A Phase I Double Blind,Placebo-Controlled,Randomized Study of the Safety and Immunogenicity of an Adjuvanted HIV-1 Gag-Pol-Nef Fusion Protein and Adenovirus 35 Gag-RT-Int-Nef Vaccine in Healthy HIV-Uninfected African Adults

Background

Sequential prime-boost or co-administration of HIV vaccine candidates based on an adjuvanted clade B p24, RT, Nef, p17 fusion protein (F4/AS01) plus a non-replicating adenovirus 35 expressing clade A Gag, RT, Int and Nef (Ad35-GRIN) may lead to a unique immune profile, inducing both strong T-cell and antibody responses.

Methods

In a phase 1, double-blind, placebo-controlled trial, 146 healthy adult volunteers were randomized to one of four regimens: heterologous prime-boost with two doses of F4/AS01_E or F4/AS01_B followed by Ad35-GRIN; Ad35-GRIN followed by two doses of F4/AS01_B; or three co-administrations of Ad35-GRIN and F4/AS01_B. T cell and antibody responses were measured.

Results

The vaccines were generally well-tolerated, and did not cause serious adverse events. The response rate, by IFN-γ ELISPOT, was greater when Ad35-GRIN was the priming vaccine and in the co-administration groups. F4/AS01 induced CD4+ T-cells expressing primarily CD40L and IL2 +/- TNF-α, while Ad35-GRIN induced predominantly CD8+ T-cells expressing IFN-γ +/- IL2 or TNF-α. Viral inhibition was induced after Ad35-GRIN vaccination, regardless of the regimen. Strong F4-specific antibody responses were induced. Immune responses persisted at least a year after the last vaccination. The complementary response profiles, characteristic of each vaccine, were both expressed after co-administration.

Conclusion

Co-administration of an adjuvanted protein and an adenovirus vector showed an acceptable safety and reactogenicity profile and resulted in strong, multifunctional and complementary HIV-specific immune responses.

Trial Registration

ClinicalTrials.gov NCT01264445     

The evolution of tenascins and fibronectin

Tenascins are extracellular matrix glycoproteins that act both as integrin ligands and as modifiers of fibronectin-integrin interactions to regulate cell adhesion, migration, proliferation and differentiation. In tetrapods, both tenascins and fibronectin bind to integrins via RGD and LDV-type tripeptide motifs found in exposed loops in their fibronectin-type III domains. We previously showed that tenascins appeared early in the chordate lineage and are represented by single genes in extant cephalochordates and tunicates. Here we have examined the genomes of the coelacanth Latimeria chalumnae, the elephant shark Callorhinchus milii as well as the lampreys Petromyzon marinus and Lethenteron japonicum to learn more about the evolution of the tenascin gene family as well as the timing of the appearance of fibronectin during chordate evolution. The coelacanth has 4 tenascins that are more similar to tetrapod tenascins than are tenascins from ray-finned fishes. In contrast, only 2 tenascins were identified in the elephant shark and the Japanese lamprey L. japonicum. An RGD motif exposed to integrin binding is observed in tenascins from many, but not all, classes of chordates. Tetrapods that lack this RGD motif in tenascin-C have a similar motif in the paralog tenascin-W, suggesting the potential for some overlapping function. A predicted fibronectin with the same domain organization as the fibronectin from tetrapods is found in the sea lamprey P. marinus but not in tunicates, leading us to infer that fibronectin first appeared in vertebrates. The motifs that recognize LDV-type integrin receptors are conserved in fibronectins from a broad spectrum of vertebrates, but the RGD integrin-binding motif may have evolved in gnathostomes.     

Host‐associated differentiation in a highly polyphagous,sexually reproducing insect herbivore

