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Géraldine Arrode-Brusés Darlene Sheffer Ramakrishna Hegde Sukbir Dhillon Zhengian Liu Fran?ois Villinger Opendra Narayan Yahia Chebloune 《Journal of virology》2010,84(3):1243-1253
The optimization of immune responses (IR) induced by HIV DNA vaccines in humans is one of the great challenges in the development of an effective vaccine against AIDS. Ideally, this vaccine should be delivered in a single dose to immunize humans. We recently demonstrated that the immunization of mice with a single dose of a DNA vaccine derived from pathogenic SHIVKU2 (Δ4SHIVKU2) induced long-lasting, potent, and polyfunctional HIV-specific CD8+ T-cell responses (G. Arrode, R. Hegde, A. Mani, Y. Jin, Y. Chebloune, and O. Narayan, J. Immunol. 178:2318-2327, 2007). In the present work, we expanded the characterization of the IR induced by this DNA immunization protocol to rhesus macaques. Animals immunized with a single high dose of Δ4SHIVKU2 DNA vaccine were monitored longitudinally for vaccine-induced IR using multiparametric flow cytometry-based assays. Interestingly, all five immunized macaques developed broad and polyfunctional HIV-specific T-cell IR that persisted for months, with an unusual reemergence in the blood following an initial decline but in the absence of antibody responses. The majority of vaccine-specific CD4+ and CD8+ T cells lacked gamma interferon production but showed high antigen-specific proliferation capacities. Proliferative CD8+ T cells expressed the lytic molecule granzyme B. No integrated viral vector could be detected in mononuclear cells from immunized animals, and this high dose of DNA did not induce any detectable autoimmune responses against DNA. Taken together, our comprehensive analysis demonstrated for the first time the capacity of a single high dose of HIV DNA vaccine alone to induce long-lasting and polyfunctional T-cell responses in the nonhuman primate model, bringing new insights for the design of future HIV vaccines.The development of a vaccine that substantially decreases the viral load set points and reduces the transmission of HIV-1 appears to be the long-term solution to control the persistently growing epidemic of this virus in the world (10). In the past, vaccines against challenging infectious diseases, including smallpox, polio, measles, and yellow fever, have been the most effective strategies for fighting these human pandemics. However, and unlike these traditional vaccines that mostly rely on the production of neutralizing antibodies (Abs) for protection from pathogenic infections, the control of HIV infection strongly depends on the development of high-frequency, broadly targeted, polyfunctional T-cell responses specific to the virus (11, 28, 45). Live-attenuated simian immunodeficiency virus (SIV)/HIV vaccines so far have been the best inducers of potent T-cell responses that correlate with protection against AIDS following challenge with pathogenic strains in nonhuman primate (NHP) models (24, 39, 47, 61), although the exact correlates of such protection remain to be fully delineated. However, the persistence, integration, and possible reversion to pathogenic forms of these replication-competent vaccines comprise a risk that will not be acceptable for their use in humans.Instead, the use of DNA-based vaccines as a strategy to induce protective responses to control infectious diseases, including HIV-1/AIDS, is very attractive, based on its safety, the absence of infection even in immunocompromised recipients, and its capacity to induce both humoral and T-cell immune responses. For many years, numerous plasmid DNAs encoding HIV proteins have been developed and tested in animal models, and some of them have been tested in humans (14, 18, 42, 49). However, unlike that in rodents, the immune responses induced in humans and NHPs by these DNA vaccines were dramatically weak despite successive immunizations with multiple doses of DNA (30). To circumvent this limitation, new strategies currently are used to improve the immunogenicity of DNA vaccines, including the incorporation of signal-to-target dendritic cells (43), the codon optimization of HIV antigens (Ag) (14), the coexpression of adjuvant (15), and new tools that optimize the delivery of DNA in target cells in the muscle (34).We have developed a noninfectious DNA vaccine derived from the highly pathogenic SHIVKU2 expressing seven proteins of HIV under the control of the SIV 5′ long terminal repeat (LTR) promoter (35). This design mimics the natural expression of the viral proteins and leads to the formation of numerous viral-like particles that are extruded out of expressing cells (4). Repeated low-dose immunizations with this vaccine without heterologous boost protected macaques from progression to AIDS following challenge with pathogenic SHIV. However, enzyme-linked immunospot (ELISPOT) assay responses to HIV antigens before challenge were sporadic and weak (35, 54). In contrast, T-cell responses specific to HIV antigens induced by our construct in immunized mice were substantially higher (21). Using the mouse model, we developed a more sensitive immunity-monitoring assay that measures proliferative capacity, cytotoxic potential, and other immune functions (gamma interferon [IFN-γ] and interleukin-2 [IL-2] secretion) and provides more robust indications regarding the immunogenicity induced by the vaccine. We reported that the intramuscular immunization of mice with a single dose of this HIV DNA vaccine induced long-lasting and polyfunctional CD8+ T-cell