Cytomegalovirus vectors expressing Plasmodium knowlesi antigens induce immune responses that delay parasitemia upon sporozoite challenge.

The development of a sterilizing vaccine against malaria remains one of the highest priorities for global health research. While sporozoite vaccines targeting the pre-erythrocytic stage show great promise, it has not been possible to maintain efficacy long-term, likely due to an inability of these vaccines to maintain effector memory T cell responses in the liver. Vaccines based on human cytomegalovirus (HCMV) might overcome this limitation since vectors based on rhesus CMV (RhCMV), the homologous virus in rhesus macaques (RM), elicit and indefinitely maintain high frequency, non-exhausted effector memory T cells in extralymphoid tissues, including the liver. Moreover, RhCMV strain 68-1 elicits CD8+ T cells broadly recognizing unconventional epitopes exclusively restricted by MHC-II and MHC-E. To evaluate the potential of these unique immune responses to protect against malaria, we expressed four Plasmodium knowlesi (Pk) antigens (CSP, AMA1, SSP2/TRAP, MSP1c) in RhCMV 68-1 or in Rh189-deleted 68-1, which additionally elicits canonical MHC-Ia-restricted CD8+ T cells. Upon inoculation of RM with either of these Pk Ag expressing RhCMV vaccines, we obtained T cell responses to each of the four Pk antigens. Upon challenge with Pk sporozoites we observed a delayed appearance of blood stage parasites in vaccinated RM consistent with a 75-80% reduction of parasite release from the liver. Moreover, the Rh189-deleted RhCMV/Pk vectors elicited sterile protection in one RM. Once in the blood, parasite growth was not affected. In contrast to T cell responses induced by Pk infection, RhCMV vectors maintained sustained T cell responses to all four malaria antigens in the liver post-challenge. The delayed appearance of blood stage parasites is thus likely due to a T cell-mediated inhibition of liver stage parasite development. As such, this vaccine approach can be used to efficiently test new T cell antigens, improve current vaccines targeting the liver stage and complement vaccines targeting erythrocytic antigens.

Using infective mosquitoes to challenge monkeys with Plasmodium knowlesi in malaria vaccine studies.

BACKGROUND: When rhesus monkeys (Macaca mulatta) are used to test malaria vaccines, animals are often challenged by the intravenous injection of sporozoites. However, natural exposure to malaria comes via mosquito bite, and antibodies can neutralize sporozoites as they traverse the skin. Thus, intravenous injection may not fairly assess humoral immunity from anti-sporozoite malaria vaccines. To better assess malaria vaccines in rhesus, a method to challenge large numbers of monkeys by mosquito bite was developed. METHODS: Several species and strains of mosquitoes were tested for their ability to produce Plasmodium knowlesi sporozoites. Donor monkey parasitaemia effects on oocyst and sporozoite numbers and mosquito mortality were documented. Methylparaben added to mosquito feed was tested to improve mosquito survival. To determine the number of bites needed to infect a monkey, animals were exposed to various numbers of P. knowlesi-infected mosquitoes. Finally, P. knowlesi-infected mosquitoes were used to challenge 17 monkeys in a malaria vaccine trial, and the effect of number of infectious bites on monkey parasitaemia was documented. RESULTS: Anopheles dirus, Anopheles crascens, and Anopheles dirus X (a cross between the two species) produced large numbers of P. knowlesi sporozoites. Mosquito survival to day 14, when sporozoites fill the salivary glands, averaged only 32% when donor monkeys had a parasitaemia above 2%. However, when donor monkey parasitaemia was below 2%, mosquitoes survived twice as well and contained ample sporozoites in their salivary glands. Adding methylparaben to sugar solutions did not improve survival of infected mosquitoes. Plasmodium knowlesi was very infectious, with all monkeys developing blood stage infections if one or more infected mosquitoes successfully fed. There was also a dose-response, with monkeys that received higher numbers of infected mosquito bites developing malaria sooner. CONCLUSIONS: Anopheles dirus, An. crascens and a cross between these two species all were excellent vectors for P. knowlesi. High donor monkey parasitaemia was associated with poor mosquito survival. A single infected mosquito bite is likely sufficient to infect a monkey with P. knowlesi. It is possible to efficiently challenge large groups of monkeys by mosquito bite, which will be useful for P. knowlesi vaccine studies.

