Synthesis and immunological activities of novel agonists of toll-like receptor 9

Novel agonists of TLR9 with two 5′-ends and synthetic immune stimulatory motifs, referred to as immune modulatory oligonucleotides (IMOs) are potent agonists of TLR9. In the present study, we have designed and synthesized 15 novel IMOs by incorporating specific chemical modifications and studied their immune response profiles both in vitro and in vivo. Analysis of the immunostimulatory profiles of these IMOs in human and NHP cell-based assays suggest that changes in the number of synthetic immunostimulatory motifs gave only a subtle change in immune stimulation of pDCs as indicated by IFN-α production and pDC maturation while the addition of self-complementary sequences produced more dramatic changes in both pDC and B cell stimulation. All IMOs induced cytokine production in vivo immediately after administration in mice. Representative compounds were also compared for the ability to stimulate cytokine production in vivo (IFN-α and IP-10) in rhesus macaques after intra-muscular administration.     

Mutants ofEscherichia coli sensitive to hydrogen peroxide

Mutants sensitive to moderate H₂O₂ concentrations were selected in a HfrphoA (S33)Escherichia coli strain after mutagenesis withN-methyl,N-nitro,N-nitrosoguanidine (NG). Of the sensitive strains, 31% were catalase-deficient and retained glutathione reductase levels similar to those of the parental strain, whereas 69% still had normal catalase and glutathione reductase activities. Mutants supersensitive to low H₂O₂ concentrations were selected in a catalase-deficient strain (CGR201) after mutagenesis with NG. Of these, 20% were glutathione reductase-deficient, and the remaining 80% were unaffected in this enzymatic activity. Compared with the parental strain S33, H₂O₂ was 5 to 12 times more toxic for the sensitive mutants, and 19 to 21 times for the supersensitive ones.     

Vaccination with Ad5 vectors expands Ad5-specific CD8 T cells without altering memory phenotype or functionality

Background

Adenoviral (Ad) vaccine vectors represent both a vehicle to present a novel antigen to the immune system as well as restimulation of immune responses against the Ad vector itself. To what degree Ad-specific CD8⁺ T cells are restimulated by Ad vector vaccination is unclear, although such knowledge would be important as vector-specific CD8⁺ T cell expansion could potentially further limit Ad vaccine efficacy beyond Ad-specific neutralizing antibody alone.

Methodology/Principal Findings

Here we addressed this issue by measuring human Adenovirus serotype 5 (Ad5)-specific CD8⁺ T cells in recipients of the Merck Ad5 HIV-1 vaccine vector before, during, and after vaccination by multicolor flow cytometry. Ad5-specific CD8⁺ T-cells were detectable in 95% of subjects prior to vaccination, and displayed primarily an effector-type functional profile and phenotype. Peripheral blood Ad5-specific CD8⁺ T-cell numbers expanded after Ad5-HIV vaccination in all subjects, but differential expansion kinetics were noted in some baseline Ad5-neutralizing antibody (Ad5 nAb) seronegative subjects compared to baseline Ad5 nAb seropositive subjects. However, in neither group did vaccination alter polyfunctionality, mucosal targeting marker expression, or memory phenotype of Ad5-specific CD8⁺ T-cells.

Conclusions

These data indicate that repeat Ad5-vector administration in humans expands Ad5-specific CD8⁺ T-cells without overtly affecting their functional capacity or phenotypic properties. This is a secondary analysis of samples collected during the 016 trial. Results of the Merck 016 trial safety and immunogenicity have been previously published in the journal of clinical infectious diseases [1].