Insect herbivores may undergo genetic divergence on their host plants through host‐associated differentiation (HAD). Much of what we know about HAD involves insect species with narrow host ranges (i.e., specialists) that spend part or all their life cycle inside their hosts, and/or reproduce asexually (e.g., parthenogenetic insects), all of which are thought to facilitate HAD. However, sexually reproducing polyphagous insects can also exhibit HAD. Few sexually reproducing insects have been tested for HAD, and when they have insects from only a handful of potential host‐plant populations have been tested, making it difficult to predict how common HAD is when one considers the entire species' host range. This question is particularly relevant when considering insect pests, as host‐associated populations may differ in traits relevant to their control. Here, we tested for HAD in a cotton (Gossypium hirsutum) pest, the cotton fleahopper (CFH) (Pseudatomoscelis seriatus), a sexually reproducing, highly polyphagous hemipteran insect. A previous study detected one incidence of HAD among three of its host plants. We used Amplified fragment length polymorphism (AFLP) markers to assess HAD in CFH collected from an expanded array of 13 host‐plant species belonging to seven families. Overall, four genetically distinct populations were found. One genetically distinct genotype was exclusively associated with one of the host‐plant species while the other three were observed across more than one host‐plant species. The relatively low degree of HAD in CFH compared to the pea aphid, another hemipteran insect, stresses the likely importance of sexual recombination as a factor increasing the likelihood of HAD.     

Effects of cerebrolysin on motor-neuron-like NSC-34 cells

Although the peripheral nervous system is capable of regeneration, this capability is limited. As a potential means of augmenting nerve regeneration, the effects of cerebrolysin (CL) – a proteolytic peptide fraction – were tested in vitro on the motor-neuron-like NSC-34 cell line and organotypic spinal cord cultures. Therefore, NSC-34 cells were subjected to mechanical stress by changing media and metabolic stress by oxygen glucose deprivation. Afterwards, cell survival/proliferation using MTT and BrdU-labeling (FACS) and neurite sprouting using ImageJ analysis were evaluated. Calpain-1, Src and α-spectrin protein expression were analyzed by Western blot. In organotypic cultures, the effect of CL on motor neuron survival and neurite sprouting was tested by immunohistochemistry.     

Pancreatic polypeptide inhibits somatostatin secretion

Pancreatic polypeptide (PP) is a major agonist for neuropeptide Y4 receptors (NPY4R). While NPY4R has been identified in various tissues, the cells on which it is expressed and its function in those cells has not been clearly delineated. Here we report that NPY4R is present in all somatostatin-containing cells of tissues that we tested, including pancreatic islets, duodenum, hippocampus, and hypothalamus. Its agonism by PP decreases somatostatin secretion from human islets. Mouse embryonic hippocampal (mHippo E18) cells expressed NPY4Rs and their activation by PP consistently decreased somatostatin secretion. Furthermore, central injection of PP in mice induced c-Fos immunoreactivity in somatostatin-containing cells in the hippocampus compared with PBS-injected mice. In sum, our results identify PP as a pivotal modulator of somatostatin secretion.     

C-type natriuretic peptide (CNP) signal peptide fragments are present in the human circulation

Background

Signal peptides may be novel biomarkers in cardiovascular diseases.

Methods

We developed a novel immunoassay to the signal peptide of preproCNP (CNPsp) and used this to document circulating venous concentrations of CNPsp in normal healthy volunteers (n = 109), regional plasma CNPsp concentrations in patients undergoing clinically indicated catheterisation (n = 24) and temporal CNPsp concentrations in patients with ST-elevation myocardial infarction (STEMI) <4 h after symptom onset (n = 8). The structure/sequence of circulating CNPsp was confirmed by tandem mass spectrometry (MS/MS).

Results

In normal human plasma, CNPsp was detectable at levels higher than NT-proCNP (74 ± 17 vs. 20 ± 5.5 pmol/L). There was no correlation between NTproCNP and CNPsp, but plasma concentrations of sibling signal peptides – CNPsp and BNPsp – were strongly correlated (r = 0.532, P < 0.001). In patients undergoing catheterisation, there were significant arterio-venous step-ups in CNPsp concentrations across the heart (P < 0.01) and kidney (P < 0.01). Arterial concentrations of CNPsp significantly correlated with heart rate (r = 0.446, P < 0.05). In STEMI patients, plasma concentrations of CNPsp showed a biphasic elevation pattern between 6 and 12 h after symptom onset, with 12 h values significantly elevated (∼3-fold) compared with levels at presentation (P < 0.05). MS/MS verified circulating CNPsp to be preproCNP(14–23) and preproCNP(16–23) peptides.

Conclusions

This is the first report of a circulating preproCNP derived signal peptide. Given the clear cardiac and renal secretion profiles of CNPsp and its response in STEMI patients, further studies on potential biological functions and biomarker applications of CNPsp in cardiovascular disease are warranted.     