Plasmodium inui infection reduces the efficacy of a simian immunodeficiency virus DNA vaccine in a rhesus macaque model through alteration of the vaccine-induced immune response.

Human immunodeficiency virus type 1 and malaria are co-endemic in many areas. We evaluated the effects of Plasmodium inui infection on the performance of a simian immunodeficiency virus (SIV) DNA vaccine. Rhesus macaques were infected with P. inui by transfusion of whole blood from a persistently infected animal. Animals with and animals without P. inui infection were then vaccinated 4 times with an SIV DNA vaccine encoding SIVgag, SIVpol, and SIVenv. Animals were subsequently challenged with thirty 50% rhesus monkey infectious doses of SIVmac251 6 weeks after the last vaccination. P. inui-infected immunized animals showed a significantly higher viral load than animals without P. inui infection (P = .010, by the Wilcoxon rank sum test). The higher viral loads in the P. inui-infected animals were durable and were observed at all sampling time points across the study (P = .00245, by the Wilcoxon rank test). The P. inui-infected animals also had correspondingly lower CD4(+) cell counts. There were fewer vaccine-specific CD4(+) and CD8(+) cells in the P. inui-infected animals, compared with uninfected animals. Of importance, P. inui infection seemed to decrease the number of CD8(+) cells that could proliferate or secrete interferon γ, although the number of CD8(+) cells capable of secreting tumor necrosis factor α following in vitro stimulation was increased. This study demonstrated that P. inui infection had an influence on the immune response to an SIV DNA vaccine and decreased the vaccine's efficacy.

Protective CD8+ T lymphocytes in primates immunized with malaria sporozoites.

Live attenuated malaria vaccines are more potent than the recombinant protein, bacterial or viral platform vaccines that have been tested, and an attenuated sporozoite vaccine against falciparum malaria is being developed for humans. In mice, attenuated malaria sporozoite vaccines induce CD8(+) T cells that kill parasites developing in the liver. We were curious to know if CD8(+) T cells were also important in protecting primates against malaria. We immunized 9 rhesus monkeys with radiation attenuated Plasmodium knowlesi sporozoites, and found that 5 did not develop blood stage infections after challenge with live sporozoites. We then injected 4 of these protected monkeys with cM-T807, a monoclonal antibody to the CD8 molecule which depletes T cells. The fifth monkey received equivalent doses of normal IgG. In 3 of the 4 monkeys receiving cM-T807 circulating CD8(+) T cells were profoundly depleted. When re-challenged with live sporozoites all 3 of these depleted animals developed blood stage malaria. The fourth monkey receiving cM-T807 retained many circulating CD8(+) T cells. This monkey, and the vaccinated monkey receiving normal IgG, did not develop blood stage malaria at re-challenge with live sporozoites. Animals were treated with antimalarial drugs and rested for 4 months. During this interval CD8(+) T cells re-appeared in the circulation of the depleted monkeys. When all vaccinated animals received a third challenge with live sporozoites, all 5 monkeys were once again protected and did not develop blood stage malaria infections. These data indicate that CD8(+) T cells are important effector cells protecting monkeys against malaria sporozoite infection. We believe that malaria vaccines which induce effector CD8+ T cells in humans will have the best chance of protecting against malaria.

Orosensory stimulation effects on human saliva proteome.