responses directed against all HIV antigens expressed by the construct. Interestingly, in the absence of any additional immunization, we observed a primary peak of immune responses (IR) within 2 to 4 weeks postinfection (p.i.), followed by a contraction phase and then the late reemergence of responses after 14 to 20 weeks p.i. and lasting until the end of the experiment (more than 63 weeks p.i.). This is a typical pattern of vaccine-specific T-cell responses induced by nonpersistent vectors that progressively elicit secondary lymphoid tissue-based memory T cells as the expressed antigen becomes rare (9, 38, 58). Importantly, the major proportion of these HIV-specific CD8+ T cells was not producing IFN-γ but proliferated vigorously following antigen stimulation and produced the lytic molecule granzyme B (5). The contribution of this type of antigen-specific T cell to viral control remains to be fully elucidated.The surprising lack of efficacy of the human STEP trial conducted by Merck using the Ad-5 vectors expressing HIV antigens that elicit sustained effector T-cell responses has been disappointing for strategies designed to induce T-cell responses to prevent HIV-1 infections (29, 48, 53, 59). However, we learned from this failure that the characteristics of the T-cell responses induced by candidate vaccines could be critical for immediate as well as long-term protection (20). To address this issue, we developed a multiparametric flow-cytometric assay in the NHP model that was similar to that used in the mouse model. Using this assay, we performed a longitudinal characterization of HIV-specific T-cell IR induced in rhesus macaques immunized with a single high dose of Δ4SHIVKU2 DNA vaccine given intramuscularly (i.m.). We also assessed the antibody responses against HIV antigens. We also addressed potential safety concerns, since this is the first report using one high dose of DNA in NHP, and we tested the animals for the integration of the vaccine genome into that of the circulating mononuclear cells and assessed whether anti-DNA antibodies were produced in all immunized monkeys. 相似文献
145.
Meenakshi Bhardwaj Sanjogta Uppal Sunita Jain Pushpa Kharb Ravinder Dhillon Rajinder K. Jain 《Journal of plant biochemistry and biotechnology.》2010,19(2):255-258
A collection of male and female plants of ten Jojoba [Simmondsia chinensis (Link) Schneider] genotypes was analyzed with 50 RAPID and 55 ISSR markers to compare the efficiency and utility of these techniques for detecting genetic polymorphism. RAPID and ISSR analysis yielded 442 and 566 scorable amplified products, respectively, of which 60.7 and 69.3% were polymorphic. ISSRs revealed efficiency over RAPDs due to high EMR (effective multiplex ratio), DI (diversity index, mean PIC per primer) and MI (marker index) values. Jaccard similarity matrices among male plants, among female plants and between male and female plants of the ten jojoba genotypes varied between 0.705-0.784. Dendrograms generated by cluster analysis (UPGMA, NTSYS-pc) supported by bootstrap values using RAPID and ISSR datasets led to grouping of most of male and females genotypes in separate clusters. While pattern of clustering remained more or less same, the two dendrograms did differ with respect to the grouping of a few male and female genotypes. The value of the Mantel test shows poor correlation (r = 0.41) between ISSR and RAPID marker datasets. 相似文献
146.
Panagiotis Douvaras Richard L. Mort Dominic Edwards Kanna Ramaesh Baljean Dhillon Steven D. Morley Robert E. Hill John D. West 《PloS one》2013,8(8)
We aimed to test previous predictions that limbal epithelial stem cells (LESCs) are quantitatively deficient or qualitatively defective in Pax6+/− mice and decline with age in wild-type (WT) mice. Consistent with previous studies, corneal epithelial stripe patterns coarsened with age in WT mosaics. Mosaic patterns were also coarser in Pax6+/− mosaics than WT at 15 weeks but not at 3 weeks, which excludes a developmental explanation and strengthens the prediction that Pax6+/− mice have a LESC-deficiency. To investigate how Pax6 genotype and age affected corneal homeostasis, we compared corneal epithelial cell turnover and label-retaining cells (LRCs; putative LESCs) in Pax6+/− and WT mice at 15 and 30 weeks. Limbal BrdU-LRC numbers were not reduced in the older WT mice, so this analysis failed to support the predicted age-related decline in slow-cycling LESC numbers in WT corneas. Similarly, limbal BrdU-LRC numbers were not reduced in Pax6+/− heterozygotes but BrdU-LRCs were also present in Pax6+/− corneas. It seems likely that Pax6+/− LRCs are not exclusively stem cells and some may be terminally differentiated CD31-positive blood vessel cells, which invade the Pax6+/− cornea. It was not, therefore, possible to use this approach to test the prediction that Pax6+/− corneas had fewer LESCs than WT. However, short-term BrdU labelling showed that basal to suprabasal movement (leading to cell loss) occurred more rapidly in Pax6+/− than WT mice. This implies that epithelial cell loss is higher in Pax6+/− mice. If increased corneal epithelial cell loss exceeds the cell production capacity it could cause corneal homeostasis to become unstable, resulting in progressive corneal deterioration. Although it remains unclear whether Pax6+/− mice have LESC-deficiency, we suggest that features of corneal deterioration, that are often taken as evidence of LESC-deficiency, might occur in the absence of stem cell deficiency if corneal homeostasis is destabilised by excessive cell loss. 相似文献
147.