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00849680","term_id":"NCT00849680"}}NCT00849680 [{"type":"clinical-trial","attrs":{"text":"NCT00849680","term_id":"NCT00849680"}}NCT00849680]     

Kcnq1 contributes to an adrenergic-sensitive steady-state K+ current in mouse heart

It has been suggested that Kcne1 subunits are required for adrenergic regulation of Kcnq1 potassium channels. However, in adult mouse hearts, which do not express Kcne1, loss of Kcnq1 causes a Long QT phenotype during adrenergic challenge, raising the possibility that native Kcnq1 currents exist and are adrenergically regulated even in absence of Kcne1. Here, we used immunoblotting and immunohistochemical staining to show that Kcnq1 protein is present in adult mouse hearts. Voltage-clamp experiments demonstrated that Kcnq1 contributes to a steady-state outward current (I(SS)) in wild-type (Kcnq1(+/+)) ventricular myocytes during isoproterenol stimulation, resulting in a significant 7.1% increase in I(SS) density (0.43+/-0.16 pA/pF, p <0.05, n =15), an effect that was absent in Kcnq1-deficient (Kcnq1(-/-)) myocytes (-0.14+/-0.13 pA/pF, n =17). These results demonstrate for the first time that Kcnq1 protein is expressed in adult mouse hearts where it contributes to a beta-adrenergic-induced component of I(SS) that does not require co-assembly with Kcne1.     

Agriculture as matchmaker of an unexpected mutualism: Great bustard disperses and enhances emergence of domestic olive seeds

By changing the habitats and altering plant traits, agriculture has severely disrupted many plant–animal mutualisms. Interestingly, however, the intensification of agricultural practices could also facilitate mutualistic relationships between species with naturally mismatching phenotypes. We illustrate the potential of the great bustard (Otis tarda), a large steppe bird, as disperser of domestic olive (Olea europaea) seeds, originally a forest species. In an area of southwestern Spain, 30% of bustard faeces included olive stones (from 1 to 13). Only 1.7% of the bustard-ingested olive seeds were broken. Moreover, using a sowing experiment, we show bustard ingestion enhanced seedling emergence, which reached 8.8%, 3.4% and 0.0% for bustard-ingested, hand-depulped, and control seeds, respectively. As expected for Mediterranean habitats, seedling mortality was very high in the first summer for all seed treatments. In 6 out of 19 non-plowed patches within our study area, we found olive saplings of different ages likely to emerge from bustard-dispersed seeds. Given the large size of domestic olive fruits, bustards are among the few local animals able to disperse their seeds and thus to assist in the forestation of field boundaries and abandoned lands. Paradoxically, because bustards are rather restricted to open habitats, their success in shaping the habitat (i.e., ‘planting’ olive trees) could lead to their own removal from the resulting forested landscape.     

Hyaluronic acid depolymerization by ascorbate-redox effects on solid state cultivation of <Emphasis Type="Italic">Streptococcus zooepidemicus</Emphasis> in cashew apple fruit bagasse

The cashew fruit (Anacardium occidentale L.) has been used as a promising agricultural resource for the production of low-molecular weight (M_W) hyaluronic acid (HA) (10⁴–10⁵ Da). The cashew juice is a rich source of vitamin C containing, 1.2–2.0 g L⁻¹. This work explores the effects of the initial concentration of the ascorbate on the solid fermentation of the juice-moisturized bagasse from the cashew apple fruit. The results show that the M_W reduction of HA is proportional to the initial ascorbate concentration. The presence of ascorbate did not influence the Streptococcus zooepidemicus metabolism. However, the HA productivity was increased from 0.18 to 0.28 mg g⁻¹ h⁻¹ when the ascorbate concentration ranged from 1.7 to 10 mg mL⁻¹. These findings contribute to the controlled production of HA in a low M_W range, which is important in cell signalization, angiogenesis and nanoparticles production.     

Efficacy of Multivalent Adenovirus-Based Vaccine against Simian Immunodeficiency Virus Challenge