Saliva flow induced by 6-gingerol (pungent), hydroxy-α/ß-sanshools (tingling), and citric acid (sour) was measured, and the time-dependent changes in the whole saliva proteome were analyzed by means of 2D-PAGE, followed by tryptic in-gel digestion and MALDI-TOF-MS peptide mass fingerprint analysis. The proteins showing significantly decreased abundance after oral 6-gingerol stimulation were identified as glutathione S-transferase P, the heat shock protein ß-1, the heat shock 70 kDa protein 1, annexin A1, and cytoplasmic ß-actin, whereas prolactin inducible proteins (PIP), short palate, lung and nasal epithelium carcinoma-associated protein 2 (SPLUNC2), zinc-α-2-glycoproteins (Zn-α-GP), and carbonic anhydrase VI (CAVI) were found with increased abundance. As the effects of this study were observed instantaneously upon stimulation, any proteome modulation is very likely to result from the release of proteins from preformed vesicles and not from de novo synthesis. The elevated levels of SPLUNC2, Zn-α-GP, and CAVI might be interpreted to trigger innate protective mechanisms in mucosal immunity and in nonimmune mucosal defense and might play an important role during the initial stage of inflammation.

Malaria infection by sporozoite challenge induces high functional antibody titres against blood stage antigens after a DNA prime, poxvirus boost vaccination strategy in Rhesus macaques.

BACKGROUND: A DNA prime, poxvirus (COPAK) boost vaccination regime with four antigens, i.e. a combination of two Plasmodium knowlesi sporozoite (csp/ssp2) and two blood stage (ama1/msp142) genes, leads to self-limited parasitaemia in 60% of rhesus monkeys and survival from an otherwise lethal infection with P. knowlesi. In the present study, the role of the blood stage antigens in protection was studied in depth, focusing on antibody formation against the blood stage antigens and the functionality thereof. METHODS: Rhesus macaques were immunized with the four-component vaccine and subsequently challenged i.v. with 100 P. knowlesi sporozoites. During immunization and challenge, antibody titres against the two blood stage antigens were determined, as well as the in vitro growth inhibition capacity of those antibodies. Antigen reversal experiments were performed to determine the relative contribution of antibodies against each of the two blood stage antigens to the inhibition. RESULTS: After vaccination, PkAMA1 and PkMSP119 antibody titres in vaccinated animals were low, which was reflected in low levels of inhibition by these antibodies as determined by in vitro inhibition assays. Interestingly, after sporozoite challenge antibody titres against blood stage antigens were boosted over 30-fold in both protected and not protected animals. The in vitro inhibition levels increased to high levels (median inhibitions of 59% and 56% at 6 mg/mL total IgG, respectively). As growth inhibition levels were not significantly different between protected and not protected animals, the ability to control infection appeared cannot be explained by GIA levels. Judged by in vitro antigen reversal growth inhibition assays, over 85% of the inhibitory activity of these antibodies was directed against PkAMA1. CONCLUSIONS: This is the first report that demonstrates that a DNA prime/poxvirus boost vaccination regimen induces low levels of malaria parasite growth inhibitory antibodies, which are boosted to high levels upon challenge. No association could, however, be established between the levels of inhibitory capacity in vitro and protection, either after vaccination or after challenge.

Effect of AFO design on walking after stroke: impact of ankle plantar flexion contracture.

This study was conducted to compare the effects of three ankle-foot orthosis (AFO) designs on walking after stroke and determine whether an ankle plantar flexion contracture impacts response to the AFOs. A total of 30 individuals, ranging from 6-215 months post-stroke, were tested in four conditions: shoes only (SH), dorsi-assist/dorsi-stop AFO (DA-DS), plantar stop/free dorsiflexion AFO (PS), and rigid AFO (Rigid). Kinematics, kinetics, and electromyographic (EMG) activity were recorded from the hemiparetic lower extremity while participants walked at a self-selected pace. Gait parameters were compared between conditions and between participants with and without a moderate ankle plantar flexion contracture. All AFOs increased ankle dorsiflexion in swing and early stance. Anterior tibialis EMG was reduced only in the PS AFO. Both PS and Rigid AFOs restricted ankle plantar flexion and increased knee flexion in loading. Peak ankle dorsiflexion in stance and soleus EMG intensity were greatest in the PS AFO. The Rigid AFO tended to restrict dorsiflexion in stance and knee flexion in swing only in participants without a plantar flexion contracture. Individuals without a contracture benefit from an AFO that permits dorsiflexion mobility in stance and those with quadriceps weakness may more easily tolerate an AFO with plantar flexion mobility in loading.

Sterile protection against Plasmodium knowlesi in rhesus monkeys from a malaria vaccine: comparison of heterologous prime boost strategies.

