coli [16], [21], [22], [23] and [24]. Meanwhile, the kanamycin concentration and the interaction of IPTG with kanamycin was not found to have any appreciable effect on cell growth within the ranges under study. The protein concentration stayed at similar levels under all the conditions tested in the factorial design (Table 1). This means that when the effects Selleck Romidepsin of the variables on protein concentration were analyzed, it could be concluded that neither the IPTG concentration nor the kanamycin concentration nor the interaction of IPTG with kanamycin had any appreciable influence on protein expression in the ranges tested, since the p-value was higher than 0.05 ( Table 3). This suggests

that under the conditions tested the inducer concentration could be reduced up to tenfold (also advantageous

because of its negative effect on cell growth) and kanamycin could even be completely eliminated from the system without this having any major impact on the protein concentration. Costs would be reduced and expression levels would remain about the same. Also, high concentrations of protein in a soluble form were expressed in all the concentrations at 37 °C and 200 rpm, which is positive, since E. coli normally expresses insoluble proteins under such conditions. The concentration of ClpP obtained in these experiments fell within the concentration range obtained in other studies in the literature, which report on experiments to optimize the expression of other proteins in E. coli using agitated flasks and batch cultivation [22], [25], [26], [27] and [28]. GS-7340 research buy Data from the literature show that higher protein concentrations from E. coli are achieved when the heterologous Electron transport chain protein is expressed and optimized in bioreactors, where conditions can be controlled [26]. Independent of the kanamycin concentration, the experiments carried out using lower concentrations of IPTG yielded higher cell growth than the others and greater plasmid stability, since the values

for Φ (fraction of plasmid-bearing cells) in experiments 1 and 2 were higher than in the others. In the experiments with lower IPTG concentrations, the fraction of plasmid-bearing cells was found to be between 37% and 56%, while in the other experiments induced with higher IPTG concentrations, Φ was found to be lower than this. In other words, in the experiments with lower IPTG concentrations there was less plasmid segregation ( Table 1). The effects of the variables on Φ are shown in Table 4. It can be concluded from these analyses, within a 95% confidence interval, that the IPTG concentration in the range tested had a negative effect on plasmid stability, while the kanamycin concentration in the system and the IPTG/kanamycin interaction had no appreciable impact on Φ in the range under study. This means that the closer the IPTG concentration was to 0.

The nature of interaction and its conformation with dock score is shown in the Table 2. Hence these compounds can be further analyzed invitro and invivo to check it’s potent against MAP kinases. BTZ-4a = 1H NMR LY2835219 (300 MHz, CDCl3) δ: 7.18–8.14 (m, 8H, Ar–H), 3.28 (s, 2H), 2.15 (s, 6H); 13C NMR (300 MHz, CDCl3) δ: 166.92, 151.37, 136.01, 132.88, 130.80, 130.66, 126.81, 126.03, 125.86, 125.74, 123.56, 83.26, 42.31, 15.03; ESI-MS, m/z calcd. for C17H16BrNS3 410.41 found [M]+ 410. BTZ-6a = 1H NMR (400 MHz, CDCl3) δ: 7.20–9.32 (m, 7H, Ar–H), 3.42 (s, 2H, CH2), 2.39 (s, 3H, CH3), 2.16 (s, 6H, 2CH3); 13C NMR (300 MHz, CDCl3) δ: 166.89,

