Generation and evaluation of clade C simian-human immunodeficiency virus challenge stocks.

UNLABELLED: The development of a panel of mucosally transmissible simian-human immunodeficiency virus (SHIV) challenge stocks from multiple virus clades would facilitate preclinical evaluation of candidate HIV-1 vaccines and therapeutics. The majority of SHIV stocks that have been generated to date have been derived from clade B HIV-1 env sequences from viruses isolated during chronic infection and typically required serial animal-to-animal adaptation for establishing mucosal transmissibility and pathogenicity. To capture essential features of mucosal transmission of clade C viruses, we produced a series of SHIVs with early clade C HIV-1 env sequences from acutely HIV-1-infected individuals from South Africa. SHIV-327c and SHIV-327cRM expressed env sequences that were 99.7 to 100% identical to the original HIV-1 isolate and did not require in vivo passaging for mucosal infectivity. These challenge stocks infected rhesus monkeys efficiently by both intrarectal and intravaginal routes, replicated to high levels during acute infection, and established chronic setpoint viremia in 13 of 17 (76%) infected animals. The SHIV-327cRM challenge stock was also titrated for both single, high-dose intrarectal challenges and repetitive, low-dose intrarectal challenges in rhesus monkeys. These SHIV challenge stocks should facilitate the preclinical evaluation of vaccines and other interventions aimed at preventing clade C HIV-1 infection. IMPORTANCE: We describe the development of two related clade C SHIV challenge stocks. These challenge stocks should prove useful for preclinical testing of vaccines and other interventions aimed at preventing clade C HIV-1 infection.

A multivalent clade C HIV-1 Env trimer cocktail elicits a higher magnitude of neutralizing antibodies than any individual component.

UNLABELLED: The sequence diversity of human immunodeficiency virus type 1 (HIV-1) presents a formidable challenge to the generation of an HIV-1 vaccine. One strategy to address such sequence diversity and to improve the magnitude of neutralizing antibodies (NAbs) is to utilize multivalent mixtures of HIV-1 envelope (Env) immunogens. Here we report the generation and characterization of three novel, acute clade C HIV-1 Env gp140 trimers (459C, 405C, and 939C), each with unique antigenic properties. Among the single trimers tested, 459C elicited the most potent NAb responses in vaccinated guinea pigs. We evaluated the immunogenicity of various mixtures of clade C Env trimers and found that a quadrivalent cocktail of clade C trimers elicited a greater magnitude of NAbs against a panel of tier 1A and 1B viruses than any single clade C trimer alone, demonstrating that the mixture had an advantage over all individual components of the cocktail. These data suggest that vaccination with a mixture of clade C Env trimers represents a promising strategy to augment vaccine-elicited NAb responses. IMPORTANCE: It is currently not known how to generate potent NAbs to the diverse circulating HIV-1 Envs by vaccination. One strategy to address this diversity is to utilize mixtures of different soluble HIV-1 envelope proteins. In this study, we generated and characterized three distinct, novel, acute clade C soluble trimers. We vaccinated guinea pigs with single trimers as well as mixtures of trimers, and we found that a mixture of four trimers elicited a greater magnitude of NAbs than any single trimer within the mixture. The results of this study suggest that further development of Env trimer cocktails is warranted.

Population genetic data for 17 STR markers from Lebanon.

Seventeen autosomal STRs were analyzed (D2S1338, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D19S433, D21S11, CSF1PO, FGA, TH01, TPOX, vWA, Penta D, and Penta E) in the Lebanese population. A total of 192 unrelated individuals were genotyped for the 15 autosomal STRs in the Promega PowerPlex 16 STR kit. An additional 275 unrelated individuals were genotyped for the Applied Biosystems AmpFlSTR Identifiler and SGM+STR kits. Allele frequencies for the shared CODIS 13 loci among the three STR kits tested were not significantly different among individuals within the Lebanese population. Forensic and population genetic parameters for the 17 loci were calculated. We also compared the allele frequencies from this population with other populations in the same geographic vicinity.

Mitochondrial DNA control region variation in a population sample from Hong Kong, China.

Entire mitochondrial control region sequences were generated from 377 unrelated individuals from urban Hong Kong. In line with other control region datasets from China, the sample from Hong Kong exhibited significant genetic diversity that was reflected in a random match probability of 0.19% and a mean pairwise difference of 13.14. A total of 305 haplotypes were identified, of which 262 were unique. These sequences will be made publicly available to serve as forensic mtDNA reference data for China.

Mitochondrial control region sequences from an Egyptian population sample.

Entire mitochondrial control region data was generated for 277 unrelated Egyptian individuals. High-throughput robotics, a redundant sequencing approach, and several quality control checks were implemented to generate a high-quality database. The data presented here will augment the limited Egyptian mtDNA reference data currently available for forensic comparisons.