Using newer vaccine platforms which have been effective against malaria in rodent models, we tested five immunization regimens against Plasmodium knowlesi in rhesus monkeys. All vaccines included the same four P. knowlesi antigens: the pre-erythrocytic antigens CSP, SSP2, and erythrocytic antigens AMA1, MSP1. We used four vaccine platforms for prime or boost vaccinations: plasmids (DNA), alphavirus replicons (VRP), attenuated adenovirus serotype 5 (Ad), or attenuated poxvirus (Pox). These four platforms combined to produce five different prime/boost vaccine regimens: Pox alone, VRP/Pox, VRP/Ad, Ad/Pox, and DNA/Pox. Five rhesus monkeys were immunized with each regimen, and five Control monkeys received a mock vaccination. The time to complete vaccinations was 420 days. All monkeys were challenged twice with 100 P. knowlesi sporozoites given IV. The first challenge was given 12 days after the last vaccination, and the monkeys receiving the DNA/Pox vaccine were the best protected, with 3/5 monkeys sterilely protected and 1/5 monkeys that self-cured its parasitemia. There was no protection in monkeys that received Pox malaria vaccine alone without previous priming. The second sporozoite challenge was given 4 months after the first. All 4 monkeys that were protected in the first challenge developed malaria in the second challenge. DNA, VRP and Ad5 vaccines all primed monkeys for strong immune responses after the Pox boost. We discuss the high level but short duration of protection in this experiment and the possible benefits of the long interval between prime and boost.

High-resolution two-dimensional electrophoresis.

Two-dimensional gel electrophoresis (2-DE) with immobilized pH gradients (IPGs) combined with protein identification by mass spectrometry is currently the workhorse for the majority of ongoing proteome projects. Although alternative/complementary technologies, such as MudPIT, ICAT, or protein arrays, have emerged recently, there is up to now no technology that matches 2-DE in its ability for routine parallel expression profiling of large sets of complex protein mixtures. 2-DE delivers a map of intact proteins, which reflects changes in protein expression level, isoforms, or post-translational modifications. High-resolution 2-DE can resolve up to 5,000 proteins simultaneously ( approximately 2,000 proteins routinely), and detect and quantify <1 ng of protein per spot. Today's 2-DE technology with IPGs has largely overcome the former limitations of carrier ampholyte-based 2-DE with respect to reproducibility, handling, resolution, and separation of very acidic or basic proteins. Current research to further advance 2-DE technology has focused on improved solubilization/separation of hydrophobic proteins, display of low abundance proteins, and reliable protein quantitation by fluorescent dye technologies. Here, we provide a comprehensive protocol of the current high-resolution 2-DE technology with IPGs for proteome analysis and describe in detail the individual steps of this technique, i.e., sample preparation and protein solubilization, isoelectric focusing in IPG strips, IPG strip equilibration, and casting and running of multiple SDS gels. Last but not the least, a section on how to circumvent the major pitfalls is included.

Protein detection and quantitation technologies for gel-based proteome analysis.

Numerous protein detection and quantitation methods for gel-based proteomics have been devised that can be classified in three major categories: (1) Universal (or "general") detection techniques, which include staining with anionic dyes (e.g., Coomassie brilliant blue), reverse (or "negative") staining with metal cations (e.g., imidazole-zinc), silver staining, fluorescent staining or labeling, and radiolabeling, (2) specific staining methods for the detection of post-translational modifications (e.g., glycosylation or phosphorylation), and (3) differential display techniques for the separation of multiple, covalently tagged samples in a single two-dimensional electrophoresis (2-DE) gel, followed by consecutive and independent visualization of these proteins to minimize methodical variations in spot positions and in protein abundance, to simplify image analysis, as well as to improve protein quantitation by including an internal standard. The most important properties of protein detection methods applied in proteome analysis include high sensitivity (i.e., low detection limit), wide linear dynamic range for quantitative accuracy, reproducibility, cost-efficiency, ease of use, and compatibility with downstream protein identification or characterization technologies, such as mass spectrometry (MS). Regrettably, no single detection method meets all these requirements, albeit fluorescence-based technologies are currently favored for most applications; hence, the major focus of this chapter is on fluorescent-dye-based protein detection and quantitation techniques. Although satisfying results with respect to sensitivity and reproducibility are also obtained by methods based on radioactive labeling of proteins (which is still unsurpassed in terms of sensitivity), radiolabeling is, however, largely impractical for routine proteomic profiling because of the costs and the health and safety concerns associated with handling radioactive compounds.