151.50, 149.94, 148.51, 137.36, 135.83, 135.07, 134.45, 125.64, 125.12, 123.05, 122.34, 82.69, 42.03, 20.99, 14.50; ESI-MS, m/z calcd. for C17H18N2S3 346.53 found [M+H]+ 347.5. BTZ-6b = 1H NMR (300 MHz, CDCl3) δ: 7.12–9.21 (m, 7H, Ar–H), 3.91 (s, 3H, OCH3), 3.21 (s, 2H, CH2), 2.18 (s, 6H, 2CH3); 13C NMR (300 MHz, CDCl3) δ: 166.35, 157.25, 151.42, 148.81, 136.23, 130.30, 124.32, 124.16, 112.94, 112.38, 82.99, 56.31, 41.80, selleck chemical 14.40; ESI-MS, m/z calcd. for C17H18N2OS3 362.53 found [M+H]+ 363.5. BTZ-19 = 1H NMR (400 MHz,CDCl3) δ: 7.05–7.91 (m, 7H, Ar–H), 3.83 (s, 3H, OCH3), 3.25 (s, 2H, CH2), 2.42 (s, 3H, CH3), 2.15 (s, 6H, 2CH3); 13C NMR (400 MHz, CDCl3) δ: 167.45, 156.51, 145.89, 141.11, 136.56, 129.20, 127.39, 126.70, 124.14, 119.06, 116.73, 82.23, 55.64, 42.07, 21.45, 14.70; ESI-MS, m/z calcd. for C19H21NOS3 375.57 found [M+H]+ 376.5. BTZ-20 = 1H NMR (400 MHz, CDCl3) δ: 7.14–8.15 (m, 12H, Ar–H),

3.85 (s, 3H, OCH3), 3.30 Carnitine dehydrogenase (s, 2H, CH2), 2.17 (s, 6H, 2CH3); 13C NMR (400 MHz, CDCl3) δ: 167.17, 156.64, 145.82, 143.36, 140.28, 138.09, 128.86, 127.91, 127.79, 127.09, 126.80, 124.22, 119.10, 116.77, 113.20, 101.56, 82.33, 55.66, 42.13, 14.72; ESI-MS, m/z calcd. for C23H21NS3 437.09 found [M+H]+ 438.8. BTZ-14a = 1H NMR (400 MHz, CDCl3) δ: 7.12–7.65 (m, 6H, Ar–H), 3.12 (s, 2H, CH2), 2.35 (s, 3H, CH3), 2.12 (s, 6H, 2CH3); 13C NMR (400 MHz, CDCl3) δ: 161.91, 151.75, 143.37, 136.25, 134.75, 131.34, 130.58, 129.53, 125.83, 123.46, 81.28, 43.79, 21.05, 14.98; ESI-MS, m/z calcd. for C16H17NS4 351.0 found [M+H]+ 352.0. BTZ-14b = 1H NMR (400 MHz, CDCl3) δ: 6.81–7.62 (m, 6H, Ar–H), 3.88 (s, 3H, OCH3), 3.54 (s, 2H, CH2), 2.20 (s, 6H, 2CH3); 13C NMR (400 MHz, CDCl3) δ: 163.64, 157.59, 152.23, 144.34, 134.63, 131.72, 130.94, 129.83, 123.53, 115.56, 114.92, 81.12, 57.02, 43.11, 14.82; ESI-MS, m/z calcd.

Similar concerns apply to thin subsidies (lowering the price of healthier products). To date only a couple of experimental studies examining these types of strategies in retail environments are available, including a New Zealand supermarket trial (Ni Mhurchu et al., Selleck ERK inhibitor 2010) and a Dutch trial in a computerized retail

environment (Waterlander et al., 2012). Both studies found that the reduced prices of healthier foods led to higher purchases of these products. Recently, Andreyeva and colleagues published a review on the price elasticity1 of food. They concluded that food is elastic and that the highest price elasticity was found for food away from home, soft drinks, juice, meats, and fruit (Andreyeva et al., 2010). These results show that thin subsidies see more are promising to stimulate healthier food purchases. Nevertheless, studies also reported that discounting healthy foods leads to more calorie purchases (Epstein et al., 2010) or is counterproductive because consumers used the saved money to buy unhealthier products (Giesen et al., 2011b). Previous studies

