Effect of preexisting immunity to adenovirus human serotype 5 antigens on the immune responses of nonhuman primates to vaccine regimens based on human- or chimpanzee-derived adenovirus vectors

In this study we compared a prime-boost regimen with two serologically distinct replication-defective adenovirus (Ad) vectors derived from chimpanzee serotypes C68 and C1 expressing Gag, Pol, gp140, and Nef of human immunodeficiency virus type 1 with a regimen in which replication-defective Ad vectors of the human serotype 5 (AdHu5) were given twice. Experiments were conducted in rhesus macaques that had or had not been preexposed to antigens of AdHu5. There was no significant difference in T-cell responses tested from peripheral blood of the different groups, although responses were overall highest in nonpreexposed animals immunized with the chimpanzee Ad vectors. Preexisting immunity to AdHu5 completely inhibited induction of transgene product-specific antibodies by the AdHu5 vectors without affecting antibody responses to the chimpanzee vectors. Upon euthanasia, T-cell responses were tested from a number of tissues. Preexisting immunity to AdHu5, commonly found in humans, changed the homing pattern of vaccine-induced T cells. In AdHu5-preexposed animals vaccinated with the chimpanzee Ad vectors, frequencies of transgene-specific T cells were higher in spleens than in blood, and in most preexposed animals vaccinated either with AdHu5 vectors or chimpanzee adenovirus vectors, frequencies of such T cells were exceptionally high in livers. The latter results indicate that analysis of T-cell responses solely from blood mononuclear cells of vaccine recipients may not suffice to compare the potencies of different vaccine regimens.     

Text-derived concept profiles support assessment of DNA microarray data for acute myeloid leukemia and for androgen receptor stimulation

Background  

High-throughput experiments, such as with DNA microarrays, typically result in hundreds of genes potentially relevant to the process under study, rendering the interpretation of these experiments problematic. Here, we propose and evaluate an approach to find functional associations between large numbers of genes and other biomedical concepts from free-text literature. For each gene, a profile of related concepts is constructed that summarizes the context in which the gene is mentioned in literature. We assign a weight to each concept in the profile based on a likelihood ratio measure. Gene concept profiles can then be clustered to find related genes and other concepts.     

Lymphokines induce specificity degradation in virus-induced cytolytic T-cell clones

The target cell specificity of antiviral cytolytic T-lymphocyte (CTL) clones is influenced by in vitro tissue culture conditions. We have demonstrated that high concentrations of lymphokines such as recombinant interleukin-2 (rIL-2) induce killer T cells to lose virus specificity. Our experiments do not indicate that the cells become like natural killer (NK) cells. The loss of specificity is more general and can be reversed upon culture in more physiological concentrations of lymphokines. The implications of these observations for the pathogenesis of viral infection is discussed.     

Liver microsomal cytochromes P450‐dependent alkoxyphenoxazone O‐dealkylation in vitro by alligator and rat: Activities,inhibition, substrate preference,and discrimination factors

Six substituted alkoxyphenoxazones (resorufins) and four inhibitors of P450‐dependent mixed‐function oxygenases (MFO) were used to probe the breadth and extent of P450 metabolism induced by pretreatment with five xenobiotic chemicals in liver microsomes of the American alligator, Alligator mississippiensis. Phenobarbital (PB), 3‐methylcholanthrene (3MC), and PB–3MC co‐pretreatment elicited major induction of alligator MFO activity measured by alkoxyresorufin O‐dealkylation (AROD). The induced levels of activities observed with appropriate substrate, 7‐ethoxy, 7‐methoxy, 2‐phenylbenzyloxy, 7‐pentoxy, or 7‐benzyloxyresorufin (EROD, MROD, PBROD, PROD and BROD, respectively), were 10 to 100 times lower in alligator as compared to rat. The exception was a higher level of isopropoxyresorufin O‐dealkylation (IPROD) in alligator. The induction regimes used in alligator and rat revealed marked differences in substrate preference, discrimination factors (DF) for various inducible P450 isoforms. EROD, a classic indicator of CYP1A activity in rat, had a low DF in alligator. MROD was the best discriminator in alligator of CYP1A‐type induction. In contrast to rats, pretreatment of alligators with Aroclor 1254, 2,2′,4,4′ tetrachlorobiphenyl, and clofibrate caused minor alterations in AROD relative to untreated controls. The inhibitors, α‐napthaflavone, 1‐ethynylpyrene, SKF 525A, and 9‐ethynylphenanthrene, inhibited AROD activity of the expected P450 isoform. For example, 10 μM α‐napthaflavone inhibited liver microsomal EROD catalyzed by 3MC‐inducible isoforms from alligator by 90% and from rat by 97%. Similarly, 10 μM SKF 525A inhibited PROD catalyzed by PB‐inducible isoforms by 63% and 79% in alligator and rat liver microsomes, respectively. To the best of our knowledge, the present studies are the first to show PB induction of P450 activities typical of the mammalian CYP2 family and their inhibition with classical inhibitors in alligator liver. While our data indicate metabolism of P450 substrates with preferences to certain isoforms, it remains to be established which isoforms exert catalytic function in alligator and whether these are homologues or orthologues of mammalian isoforms. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 13: 17–27, 1999     

Induction of genital immunity by DNA priming and intranasal booster immunization with a replication-defective adenoviral recombinant.

Mice immunized through different routes such as i.m., intradermally, or intratracheally with a DNA vaccine to rabies virus developed high titers of serum Ab but only borderline levels of mucosal Abs determined from vaginal secretions. DNA vaccines given by either route enhanced vaginal IgA and IgG2a secretion upon a subsequent intranasal booster immunization with an E1-deleted adenoviral recombinant expressing the same Ag of rabies virus. DNA vaccine priming reduced the Ab response to the adenoviral Ags and counterbalanced the impaired B cell response to the rabies virus Ag expressed by the adenoviral recombinant in mice preimmune to adenovirus. The vaginal B cell response could further be enhanced by using the Th2-type cytokines IL-4 or IL-5 as genetic adjuvants concomitantly with the DNA vaccine before intranasal booster immunization with the recombinant vaccine.     

Investigating the utility of combining Φ29 whole genome amplification and highly multiplexed single nucleotide polymorphism BeadArray™ genotyping

Background

Sustainable DNA resources and reliable high-throughput genotyping methods are required for large-scale, long-term genetic association studies. In the genetic dissection of common disease it is now recognised that thousands of samples and hundreds of thousands of markers, mostly single nucleotide polymorphisms (SNPs), will have to be analysed. In order to achieve these aims, both an ability to boost quantities of archived DNA and to genotype at low costs are highly desirable. We have investigated Φ29 polymerase Multiple Displacement Amplification (MDA)-generated DNA product (MDA product), in combination with highly multiplexed BeadArray? genotyping technology. As part of a large-scale BeadArray genotyping experiment we made a direct comparison of genotyping data generated from MDA product with that from genomic DNA (gDNA) templates.

Results

Eighty-six MDA product and the corresponding 86 gDNA samples were genotyped at 345 SNPs and a concordance rate of 98.8% was achieved. The BeadArray sample exclusion rate, blind to sample type, was 10.5% for MDA product compared to 5.8% for gDNA.

Conclusions

We conclude that the BeadArray technology successfully produces high quality genotyping data from MDA product. The combination of these technologies improves the feasibility and efficiency of mapping common disease susceptibility genes despite limited stocks of gDNA samples.