Investigation of heteroplasmy in the human mitochondrial DNA control region: a synthesis of observations from more than 5000 global population samples.

Instances of point and length heteroplasmy in the mitochondrial DNA control region were compiled and analyzed from over 5,000 global human population samples. These data represent observations from a large and broad population sample, representing nearly 20 global populations. As expected, length heteroplasmy was frequently observed in the HVI, HVII and HVIII C-stretches. Length heteroplasmy was also observed in the AC dinucleotide repeat region, as well as other locations. Point heteroplasmy was detected in approximately 6% of all samples, and while the vast majority of heteroplasmic samples comprised two molecules differing at a single position, samples exhibiting two and three mixed positions were also observed in this data set. In general, the sites at which heteroplasmy was most commonly observed correlated with reported control region mutational hotspots. However, for some sites, observations of heteroplasmy did not mirror established mutation rate data, suggesting the action of other mechanisms, both selective and neutral. Interestingly, these data indicate that the frequency of heteroplasmy differs between particular populations, perhaps reflecting variable mutation rates among different mtDNA lineages and/or artifacts of particular population groups. The results presented here contribute to our general understanding of mitochondrial DNA control region heteroplasmy and provide additional empirical information on the mechanisms contributing to mtDNA control region mutation and evolution.

Characterization of neutralizing antibody responses elicited by clade A envelope immunogens derived from early transmitted viruses.

The vast majority of studies with candidate immunogens based on the human immunodeficiency virus envelope (Env) have been conducted with Env proteins derived from clade B viruses isolated during chronic infection. Whether non-clade B Env protein immunogens will elicit antibodies with epitope specificities that are similar to those of antibodies elicited by clade B Envs and whether the antibodies elicited by Envs derived from early transmitted viruses will be similar to those elicited by Envs derived from viruses isolated during chronic infection are currently unknown. Here we performed immunizations with four clade A Envs, cloned directly from the peripheral blood of infected individuals during acute infection, which differed in lengths and extents of glycosylation. The antibody responses elicited by these four Envs were compared to each other and to those elicited by a well-characterized clade B Env immunogen derived from the SF162 virus, which was isolated during chronic infection. Only one clade A Env, the one with the fewer glycosylation sites, elicited homologous neutralizing antibodies (NAbs); these did not target the V1, V2, or V3 regions. In contrast, all four clade A Envs elicited anti-V3 NAbs against "easy-to-neutralize" clade B and clade A isolates, irrespective of the variable region length and extent of glycosylation of the Env used as an immunogen. These anti-V3 NAbs did not access their epitopes on homologous and heterologous clade A, or B, neutralization-resistant viruses. The length and extent of glycosylation of the variable regions on the clade A Env immunogens tested did not affect the breadth of the elicited NAbs. Our data also indicate that the development of cross-reactive NAbs against clade A viruses faces similar hurdles to the development of cross-reactive anti-clade B NAbs.

Mitochondrial control region sequences from northern Greece and Greek Cypriots.

Entire mitochondrial control region data were generated for population samples of 319 unrelated individuals from northern Greece and 91 unrelated individuals from Cyprus. The samples from northern Greece have been previously typed for 15 nuclear short tandem repeat (STR; Kovatsi et al., Forensic Sci. Int. 159:61-63, 2006).

Development and expansion of high-quality control region databases to improve forensic mtDNA evidence interpretation.

In an effort to increase the quantity, breadth and availability of mtDNA databases suitable for forensic comparisons, we have developed a high-throughput process to generate approximately 5000 control region sequences per year from regional US populations, global populations from which the current US population is derived and global populations currently under-represented in available forensic databases. The system utilizes robotic instrumentation for all laboratory steps from pre-extraction through sequence detection, and a rigorous eight-step, multi-laboratory data review process with entirely electronic data transfer. Over the past 3 years, nearly 10,000 control region sequences have been generated using this approach. These data are being made publicly available and should further address the need for consistent, high-quality mtDNA databases for forensic testing.

Involvement of HTLV-I Tax and CREB in aneuploidy: a bioinformatics approach.