show that both taxing and subsidizing strategies have positive (e.g., more healthy food purchases), but also potentially negative side effects (e.g., more calories, lower fruit purchases). Therefore, the best suggestion may be to combine both strategies (Ni Mhurchu, 2010, Nnoaham et al., 2009 and Powell and Chaloupka, 2009). Therefore, this study aimed to examine both single and combined effects of lowering the prices of healthier foods and (simultaneously) increasing the prices of unhealthier foods on food purchases. It is hypothesized that the most favorable nutrient purchases will be found when combining the greatest discounts on healthier foods with the greatest

tax increase on unhealthier foods. This study used a unique 3-D web-based supermarket (Fig. 1). The main features are described below; additional information can be found elsewhere (Waterlander et al., 2011). The web-based Bay 11-7085 supermarket was designed in the image of an existent branch of the Dutch market leader supermarket. Photographs of genuine products were used to compose product images and prices were made available through shelf labeling. Food prices were based on the prices of the two Dutch market leaders, and the stock was also based on an existing supermarket. It was decided to create a representative product selection based on the 38 different food categories as used on the website of the market leader supermarket (Albert Heijn Online Shop, 2010). Within each product category, a sample representing around 10% of the regular assortment was selected by choosing popular and frequently consumed products. In total, the web-based supermarket contained 512 different food products modeling the actual distribution of store products and categories (Table 1). The stock did not take in specific brands or different package sizes.

e., 60% of antigen-specific lysis by in vivo CTL) responses”. The correct sentence should be “Mucosal immunization of C57BL/6 mice with OVA using c-di-IMP as adjuvant also led to the stimulation of strong in vivo CTL responses (i.e., 60% of antigen-specific lysis)”. “
“Infection with many vector-borne pathogens including Theileria spp., Anaplasma spp., Babesia spp., Borrelia spp., and Plasmodium spp. results in long-term persistent infection due to the pathogen’s ability to evade the host immune response. This

ability is in large part due to generation of outer membrane protein antigenic variants. For example, infection with Anaplasma marginale, a bacterial pathogen of cattle, generally results in life-long persistence in the mammalian host. Persistence is attributed primarily to rapid shifts in the surface coat structure and specifically variation in the highly immunogenic major surface protein Dolutegravir 2 (Msp2). The expressed copy of Msp2 is composed of a central hypervariable region that is flanked by highly conserved regions ( Fig. 1a and b). The variation is generated by gene conversion in which

one of multiple msp2 donor alleles is recombined into a single, operon-linked expression site [1], [2] and [3]. The donor alleles have 5′ and 3′ regions which are identical to the expression site copy and flank a unique allele-specific hypervariable domain [1] and [4]. These donor alleles are termed functional selleck pseudogenes as their 5′ and 3′

regions are truncated, they lack the function elements for in situ transcription, and are Resminostat only expressed following recombination into the single expression site [1] and [4]. During infection, Msp2 represents dominant antigens recognized by sera from cattle infected with A. marginale. The anti-Msp2 specific antibody response is predominantly directed toward the hypervariable region rather than the flanking conserved regions [5] and [6]. However, the hypervariable region of newly emergent variants is not recognized by existing antibody [7] and [8]. Thus, generation of Msp2 variants allows for immune escape and long-term pathogen persistence [8] and [9]. In contrast to infection, where clearance does not occur, immunization with either purified A. marginale outer membranes or cross-linked outer membrane protein complexes induces complete protection against infection in 40–70% of vaccinees, and protection against anemia and high-level bacteremia in nearly all animals [7], [10] and [11]. Protection correlates with high IgG antibody titers against surface-exposed polypeptides, including Msp2 [7]. While protection associates with the IgG response to outer membrane proteins, the specific epitope targets and characteristics of this protective immune response remain unknown.