2-DE with IPGs.

In order to overcome the limitations of carrier ampholyte generated pH gradients, IPGs were developed in the late 1970s. However, the 2-DE pattern we included in the first publication on IEF with IPGs [Bjellqvist et al., J. Biochem. Biophys. Methods 1982, 6, 317-339] was far from being competitive to O'Farrell's high-resolution 2-DE with carrier ampholytes. Our 2-DE pattern in this article was, more or less, only a proof of principle. It was, however, the beginning of a long journey of stepwise improved 2-DE protocols we developed in our laboratory and summarized in the reviews published in Electrophoresis 1988, 9, 531-546 and in Electrophoresis 2000, 21, 1037-1053. Milestones were the design of the IPG strip, and the "reduction-alkylation equilibration protocol" of IPG strips after IEF for the efficient transfer of proteins from first to second dimension. The protocol of 2-DE with IPGs has been constantly refined, e.g. by the generation of tailor-made IPGs with different pH intervals from the acidic to the basic extremes (pH 2.5-12), and extended separation distances for improved resolution. In the present review, a historical outline from the technical difficulties encountered during the development of 2-DE with IPGs, to the establishment of the actual "standard protocol" will be given, as well as the modified procedures for the separation of very acidic, very alkaline, low-abundance and hydrophobic proteins, followed by a brief discussion of the advantages and technical challenges of gel-based proteomic technologies.

A proteome analysis of the cadmium and mercury response in Corynebacterium glutamicum.

Cadmium and mercury are well-known toxic heavy metals, but the basis of their toxicity is not well understood. In this study, we analyzed the cellular response of Corynebacterium glutamicum to sublethal concentrations of cadmium and mercury ions using 2-DE and MS. Mercury induced the over-expression of 13 C. glutamicum proteins, whereas 35 proteins were induced, and 8 proteins were repressed, respectively, under cadmium stress. The principal response to these metals was protection against oxidative stress, as demonstrated by upregulation of, e.g., Mn/Zn superoxide dismutase. Thioredoxin and oxidoreductase responded most strongly to cadmium and mercury. The increased level of heat-shock proteins, enzymes involved in energy metabolism, as well as in lipoic acid and terpenoid biosynthesis after the treatment of cells with cadmium was also registered. Identification of these proteins and their mapping into specific cellular processes enable a global understanding of the way in which C. glutamicum adapts to heavy-metal stress and may help to gain deeper insight into the toxic mechanism of these metals.

Sample solublization buffers for two-dimensional electrophoresis.

Before two-dimensional electrophoresis (2-DE), proteins of the sample must be denatured, reduced, disaggregated, and solubilized. Sample solubilization is usually carried out in a buffer containing chaotropes (typically 9.5 M urea, or 5-8 M urea and 2 M thiourea), 2-4% nonionic and/or zwitterionic detergent(s), reducing agent(s), carrier ampholytes and, depending on the type of sample, protease inhibitors. In this chapter, the major constituents of sample solubilization/lysis buffers will be briefly reviewed, some general sample preparation guidelines will be given, and the most common protein solubilization cocktails will be described.

Sample prefractionation in granulated sephadex IEF gels.