BACKGROUND: Adult T-cell leukemia (ATL) is a complex and multifaceted disease associated with human T-cell leukemia virus type 1 (HTLV-I) infection. Tax, the viral oncoprotein, is considered a major contributor to cell cycle deregulation in HTLV-I transformed cells by either directly disrupting cellular factors (protein-protein interactions) or altering their transcription profile. Tax transactivates these cellular promoters by interacting with transcription factors such as CREB/ATF, NF-kappaB, and SRF. Therefore by examining which factors upregulate a particular set of promoters we may begin to understand how Tax orchestrates leukemia development. RESULTS: We observed that CTLL cells stably expressing wild-type Tax (CTLL/WT) exhibited aneuploidy as compared to a Tax clone deficient for CREB transactivation (CTLL/703). To better understand the contribution of Tax transactivation through the CREB/ATF pathway to the aneuploid phenotype, we performed microarray analysis comparing CTLL/WT to CTLL/703 cells. Promoter analysis of altered genes revealed that a subset of these genes contain CREB/ATF consensus sequences. While these genes had diverse functions, smaller subsets of genes were found to be involved in G2/M phase regulation, in particular kinetochore assembly. Furthermore, we confirmed the presence of CREB, Tax and RNA Polymerase II at the p97Vcp and Sgt1 promoters in vivo through chromatin immunoprecipitation in CTLL/WT cells. CONCLUSION: These results indicate that the development of aneuploidy in Tax-expressing cells may occur in response to an alteration in the transcription profile, in addition to direct protein interactions.

The HTLV-I Tax oncoprotein targets the retinoblastoma protein for proteasomal degradation.

Human T-cell leukemia virus type-I (HTLV-I), the etiologic agent of adult T-cell leukemia (ATL), is estimated to affect 10-20 million people worldwide. The transforming ability of HTLV-I has been largely attributed to the viral protein Tax, which modulates the activity of several well-known cell cycle regulators. An important cell cycle regulator, the retinoblastoma (Rb) protein, is often inactivated in many cancers including virally induced cancers. Upon examination of Rb status, we observed a decrease in Rb protein expression in HTLV-1-infected cell lines as well as in ex vivo ATL patient samples. Transient transfection assays indicated that decreased Rb protein levels were Tax dependent. Here, we demonstrate for the first time that Tax directly associates with Rb. This interaction was localized within the B pocket of Rb and the C-terminus of Tax (aa 245-353). Within the C-terminus of Tax, we have identified an LXCXE-like motif, that when mutated resulted in the loss of Tax/Rb interaction. Furthermore, through the use of proteasome inhibitors, such as MG-132, in vivo and proteasome degradation assays in vitro, we found that Tax destabilizes the hypo-phosphorylated (active) form of Rb via the proteasome pathway. Therefore, we propose a model whereby Tax targets Rb to the proteasome by acting as a molecular bridge bringing Rb into contact with the proteasome for degradation.

Mechanisms of HTLV-1 transformation.

HTLV-1 is the etiological agent of the fatal disease adult T-cell leukemia. The virus encodes many proteins including several accessory proteins, p12I, p13II, p27I, and p30II, whose roles have recently begun to be elucidated. These accessory proteins are important in T-cell activation, transcriptional regulation, viral persistence, and virus assembly. The viral oncogene Tax is thought to be largely responsible for tumorigenesis, although the precise mechanisms underlying transformation are not completely understood. Tax has a profound impact on transcription, cell growth regulation, genomic stability and apoptosis. This review will provide possible contributions of the accessory proteins to transformation as well as highlight the alterations of the above-mentioned cellular events by Tax. Animal models of both Tax and the accessory proteins are also included based on the essential information on the transformation process in vivo that they provide.

The role of cyclin D2 and p21/waf1 in human T-cell leukemia virus type 1 infected cells.

BACKGROUND: The human T-cell leukemia virus type 1 (HTLV-1) Tax protein indirectly influences transcriptional activation, signal transduction, cell cycle control, and apoptosis. The function of Tax primarily relies on protein-protein interactions. We have previously shown that Tax upregulates the cell cycle checkpoint proteins p21/waf1 and cyclin D2. Here we describe the consequences of upregulating these G1/S checkpoint regulators in HTLV-1 infected cells. RESULTS: To further decipher any physical and functional interactions between cyclin D2 and p21/waf1, we used a series of biochemical assays from HTLV-1 infected and uninfected cells. Immunoprecipitations from HTLV-1 infected cells showed p21/waf1 in a stable complex with cyclin D2/cdk4. This complex is active as it phosphorylates the Rb protein in kinase assays. Confocal fluorescent microscopy indicated that p21/waf1 and cyclin D2 colocalize in HTLV-1 infected, but not in uninfected cells. Furthermore, in vitro kinase assays using purified proteins demonstrated that the addition of p21/waf1 to cyclin D2/cdk4 increased the kinase activity of cdk4. CONCLUSION: These data suggest that the p21/cyclin D2/cdk4 complex is not an inhibitory complex and that p21/waf1 could potentially function as an assembly factor for the cyclin D2/cdk4 complex in HTLV-1 infected cells. A by-product of this assembly with cyclin D2/cdk4 is the sequestration of p21/waf1 away from the cyclin E/cdk2 complex, allowing this active cyclin-cdk complex to phosphorylate Rb pocket proteins efficiently and push cells through the G1/S checkpoint. These two distinct functional and physical activities of p21/waf1 suggest that RNA tumor viruses manipulate the G1/S checkpoint by deregulating cyclin and cdk complexes.