INH-C17 showed synergism with RIF but additive/indifferent interaction with STR. This could be due the structure Dasatinib mw of INH-C17 which might be hindered by the cell wall in the presence of STR. However, author could not obtain a better explanation for such phenomenon. Moreover, not all in vitro drug interactions could be acknowledged meticulously for predicting efficiency of these drugs in combination in clinical practices against TB as these interactions can only provide information about synergistic, additive/indifferent, or antagonistic actions of the drugs in inhibiting the bacterial growth. Therefore, this in vitro study should be further assessed with in vivo studies for

clinical significance against TB. The lipophilic derivatives, INH-C16, INH-C17 and

click here INH-C18 showed a better anti-TB activity against M. tuberculosis H37Rv and interacted positively with the first-line drugs. Therefore, they have the potential to be drug leads worthy of further investigations as anti-TB drugs. All authors have none to declare. We are grateful to the Ministry of Science and Technology, Malaysia for providing financial support to carry out this research (FRGS: 203/PFARMASI/671157). Thaigarajan Parumasivam was endowed with a USM Fellowship from Universiti Sains Malaysia. “
“Among the protozoan, bacterial, viral and fungal pathogen bacterial infection is more prevalent in the silkworm, Bombyx mori and constitutes about 60–70% of total silk crop loss in Japan 1 and India. 2 and 3 Among bacterial species those are linked to spread disease in B. mori during rearing majorly belongs to the genus Bacillus sp. such as Bacillus cuboniaus, 4Bacillus bombysepticus, 5Bacillus mycoides, and Bacillus leterosporus. 6 The mortality attributable to eight genotypes of Bacillus thuringiensis in all the larval stages of B. mori within 3 h post inoculation

has been reported by Selvakumar, 7 PAK6 where B. thuringiensis endotoxin known to damage the gut lining to cause gut paralysis and the larval death in silkworm occurs due to starvation. 8, 9, 10 and 11 The beta endotoxin of Staphylococcus aureus, Pseudomonas aeruginosa and Bacillus cereus causes toxidermia, a septicemia and death in the silkworm larvae. 12 While, the cause of latent bacterial infection via transovarial transmission and it’s persistence in the silkworm eggs is not reported earlier. During screening of surface sterilized silkworm egg homogenate for the presence of bacterial species, several colonies of Bacillus species were evidenced from egg homogenate inoculated on nutrient agar plates. It was subsequently sub cultured, purified and identified as Bacillus subtilis. To understand the mode of infection and mechanism of transmission of B. subtilis in the eggs, the infection experiments were carried out.

2 μM of rVCP and 1 μM of mAb in a total volume of 25 μl and incubated for 5 min at 22 °C. The remaining C3-convertase activity was determined by measuring hemolysis after incubating the reaction mixture for 30 min at 37 °C with Nutlin-3 1:100 diluted guinea pig serum containing 40 mM EDTA. Hemolysis was measured at 405 nm. The kinetics of binding of the mAbs to VCP was

determined on the SPR-based biosensor BIACORE 2000 (Biacore AB, Uppsala, Sweden). All the experiments were performed in PBS-T (10 mM sodium phosphate, 145 mM NaCl, pH 7.4 containing 0.05% Tween 20) at 25 °C. About 2600 RUs of each of the mAbs was immobilized on test flow cells (Fc-2, Fc-3 or Fc-4) of a CM5 chip using amine-coupling chemistry and non-immobilized flow cell (Fc-1) served as the control flow cell [40]. Various concentrations of rVCP were then flown over the chip at 30 μl/min for 120 s and dissociation was followed for an additional 180 s. The chip was regenerated by injecting brief pulses of 0.2 M sodium carbonate, pH 9.5. Data obtained Ibrutinib order for the control flow cell were subtracted from those obtained for test flow

cell and evaluated using BIAevaluation software version 4.1 using global fitting. The half-life of two of the VCP neutralizing mAbs (NCCS 67.5 and 67.9) in rabbits was assessed by radiolabeling the antibodies with 131I. One hundred microliters of the labeled mAbs at a dose of 100–200 μCi (∼65–100 μg) were injected intradermally on backs of New Zealand White rabbits