The prophylactic efficacies of several multivalent replication-incompetent adenovirus serotype 5 (Ad5) vaccines were examined in rhesus macaques using an intrarectal high-dose simian immunodeficiency virus SIVmac239 challenge model. Cohorts of Mamu-A*01⁺/B*17⁻ Indian rhesus macaques were immunized with one of several combinations of Ad5 vectors expressing Gag, Pol, Nef, and Env gp140; for comparison, a Mamu-A*01⁺ cohort was immunized using the Ad5 vector alone. There was no sign of immunological interference between antigens in the immunized animals. In general, expansion of the antigen breadth resulted in more favorable virological outcomes. In particular, the order of efficacy trended as follows: Gag/Pol/Nef/Env ≈ Gag/Pol > Gag ≈ Gag/Pol/Nef > Nef. However, the precision in ranking the vaccines based on the study results may be limited by the cohort size, and as such, may warrant additional testing. The implications of these results in light of the recent discouraging results of the phase IIb study of the trivalent Ad5 HIV-1 vaccine are discussed.There is a significant body of evidence suggesting that anti-human immunodeficiency virus type 1 (HIV-1) cellular immunity plays a prominent role in controlling viral infection and progression to disease (15, 32, 33). This stimulated substantial research into vaccines capable of eliciting this type of immunity, and several vaccine candidates (5, 6, 8-13, 22, 29-31, 35) have reached various stages of clinical development. However, the viability of this general vaccine approach was recently undermined by the findings in a phase II trial (called the Step Study) that immunization with a replication-defective adenovirus serotype 5 (Ad5) vaccine expressing HIV-1 clade B Gag, Pol, and Nef was not effective in either reducing acquisition rates and/or lowering set point viral loads in infected subjects (2, 25). In fact, more infections were originally observed in the vaccine group than in the placebo arm (2).The outcomes of the Step Study led to several important questions. Do the results argue against the concept of a HIV-1 vaccine based on the induction of specific T lymphocytes? On the other hand, if cytotoxic T-lymphocyte (CTL) responses are intrinsically valuable for an effective vaccine, what are the shortcomings in the vaccine-induced immunity that contributed to the lack of efficacy in the Step trial? What is the predictive value of preclinical challenge studies for selection of future clinical vaccine candidates? The potential role of CTL responses in an effective vaccine is also challenged by the recently reported phase III study results for the ALVAC vCP1521 prime-AIDSVAX B/E boost vaccine. The efficacy of this vaccine in a low-risk population was recently shown to trend toward prevention of HIV acquisition and not reduction of viral loads (30). Unlike the Step study vaccine, the ALVAC/AIDSVAX vaccine approach utilized a heterologous prime-boost regimen and contained an Env component that may have contributed to the type of outcome observed here. A better understanding of the immune correlates for this vaccine may be possible following further experimental investigations of the samples collected from the phase III study and earlier-stage trials.Despite the proven efficacy of Ad5 vaccination against simian-human immunodeficiency virus 89.6P (SHIV89.6P) challenge, subsequent primate studies provided equivocal results. In a homologous prime-boost regimen, Ad5 vaccine expressing Gag was ineffective against a high-dose simian immunodeficiency virus SIVmac239 challenge (4, 24). The same study compared this regimen with the DNA prime/Ad5 boost regimen that was found to be efficacious in Mamu-A*01⁺ monkeys; the level of protection in the overall study was correlated with the breadth of epitopes recognized and the frequency of induced antigen-specific CTLs. In this study, we examine whether the expansion of antigens to include Pol, Nef, and Env gp140 using the Ad5/Ad5 regimen would improve the outcome against the same high-dose SIV challenge. Of particular interest is the combination of Gag, Pol, and Nef, for which the homologous human vaccine was utilized in the Step study (29).     

Genetic impact of vaccination on breakthrough HIV-1 sequences from the STEP trial

We analyzed HIV-1 genome sequences from 68 newly infected volunteers in the STEP HIV-1 vaccine trial. To determine whether the vaccine exerted selective T cell pressure on breakthrough viruses, we identified potential T cell epitopes in the founder sequences and compared them to epitopes in the vaccine. We found greater distances to the vaccine sequence for sequences from vaccine recipients than from placebo recipients. The most significant signature site distinguishing vaccine from placebo recipients was Gag amino acid 84, a site encompassed by several epitopes contained in the vaccine and restricted by human leukocyte antigen (HLA) alleles common in the study cohort. Moreover, the extended divergence was confined to the vaccine components of the virus (HIV-1 Gag, Pol and Nef) and not found in other HIV-1 proteins. These results represent what is to our knowledge the first evidence of selective pressure from vaccine-induced T cell responses on HIV-1 infection in humans.