Proteomic identification of interactions between histones and plasma proteins: Implications for cytoprotection

Extracellular histones released from cells during acute inflammation contribute to organ failure and death in a mouse model of sepsis, and histones are known to exert in vitro cytotoxicity in the absence of serum. Since addition of histones to serum and plasma is known to induce protein aggregation, we reasoned that plasma proteins may afford protection from cytotoxicity. We found that MODE‐K mouse small intestinal epithelial cells were protected from histone‐induced toxicity in the presence of 10% FCS. Therefore, the main aim of this study was to identify histone‐interacting plasma proteins that might be involved in cytoprotection. The precipitate formed following addition of calf thymus histones to human EDTA plasma was characterised by shotgun proteomics, identifying a total of 36 protein subunits, including complement components, coagulation factors, protease inhibitors and apolipoproteins. The highly sulphated glycosaminoglycan heparin inhibited histone‐induced plasma protein aggregation. Moreover, histones bound to heparin agarose were capable of pulling down plasma proteins from solution, indicating their effective cross‐linking properties. It was particularly notable that inter‐α‐trypsin inhibitor was prominent among the histone‐precipitated proteins, since it contains a chondroitin sulphate glycan chain, and suggests a potential role for this protein in histone sequestration during acute inflammation in vivo.     

Development of a host-genotype-independent counterselectable marker and a high-frequency conjugative delivery system and their use in genetic analysis of Enterococcus faecalis

Enterococcus faecalis is a gram-positive commensal bacterium of the gastrointestinal tract. E. faecalis is also an opportunistic pathogen that frequently exhibits resistance to available antibiotics. Despite the clinical significance of the enterococci, genetic analysis has been restricted by limitations inherent in the available genetic tools. To facilitate genetic manipulation of E. faecalis, we developed a conjugative delivery system for high-frequency introduction of cloned DNA into target strains of E. faecalis and a host-genotype-independent counterselectable marker for use in markerless genetic exchange. We used these tools to construct a collection of E. faecalis mutant strains carrying defined mutations in several genes, including ccfA, eep, gelE, sprE, and an alternative sigma factor (sigH). Furthermore, we combined these mutations in various permutations to create double mutants, triple mutants, and a quadruple mutant of E. faecalis that enabled tests of epistasis to be conducted on the pheromone biosynthesis pathway. Analysis of cCF10 pheromone production by the mutants revealed that both the ccfA2 and delta eep10 mutations are epistatic to mutations in gelE/sprE. To our knowledge, this represents the first example of epistasis analysis applied to a chromosomally encoded biosynthetic pathway in enterococci. Thus, the advanced tools for genetic manipulation of E. faecalis reported here enable efficient and sophisticated genetic analysis of these important pathogens.     

Development of a novel method to determine very low density lipoprotein kinetics

Isotopic tracer methods of determining triglyceride-rich lipoprotein (TRL) kinetics are costly, time-consuming, and labor-intensive. This study aimed to develop a simpler and cost-effective method of obtaining TRL kinetic data, based on the fact that chylomicrons compete with large VLDL (VLDL(1); S(f) = 60-400) for the same catalytic pathway. Ten healthy subjects [seven men; fasting triglyceride (TG), 44.3-407.6 mg/dl; body mass index, 21-35 kg/m(2)] were given an intravenous infusion of a chylomicron-like TG emulsion (Intralipid; 0.1 g/kg bolus followed by 0.1 g/kg/h infusion) for 75-120 min to prevent the clearance of VLDL(1) by lipoprotein lipase. Multiple blood samples were taken during and after infusion for separation of Intralipid, VLDL(1), and VLDL(2) by ultracentrifugation. VLDL(1)-apolipoprotein B (apoB) and TG production rates were calculated from their linear increases in the VLDL(1) fraction during the infusion. Intralipid-TG clearance rate was determined from its exponential decay after infusion. The production rates of VLDL(1)-apoB and VLDL(1)-TG were (mean +/- SEM) 25.4 +/- 3.9 and 1,076.7 +/- 224.7 mg/h, respectively, and the Intralipid-TG clearance rate was 66.9 +/- 11.7 pools/day. Kinetic data obtained from this method agree with values obtained from stable isotope methods and show the expected relationships with indices of body fatness and insulin resistance (all P < 0.05). The protocol is relatively quick, inexpensive, and transferable to nonspecialist laboratories.