and imaged using an ELGEMS “Millennium MPS” gamma ray camera (GE, USA) at the Department of Veterinary Medicine and Veterinary Nuclear Medicine Center (Mumbai, India). A maximum of 250 kilocounts was set for acquiring images and a medium energy collimator was used to capture emerging radiations. The images were acquired at various time points Adenosine and analyzed using GENIE acquisition user interface software (GE, USA). The first image acquired immediately after the injection was considered as zero time point. The data obtained were normalized by considering the counts obtained at the zero time point as 100%. To re-examine the VCP domains responsible for complement modulation and to understand the in vivo relevance of these complement regulatory functions in VACV pathogenesis, we raised a panel of mAbs against VCP by immunizing BALB/c mice followed by fusion, and cloning and subcloning of the candidate hybrids. The monoclonal antibodies thus generated largely belonged to IgG1κ isotype. Four antibodies, namely NCCS 67.5, NCCS 67.9, NCCS 67.11 and NCCS 67.13, all belonging to IgG1κ isotype, were chosen for further characterization as they differentially inhibited the functional activities of VCP (see below). Of the four, mAb 67.5 uniquely displayed two distinct light chains, which could be a result of difference in their glycosylation states [51].

We addressed this uncertainty by comparing the adjuvant effect of two different VRP genomes: VRP16M or a new VRP genome

named VRP(-5) which contains a deletion in the core 26S subgenomic promoter and is genetically incapable of producing a subgenomic RNA (Fig. 1A). Mice were primed and boosted with OVA alone or OVA in the presence of a low dose of VRP16M or VRP(-5) (103 IU, which corresponds to 106 GE). (VRP IU are based on in vitro infection of BHK-21 cells; in vivo infectivity is undefined.) After the boost we measured anti-OVA IgG in the serum and anti-OVA IgA in fecal extracts. Both VRP genomes significantly increased antibody responses compared to OVA alone (Fig. 1B and C), with the VRP(-5) genome inducing a significantly stronger mucosal IgA response. These results show clearly that the

26S promoter is not required for the adjuvant effect induced CCI-779 in vivo by VRP, so for all subsequent experiments we used the VRP(-5) genome, which will be referred to as simply VRP Selleckchem 17-AAG for the rest of this report. In all previous studies of VRP adjuvant activity the VRP were injected into the footpad, but because this is an impractical route for human vaccines, we assessed whether VRP would be effective by intramuscular (i.m.) delivery. Mice were primed and boosted with OVA and VRP (105 IU) in the footpad or i.m. Anti-OVA serum IgG and fecal IgA titers were significantly increased by both routes of delivery (Fig. 1D and E), indicating that i.m. delivery of VRP is just as effective as footpad delivery. Data shown in Fig. 1 demonstrate that VRP injected into the footpad are an effective adjuvant at a relatively low dose (103 IU). To evaluate the efficacy of lower doses of VRP delivered i.m., we tested the effect of VRP on anti-OVA immunity after i.m.

injection in Balb/c mice using a range of Bay 11-7085 VRP doses between 102 and 105 IU (105 to 108 GE). Titers of anti-OVA IgG in the serum had a clear dose–response, and all tested doses of VRP significantly increased the anti-OVA titers relative to mice immunized with OVA alone (Fig. 2A). The mucosal response measured in the fecal extracts demonstrated clear induction of anti-OVA IgA antibodies at all tested VRP doses, with the strongest response at ≥104 IU (Fig. 2B). To examine the VRP dose effect on T cell responses, we primed and boosted C57Bl/6 mice i.m. with OVA alone or in the presence of increasing doses of VRP. This mouse strain was used because T cell-reactive OVA peptides are known for this mouse, and it was previously shown that the VRP adjuvant effect is intact in this strain [21]. The dose of OVA used (100 μg) was based on the previous finding that this higher dose was required for a detectable T cell response [21]. After boost, spleen cells harvested from these mice were incubated in vitro with a CD8-specific OVA peptide, and IFN-γ production was measured by intracellular staining and flow cytometric analysis.

