (n = 11). Alloreactive response in MLR to irradiated stimulator cells was compared between PBMC and CD4+CD25low responder populations after depletion of CD4+CD25high T cells. Depletion of the Treg fraction from the PBMC resulted in a 91·3% increase in the proliferative response (P < 0·05). Subsequently, the suppressive capacity of these isolated Tregs was determined in co-culture experiments with CD25low responder cells in a 1 : 5 ratio. We set the Teff proliferation as triclocarban 100%, and compared this to the proliferation after addition of sotrastaurin and after co-culture with Tregs. Tregs significantly inhibited alloproliferation in the absence (median inhibition 47%, P = 0·002) and presence of 50 ng/ml sotrastaurin (median inhibition 35%, P = 0·002). This difference in inhibition was not statistically significant (P = 0·33) (Fig. 3). Fourteen patients were treated with sotrastaurin

and seven patients were treated with neoral. Blood samples were collected pre-, 3 and 6 months after transplantation. At 6 months, 17 patients still used their study drug regimen (10 sotrastaurin versus seven neoral patients). The reasons for discontinuing the study drug were various, among them adverse events related to the use of sotrastaurin, neoral and everolimus. The absolute numbers of different lymphocyte subsets were measured using flow cytometry. The numbers of CD3+ T cells, CD4+ helper T cells, CD8+ cytotoxic T cells, CD16+56+ NK cells, CD19+ B cells and the ratio of CD4+/CD8+ T cells did not change significantly over this 6-month period (Table 2). The Treg population was defined as cells with high CD25 expression in combination with slightly less CD4 expression in combination with high FoxP3 and no or low expression of CD127 (IL-7R-α) expression (Fig.

The effect of

caspase-11-mediated lethality was similarly evident in Selleck BGB324 vivo [3, 8]. Both Casp11−/− and double Casp1−/− Casp11−/− mice were resistant to lethal septic shock, whereas Casp1−/− Casp11Tg animals all succumbed [3]. Similarly, Casp11−/− macrophages were more resistant to death compared with wild-type cells during infections with ΔFlag Salmonella or Legionella [3, 10]. However, pyroptosis induced by canonical stimuli (LPS/ATP, LPS/C. difficile toxin B or wild-type Legionella) required caspase-1, but not caspase-11, since these stimuli activate NLRP3 or NAIP/NLRC4 directly [3, 10]. The fact that Gram-negative bacteria activate the noncanonical inflammasome pathway and induce pyroptosis raised the question of whether caspase-11 might directly contribute to clearing bacterial infections. The ability of caspase-11 to restrict bacterial replication was evaluated in macrophages infected with L. pneumophila this website [4]. Casp11−/− macrophages were significantly more permissive for bacterial growth compared with wild-type macrophages. This enhanced permissiveness was related to impaired phagosome–lysosome fusion in Casp11−/− cells, which allowed bacteria to evade degradation [4]. This lack of phagosome–lysosome fusion required the catalytic activity of caspase-11 and was associated with impaired actin polymerization. Indeed, it had previously been shown that murine caspase-11 physically directs

actin-interacting protein 1 (Aip1), an activator of cofilin-mediated actin depolymerization [21]. Therefore, these results suggest that caspase-11 contributes to bacterial clearance by controlling the polymerization and depolymerization of actin, a crucial

step for phagosome–lysosome fusion. Interestingly, caspase-11-mediated phagosome–lysosome fusion proceeded only with pathogenic bacteria, but not with nonpathogenic bacteria, such as E. coli [4]. The protective role of caspase-11 during bacterial infection was also seen in vivo. A higher bacterial load was recovered from lungs of Casp11−/− mice infected with Legionella compared with that in wild-type mice [4]. Moreover, co-infection with equal numbers of Salmonella wild-type Dichloromethane dehalogenase and Salmonella ΔsilA, an attenuated mutant that is released into the cytosol, resulted in more efficient clearance of Salmonella ΔsilA in wild-type mice compared with Casp11−/− animals [20]. This suggests that caspase-11 is responsible for the clearance of Salmonella ΔsilA, whereas the wild-type Salmonella, by remaining inside the vacuoles, is not exposed to caspase-11 activity and hence cannot be eliminated by pyroptosis. In a different study using wild-type Salmonella, the number of bacteria recovered from Casp11−/− tissues was similar to that from wild-type mouse controls [8]. Interestingly, much higher bacterial loads were measured in double Casp1−/− Casp11−/− mice, which increased further in single Casp1−/− mice.

Here, we rederived ChAdV-68 [37] (also called SAdV-25, C68, and Pan9), inserted its whole genome into bacterial artificial chromosome (BAC), deleted the E1 and E3 regions, and inserted

a consensus clade B RXDX-106 Gag Tg expression cassette into its genome at the E1 locus. The resulting ChAdV68.GagB vaccine was evaluated for protective efficacy in combinations with plasmid pTH.GagB DNA and modified vaccinia virus Ankara MVA.GagB. This work extends on previously published mouse data [17, 20], and parallels rhesus macaque [11, 19, 21] and ongoing phase I/IIa clinical trial [38] studies exploring similar regimens. Although SAdV-25 had previously been cloned as an E1-deleted vector AdC68 [37, 39], we generated an independently PLX4032 derived E1

and E3 region deleted vector, here referred to as ChAdV-68, from WT SAdV-25 genomic DNA. Rather than traditional and laborious ligation-based methods, we used two new restriction site-independent approaches to precise deletion of E1 and E3 regions from the adenovirus genome as described elsewhere [40]. Briefly, the first method of Chartier et al. [41] was modified to enable E1 deletion concomitant with recombination of the viral genome into the destination plasmid (see Materials and Methods). Of four clones analyzed, all contained the viral genome and three contained the intended E1 deletion, resulting from recombination downstream rather than upstream of the E1 locus. Having used a BAC rather than a multicopy plasmid backbone, we were then able to employ GalK recombineering [42] to delete the E3 region and replace it with a unique PmeI site, an approach that exhibited 100% efficiency. Complete shotgun sequencing of the resulting E1 and E3 deleted ChAdV-68-BAC clone revealed that it was identical to the SAdV-25 reference sequence (NCBI RefSeq accession no. AC 000011) with the exception of five single-nucleotide differences at positions 8919 (C to

G, Gly to Arg in preterminal protein), 15758 (G to C, silent), 17156 (A to Amobarbital T, intergenic), 17434 (C to A, intergenic), and 35228 (G to C, His to Gln in E4). In order to directly confirm and extend published data on chimpanzee adenovirus serotype 68 vectored vaccines expressing HIV-1 clade B Gag [17-20], ChAdV68.GagB was constructed. A synthetic gene using humanized codons coding for myristoylated full-size consensus HIV-1 clade B p55Gag polypeptide (Genbank accession no. AAS19377) was coupled to an mAb epitope Pk at its C-terminus to facilitate detection and the chimeric gene GagB was inserted into the adenovirus genome at the E1 locus under control of the CMV major immediate-early promoter. To assess the ChAdV68.GagB vaccine in heterologous prime-boost regimens, vaccines expressing GagB vectored by plasmid DNA pTH.GagB and modified vaccinia virus Ankara MVA.GagB were also constructed. Expression of the GagB protein in human cells was confirmed by immunofluorescence (Fig. 1A) and on a western blot of infected/transfected cell lysates (Fig.