Van Inghelandt D Reif JC Dhillon BS Flament P Melchinger AE 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2011,123(1):11-20
Association mapping is based on linkage disequilibrium (LD) resulting from historical recombinations and helps understanding the genetic basis of complex traits. Many factors affect LD and, therefore, it must be determined empirically in the germplasm under investigation to examine the prospects of successful genome-wide association mapping. The objectives of our study were to (1) examine the extent of LD with simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers in 1,537 commercial maize inbred lines belonging to four heterotic pools, (2) compare the LD patterns determined by these two marker types, (3) evaluate the number of SNP markers needed to perform genome-wide association analyses, and (4) investigate temporal trends of LD. Mean values of the squared correlation coefficient ( $ \bar{R} $ ) were almost identical for unlinked, linked, and adjacent SSR marker pairs. In contrast, $ \bar{R} $ values were lowest for the unlinked SNP loci and highest for the SNPs within amplicons. LD decay varied across the different heterotic pools and the individual chromosomes. The SSR markers employed in the present study are not adequate for association analysis, because of insufficient marker density for the germplasm evaluated. Based on the decay of LD in the various heterotic pools, we would need between 4,000 and 65,000 SNP markers to detect with a reasonable power associations with rather large quantitative trait loci (QTL). A much higher marker density is required to identify QTL with smaller effects. However, not only the total number of markers but also their distribution among and along the chromosomes are primordial for undertaking powerful association analyses. 相似文献
148.
The genetic relationships of three Indian horse breeds—Marwari, Spiti, and Kathiawari were studied by genotyping 96 individuals
with 20 polymorphic microsatellite markers. A total of 157 alleles were detected across 20 polymorphic loci. The Marwari population
showed the highest allelic diversity (A = 5.7 and Ar = 5.14), followed by Spiti (A = 4.9 and Ar = 4.74) and Kathiawari (A = 4.1 and Ar = 3.82). The gene diversity was highest in the Spiti population (He = 0.67), followed by Marwari (He = 0.66) and Kathiawari (He = 0.59). Within population inbreeding estimates (f) in Marwari, Spiti and Kathiawari breeds were 0.18, 0.08, and 0.07, respectively, suggesting high level of inbreeding in
these breeds. Analysis of bottleneck revealed evidence of recent bottleneck in Spiti and Kathiawari populations. Pair-wise
Fst analysis, AMOVA and assignment tests demonstrated high genetic differentiation and low gene flow between populations. The
information about genetic diversity and population structure will be useful for the future development of effective breeding
management in order to preserve these Indian horse breeds. 相似文献
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150.
Dario I. Ojeda Braham Dhillon Clement K. M. Tsui Richard C. Hamelin 《Molecular ecology resources》2014,14(2):401-410
Single‐nucleotide polymorphisms (SNPs) are rapidly becoming the standard markers in population genomics studies; however, their use in nonmodel organisms is limited due to the lack of cost‐effective approaches to uncover genome‐wide variation, and the large number of individuals needed in the screening process to reduce ascertainment bias. To discover SNPs for population genomics studies in the fungal symbionts of the mountain pine beetle (MPB), we developed a road map to discover SNPs and to produce a genotyping platform. We undertook a whole‐genome sequencing approach of Leptographium longiclavatum in combination with available genomics resources of another MPB symbiont, Grosmannia clavigera. We sequenced 71 individuals pooled into four groups using the Illumina sequencing technology. We generated between 27 and 30 million reads of 75 bp that resulted in a total of 1, 181 contigs longer than 2 kb and an assembled genome size of 28.9 Mb (N50 = 48 kb, average depth = 125x). A total of 9052 proteins were annotated, and between 9531 and 17 266 SNPs were identified in the four pools. A subset of 206 genes (containing 574 SNPs, 11% false positives) was used to develop a genotyping platform for this species. Using this roadmap, we developed a genotyping assay with a total of 147 SNPs located in 121 genes using the Illumina® Sequenom iPLEX Gold. Our preliminary genotyping (success rate = 85%) of 304 individuals from 36 populations supports the utility of this approach for population genomics studies in other MPB fungal symbionts and other fungal nonmodel species. 相似文献