Furthermore, surveillance data in Kenya suggest ALRI cause a considerable burden of disease in rural and urban communities,

with the greatest burden among children PLX4032 clinical trial [10]. Although routine vaccination is a major tool in the primary prevention of influenza [11] and [12], a significant proportion of the population is reluctant to receive vaccines [13] and [14]. We examined demographic, socio-economic and geographic factors that contributed to acceptance of childhood seasonal influenza vaccination among families in rural western Kenya. Existing literature from other countries suggest important determinants of childhood vaccine uptake [15], [16], [17], [18], [19] and [20]. Analyses from demographic and surveillance systems (DSS) have found different socio-demographic factors associated with childhood vaccination; In Bangladesh, diphtheria–tetanus–pertussis and oral polio vaccination were independently associated with higher maternal age, lower maternal education and birth order

of the child [15]. In Malawi, maternal education was found to be among major determinants of the immunization status of the child [16], Moreover, findings from DSS in Ghana showed positive relationship between socio-economic status and vaccination status [17]. Cross-sectional surveys have similarly suggested important determinants of childhood vaccination; a survey in Khartoum State of Sudan observed an increased vaccination rate with an increase in the age of the children and the education level of the mother, subsequently children of older mothers were more likely to buy Sunitinib have had the correct vaccinations [18]. A survey in Ghana found distance to be the most important Phosphoprotein phosphatase factor that influences the utilization of health services [19]. Moreover,

a survey in Kenya found that immunization rate ratios were reduced with every kilometer of distance from home to vaccine clinic [20]. Researches on factors associated with vaccination among children in Africa have focused on vaccinations covered by EPI programs. None of these studies, however, draws attention on the issue raised in our work and to best of our knowledge determinants of childhood vaccination in the context of influenza vaccination remains an ignored expedition for sub-Saharan Africa. Understanding the determinants of children’s vaccine uptake in Kenya is therefore important for guiding future immunization policies. The CDC’s International Emerging Infections Program in collaboration with KEMRI has conducted population-based infectious disease surveillance (PBIDS) in Asembo Division, Siaya County since late 2005 [21]. Asembo has an area of 200 km2 and lies northeast of Lake Victoria in Nyanza Province in western Kenya. The PBIDS area comprises approximately 100 km2 with an overall population density of about 325 persons per square kilometer. The surveillance population includes approximately 25,000 persons living in 33 villages.

Anyway, these ‘negative’ observations on free hormone responses generate some novel insights. First of all, measurement of total plasma glucocorticoid hormone only Forskolin molecular weight provides limited information about the real biologically active free concentration. Second, from a homeostatic perspective, it seems that, with regard to the free glucocorticoid hormone, the organism is keen to generate stressor-specific set response levels to stress. If like in the case of long-term exercise the enhanced sympatho-adrenomedullary drive results in enhanced total plasma corticosterone

responses to physical challenges then apparently mechanisms are in place to adjust the available free hormone levels to match those in the sedentary animals. A similar mechanism is supposedly in place in case of mild psychological stressors. Identification of these mechanism(s) is important, as they are part of the nuts and bolts that constitute resilience. Consequently, disturbances in these adjusting mechanisms would result in hypo- or hyper-levels

of glucocorticoid hormone, which could lead to development of various disorders. We would like to note that in addition to exercise, gender is another example in which this www.selleckchem.com/products/dabrafenib-gsk2118436.html mechanism of free glucocorticoid adjustment may be operational. It’s known for many years that female rats and mice have substantially higher baseline and stress-induced total plasma glucocorticoid levels than their male counterparts. Using microdialysis, we found however that the free corticosterone levels at baseline and after stress were very similar between female and male rats (Droste et al., 2009a). In a sleep physiological study we studied various properties of the sleep/EEG pattern in exercising and sedentary mice including the duration of sleep episodes, sleep intensity, rapid eye movement (REM) sleep, non-REM sleep and wakefulness. These properties are indicators of sleep quality.

