Structure and possible function of a G-quadruplex in the long terminal repeat of the proviral HIV-1 genome.

The long terminal repeat (LTR) of the proviral human immunodeficiency virus (HIV)-1 genome is integral to virus transcription and host cell infection. The guanine-rich U3 region within the LTR promoter, previously shown to form G-quadruplex structures, represents an attractive target to inhibit HIV transcription and replication. In this work, we report the structure of a biologically relevant G-quadruplex within the LTR promoter region of HIV-1. The guanine-rich sequence designated LTR-IV forms a well-defined structure in physiological cationic solution. The nuclear magnetic resonance (NMR) structure of this sequence reveals a parallel-stranded G-quadruplex containing a single-nucleotide thymine bulge, which participates in a conserved stacking interaction with a neighboring single-nucleotide adenine loop. Transcription analysis in a HIV-1 replication competent cell indicates that the LTR-IV region may act as a modulator of G-quadruplex formation in the LTR promoter. Consequently, the LTR-IV G-quadruplex structure presented within this work could represent a valuable target for the design of HIV therapeutics.

Low Proviral Load is Associated with Indeterminate Western Blot Patterns in Human T-Cell Lymphotropic Virus Type 1 Infected Individuals: Could Punctual Mutations be Related?

BACKGROUND: indeterminate Western blot (WB) patterns are a major concern for diagnosis of human T-cell lymphotropic virus type 1 (HTLV-1) infection, even in non-endemic areas. OBJECTIVES: (a) to define the prevalence of indeterminate WB among different populations from Argentina; (b) to evaluate if low proviral load (PVL) is associated with indeterminate WB profiles; and (c) to describe mutations in LTR and tax sequence of these cases. RESULTS: Among 2031 samples, 294 were reactive by screening. Of them, 48 (16.3%) were WB indeterminate and of those 15 (31.3%) were PCR+. Quantitative real-time PCR (qPCR) was performed to 52 HTLV-1+ samples, classified as Group 1 (G1): 25 WB+ samples from individuals with pathologies; Group 2 (G2): 18 WB+ samples from asymptomatic carriers (AC); and Group 3 (G3): 9 seroindeterminate samples from AC. Median PVL was 4.78, 2.38, and 0.15 HTLV-1 copies/100 PBMCs, respectively; a significant difference (p=0.003) was observed. Age and sex were associated with PVL in G1 and G2, respectively. Mutations in the distal and central regions of Tax Responsive Elements (TRE) 1 and 2 of G3 were observed, though not associated with PVL.The 8403A>G mutation of the distal region, previously related to high PVL, was absent in G3 but present in 50% of WB+ samples (p = 0.03). CONCLUSIONS: indeterminate WB results confirmed later as HTLV-1 positive may be associated with low PVL levels. Mutations in LTR and tax are described; their functional relevance remains to be determined.

The first full-length endogenous hepadnaviruses: identification and analysis.

In silico screening of metazoan genome data identified multiple endogenous hepadnaviral elements in the budgerigar (Melopsittacus undulatus) genome, most notably two elements comprising about 1.3 × and 1.0 × the full-length genome. Phylogenetic and molecular dating analyses show that endogenous budgerigar hepatitis B viruses (eBHBV) share an ancestor with extant avihepadnaviruses and infiltrated the budgerigar genome millions of years ago. Identification of full-length genomes with preserved key features like ε signals could enable resurrection of ancient BHBV.

Analysis of the molecular and regulatory properties of active porcine endogenous retrovirus gamma-1 long terminal repeats in kidney tissues of the NIH-Miniature pig.

The pig genome contains the gamma 1 family of porcine endogenous retroviruses (PERVs), which are a major obstacle to the development of successful xenotransplantation from pig to human. Long terminal repeats (LTRs) found in PERVs are known to be essential elements for the control of the transcriptional activity of single virus by different transcription factors (TFs). To identify transcribed PERV LTR elements, RT-PCR and DNA sequencing analyses were performed. Twenty-nine actively transcribed LTR elements were identified in the kidney tissues of the NIH-Miniature pig. These elements were divided into two major groups (I and II), and four minor groups (I-1, I-2, I-3, and II-1), by the presence of insertion and deletion (INDEL) sequences. Group I elements showed strong transcriptional activity compared to group II elements. Four different LTR elements (PL1, PL2, PL3, and PL4) as representative of the groups were analyzed by using a transient transfection assay. The regulation of their promoter activity was investigated by treatment with M.SssI (CpG DNA methyltransferase) and garcinol (histone acetyltransferase inhibitor). The transcriptional activity of PERV LTR elements was significantly reduced by treatment with M.SssI. These data indicate that transcribed PERV LTR elements harbor sufficient promoter activity to regulate the transcription of a single virus, and the transcriptional activity of PERV LTRs may be controlled by DNA methylation events.

Quantification of endogenous and exogenous feline leukemia virus sequences by real-time PCR assays.