Prefractionation procedures not only aid in reducing sample complexity, but also permit loading of higher protein amounts within the separation range applied to two-dimensional electrophoresis (2-DE) gels and thus facilitate the detection of less abundant protein species. Hence we developed a simple, cheap, and fast prefractionation procedure based on flat-bed isoelectric focusing (IEF) in granulated Sephadex gels, containing chaotropes, zwitterionic detergents and carrier ampholytes. After IEF, up to ten Sephadex gel fractions alongside the pH gradient are obtained, and then applied directly onto the surface of the corresponding narrow pH range immobilized pH gradient (IPG) strips as first dimension of 2-DE. The major advantages of this technology are the highly efficient electrophoretic transfer of the prefractionated proteins from the Sephadex IEF fraction into the IPG strip without any sample dilution, and full compatibility with subsequent 2-DE, because the prefractionated samples have not to be eluted, concentrated or desalted, nor does the amount of the carrier ampholytes in the Sephadex fraction interfere with IEF in IPG strips. This sample prefractionation method has been successfully applied for the separation, detection and identification of low abundance proteins from pro- and eukaryotic samples.

Protection of rhesus monkeys by a DNA prime/poxvirus boost malaria vaccine depends on optimal DNA priming and inclusion of blood stage antigens.

BACKGROUND: We have previously described a four antigen malaria vaccine consisting of DNA plasmids boosted by recombinant poxviruses which protects a high percentage of rhesus monkeys against Plasmodium knowlesi (Pk) malaria. This is a multi-stage vaccine that includes two pre-erythrocytic antigens, PkCSP and PkSSP2(TRAP), and two erythrocytic antigens, PkAMA-1 and PkMSP-1(42kD). The present study reports three further experiments where we investigate the effects of DNA dose, timing, and formulation. We also compare vaccines utilizing only the pre-erythrocytic antigens with the four antigen vaccine. METHODOLOGY: In three experiments, rhesus monkeys were immunized with malaria vaccines using DNA plasmid injections followed by boosting with poxvirus vaccine. A variety of parameters were tested, including formulation of DNA on poly-lactic co-glycolide (PLG) particles, varying the number of DNA injections and the amount of DNA, varying the interval between the last DNA injection to the poxvirus boost from 7 to 21 weeks, and using vaccines with from one to four malaria antigens. Monkeys were challenged with Pk sporozoites given i.v. 2 to 4 weeks after the poxvirus injection, and parasitemia was measured by daily Giemsa stained blood films. Immune responses in venous blood samples taken after each vaccine injection were measured by ELIspot production of interferon-gamma, and by ELISA. CONCLUSIONS: 1) the number of DNA injections, the formulation of the DNA plasmids, and the interval between the last DNA injection and the poxvirus injection are critical to vaccine efficacy. However, the total dose used for DNA priming is not as important; 2) the blood stage antigens PkAMA-1 and PkMSP-1 were able to protect against high parasitemias as part of a genetic vaccine where antigen folding is not well defined; 3) immunization with PkSSP2 DNA inhibited immune responses to PkCSP DNA even when vaccinations were given into separate legs; and 4) in a counter-intuitive result, higher interferon-gamma ELIspot responses to the PkCSP antigen correlated with earlier appearance of parasites in the blood, despite the fact that PkCSP vaccines had a protective effect.

Induction of multi-antigen multi-stage immune responses against Plasmodium falciparum in rhesus monkeys, in the absence of antigen interference, with heterologous DNA prime/poxvirus boost immunization.

The present study has evaluated the immunogenicity of single or multiple Plasmodium falciparum (Pf) antigens administered in a DNA prime/poxvirus boost regimen with or without the poloxamer CRL1005 in rhesus monkeys. Animals were primed with PfCSP plasmid DNA or a mixture of PfCSP, PfSSP2/TRAP, PfLSA1, PfAMA1 and PfMSP1-42 (CSLAM) DNA vaccines in PBS or formulated with CRL1005, and subsequently boosted with ALVAC-Pf7, a canarypox virus expressing the CSLAM antigens. Cell-mediated immune responses were evaluated by IFN-gamma ELIspot and intracellular cytokine staining, using recombinant proteins and overlapping synthetic peptides. Antigen-specific and parasite-specific antibody responses were evaluated by ELISA and IFAT, respectively. Immune responses to all components of the multi-antigen mixture were demonstrated following immunization with either DNA/PBS or DNA/CRL1005, and no antigen interference was observed in animals receiving CSLAM as compared to PfCSP alone. These data support the down-selection of the CSLAM antigen combination. CRL1005 formulation had no apparent effect on vaccine-induced T cell or antibody responses, either before or after viral boost. In high responder monkeys, CD4+IL-2+ responses were more predominant than CD8+ T cell responses. Furthermore, CD8+ IFN-gamma responses were detected only in the presence of detectable CD4+ T cell responses. Overall, this study demonstrates the potential for multivalent Pf vaccines based on rational antigen selection and combination, and suggests that further formulation development to increase the immunogenicity of DNA encoded antigens is warranted.