For more information about our method of sleep recording, sleep analysis and spectrum Fossariinae analysis see Lancel et al. (1997). We observed that long-term wheel running mice showed significantly less sleep episodes, however, these episodes were of longer duration indicating a better sleep consolidation (Lancel et al., 2003). Compared with sedentary controls the exercising mice also showed less REM sleep. A 15 min social conflict resulted in an increase in non-REM sleep, enhancement of low-frequency activity in the EEG within non-REM sleep (indicating increased sleep intensity) and less wakefulness in both control and exercising mice. In the control mice however an increased REM sleep concurrently with the rise in non-REM sleep was observed. In contrast, exercising animals showed a decrease in REM sleep.

50 μg/ml of anti-H-2Kd competitive binding antibody (BD PharMigen, San Diego, USA) was added to each well to prevent dissociated tetramer from re-binding and plates were incubated at 37 °C, 5% CO2. At each time point, cells were transferred into ice-cold FACS Talazoparib in vitro buffer to stop the reaction, washed and resuspended in 100 μl of FACS buffer containing 0.5% paraformaldehyde. 100,000 events were acquired on a FACs Calibur flow cytometer (Becton-Dickinson, San Diego, USA) and analysed using Cell Quest Pro software.

In tetramer dissociation assays, lower dissociation rates or stronger MHC-I/peptide complex binding to the TCR complex of CD8 T cell, is associated with higher avidity [43]. IFN-γ or IL-2 capture ELISpot assays was used to assess IFN-γ or IL-2 HIV-specific T cell responses [40]. Briefly, 2 × 105 spleen or GN cells were added to 96-well Millipore PVDF

plates (Millipore, Volasertib MA, Ireland) coated with 5 μg/ml of mouse anti-IFN-γ or IL-2 capture antibodies (BD PharMigen, San Diego, CA), and stimulated for 12 h or 22 h respectively for IL-2 or IFN-γ ELISpot, in the presence of H-2Kd immuno-dominant CD8+ T cell epitope, Gag197–205 AMQMLKETI (synthesised at the Bio-Molecular Resource Facility at JCSMR). ConA-stimulated cells (Sigma, USA) were used as positive controls and unstimulated cells as negative controls. For both ELISpot assays, all steps were carried out exactly as described previously [20] and [40]. The graphed data are expressed as SFU (spot-forming units) per 106 T cells and represent mean values ± SD. Unstimulated cell counts were subtracted from each stimulated value before plotting the data. In all assays the background SFU counts were between 4–10 SFU for IFN-γ and 5–18 SFU for IL-2 ELISpot. Also the unimmunised animals did not show any responses following Gag197–205-AMQMLKETI stimulation. IFN-γ and TNF-α producing HIV-specific CD8 T cells, were analysed as described in Ranasinghe

et al. [20] and [40]. Briefly, 2 × 106 lymphocytes were stimulated with AMQMLKETI peptide at 37 °C for 16 h, and further incubated with Brefeldin A (eBioscience, CA, USA) for 4 h. Cells were surface-stained with CD8-Allophycocyanin (Biolegend, USA) then fixed and permeabilized prior to intracellular staining with IFN-γ-FITC and TNF-α-PE (Biolegend, USA). Total 100,000 gated events per sample were collected using FACS Calibur flow heptaminol cytometer (Becton Dickinson, San Diego, CA), and results were analysed using Cell Quest Pro software. Prior to plotting the graphs the unstimulated background values were subtracted from the data, The IFN-γ+ cell counts were less than 0.05–0.1% in unimmunised or unstimulated samples similar to our previous studies [23]. Female BALB/c mice n = 8 were i.n./i.m. prime-boost immunised using the strategies 1, 4 and 5 indicated in Table 1. ELISA was used to determine HIV-1 p55 gag-specific IgG1 and IgG2a serum antibody titres similar to as described in Ranasinghe et al. [40].