Two-dimensional electrophoresis for plant proteomics.

Two-dimensional gel electrophoresis (2-DE) with immobilized pH gradients (IPGs) combined with protein identification by mass spectrometry (MS) is currently the workhorse for proteome analysis. 2-DE allows separation of highly complex mixtures of proteins according to isoelectric point (pI), molecular mass (Mr), solubility, and relative abundance and delivers a map of intact proteins, which reflects changes in protein expression level, isoforms, or posttranslational modifications. 2-DE can resolve more than 5000 proteins simultaneously (approx 2000 proteins routinely) and can detect and quantify <1 ng of protein per spot. Today's 2-DE technology with IPGs has overcome the former limitations of carrier ampholyte-based 2-DE with respect to reproducibility, handling, resolution, and separation of very acidic and/or basic proteins. The development of IPGs between pH 2.5 and 12 has allowed the analysis of very alkaline proteins and the construction of the corresponding databases. Narrow pH range IPGs provide increased resolution (delta pI = 0.001) and, in combination with prefractionation methods, permit the detection of low abundance proteins. In this article we provide a comprehensive protocol of the current 2-DE technology for plant proteome analysis and describe in detail the individual steps of this technique.

Toll-like receptor agonists influence the magnitude and quality of memory T cell responses after prime-boost immunization in nonhuman primates.

There is a remarkable heterogeneity in the functional profile (quality) of T cell responses. Importantly, the magnitude and/or quality of a response required for protection may be different depending on the infection. Here, we assessed the capacity of different Toll like receptor (TLR)-binding compounds to influence T helper cell (Th)1 and CD8+ T cell responses when used as adjuvants in nonhuman primates (NHP) with HIV Gag as a model antigen. NHP were immunized with HIV Gag protein emulsified in Montanide ISA 51, an oil-based adjuvant, with or without a TLR7/8 agonist, a TLR8 agonist, or the TLR9 ligand cytosine phosphate guanosine oligodeoxynucleotides (CpG ODN), and boosted 12 wk later with a replication-defective adenovirus-expressing HIV-Gag (rAD-Gag). Animals vaccinated with HIV Gag protein/Montanide and CpG ODN or the TLR7/8 agonist had higher frequencies of Th1 responses after primary immunization compared to all other vaccine groups. Although the rAD-Gag boost did not elevate the frequency of Th1 memory cytokine responses, there was a striking increase in HIV Gag-specific CD8+ T cell responses after the boost in all animals that had received a primary immunization with any of the TLR adjuvants. Importantly, the presence and type of TLR adjuvant used during primary immunization conferred stability and dramatically influenced the magnitude and quality of the Th1 and CD8+ T cell responses after the rAD-Gag boost. These data provide insights for designing prime-boost immunization regimens to optimize Th1 and CD8+ T cell responses.

Guidelines for the next 10 years of proteomics.

In the last ten years, the field of proteomics has expanded at a rapid rate. A range of exciting new technology has been developed and enthusiastically applied to an enormous variety of biological questions. However, the degree of stringency required in proteomic data generation and analysis appears to have been underestimated. As a result, there are likely to be numerous published findings that are of questionable quality, requiring further confirmation and/or validation. This manuscript outlines a number of key issues in proteomic research, including those associated with experimental design, differential display and biomarker discovery, protein identification and analytical incompleteness. In an effort to set a standard that reflects current thinking on the necessary and desirable characteristics of publishable manuscripts in the field, a minimal set of guidelines for proteomics research is then described. These guidelines will serve as a set of criteria which editors of PROTEOMICS will use for assessment of future submissions to the Journal.