Endogenous retroviruses are integrated in the genome of most vertebrates. They represent footprints of ancient retroviral infection and are vertically transmitted from parents to their offspring. In the genome of all domestic cats, sequences closely related to exogenous FeLV known as endogenous feline leukemia virus (enFeLV), are present. enFeLV are incapable of giving rise to infectious virus particles. However, transcription and translation of enFeLV have been demonstrated in tissues of healthy cats and in feline cell lines. The presence of enFeLV-env has been shown in specific embryonic tissues and adult thymic cells. In addition, the enFeLV-env region recombines with FeLV subgroup A giving rise to an infectious FeLV-B virus. enFeLV envelope protein, FeLIX (FeLV infectivity X-essory protein) is also involved in mediating FeLV-T infection. In order to test the hypothesis that the enFeLV loads play a role in exogenous FeLV-A infection and pathogenesis, quantitative real-time PCR and RT-PCR assays were developed. An assay, specific to U3 region of all different subtypes of exogenous FeLV, was designed and applied to quantify exogenous FeLV proviral or viral load in cats, while three real-time PCR assays were designed to quantify U3 and env enFeLV loads (two within U3 amplifying different sequences; one within env). enFeLV loads were investigated in blood samples derived from Swiss privately owned domestic cats, specific pathogen-free (SPF) cats and European wildcats (Felis silvestris silvestris). Significant differences in enFeLV loads were observed between privately owned cats and SPF cats as well as among SPF cats originating from different catteries and among domestic cats of different breeds. When privately owned cats were compared, FeLV-infected cats had higher loads than uninfected cats. In addition, wildcats had higher enFeLV loads than domestic cats. In conclusion, the quantitative real-time PCR assays described herein are important prerequisites to quantify enFeLV proviral loads in felids and thus are important tools to investigate the role of enFeLV loads in FeLV infection.

Genome organization of the Chelonus inanitus polydnavirus: excision sites, spacers and abundance of proviral and excised segments.

Polydnaviruses are only found in symbiotic association with parasitic wasps within the families Ichneumonidae and Braconidae (ichnoviruses and bracoviruses). They have a segmented genome consisting of circular double-stranded DNA. In the proviral linear form they are integrated in the wasp's genome; in two bracoviruses, segments were found to be clustered. Proviral segments have direct terminal repeats. Segment excision has been proposed to occur through juxtaposition of these repeats by formation of a loop and recombination; one copy of the repeat then ends up in the circular segment and one in the rejoined DNA. Here we analysed the excision/circularization site of four segments of the Chelonus inanitus bracovirus (CiV) and found that they are similar to the two already known sites; on the basis of the combined data an extended excision site motif was found. Analyses of segment flanking sequences led to the first identification of one complete and several partial spacers between proviral segments in a polydnavirus. The spacer between the proviral segments CiV14 and CiV22.5 has a length of 2065 bp; the terminal repeats of CiV14 and CiV22.5 were seen to have an opposite orientation and from this a model on the spacial organization of the loops of the proviral cluster is proposed. Through various approaches it was shown that spacers are not excised or injected into the host. Measurement of relative abundances of various segments in proviral and excised form indicates for the first time that abundant segments are present in multiple copies in the proviral form.

Methylation of the human papillomavirus-18 L1 gene: a biomarker of neoplastic progression?

Epigenetic transcriptional regulation plays an important role in the life cycle of human papillomaviruses (HPVs) and the carcinogenic progression of anogenital HPV associated lesions. We performed a study designed to assess the methylation status of the HPV-18 genome, specifically of the late L1 gene, the adjacent long control region (LCR), and part of the E6 oncogene in cervical specimens with a range of pathological diagnoses. In asymptomatic infections and infections with precancerous (precursor) lesions, HPV-18 DNA was mostly unmethylated, with the exception of four samples where hypermethylation of L1 was detected. In contrast, L1 sequences were strongly methylated in all cervical carcinomas, while the LCR and E6 remained unmethylated. HeLa cells, derived from a cervical adenocarcinoma, contain chromosomally integrated HPV-18 genomes. We found that L1 is hypermethylated in these cells, while the LCR and E6 are unmethylated. Treatment of HeLa cells with the methylation inhibitor 5-Aza-2'-deoxycytidine (5-Aza-CdR) led to the expected reduction of L1 methylation. After removal of 5-Aza-CdR, L1 methylation resumed and exceeded pretreatment levels. Unexpectedly, the LCR and E6 also became methylated under these conditions, albeit at lower levels than L1. We hypothesize that L1 is preferentially methylated after integration of the HPV genome into the cellular DNA, possibly since linearization prohibits its normal transcription, while the enhancer and promoter may be protected from methylation by transcription factors. Since our data suggest that HPV-18 L1 methylation can only be detected in carcinomas, except in some few precancerous lesions and asymptomatic infections, L1 methylation may constitute a powerful molecular marker for detecting this important step of neoplastic progression.

Consistent patterns of change during the divergence of human immunodeficiency virus type 1 envelope from that of the inoculated virus in simian/human immunodeficiency virus-infected macaques.

We have analyzed changes to proviral Env gp120 sequences and the development of neutralizing antibodies (NAbs) during 1 year of simian/human immunodeficiency virus SHIV-89.6P infection in 11 Macaca nemestrina macaques. Seven macaques had significant env divergence from that of the inoculum, and macaques with greater divergence had higher titers of homologous NAbs. Substitutions in sequons encoding potential N-linked glycosylation sites (PNGs) were among the first to be established, although overall the total number of sequons did not increase significantly. The majority (19 of 23) of PNGs present in the inoculum were conserved in the sequences from all macaques. Statistically significant variations in PNGs occurred in multiple macaques within constrained regions we term "hot spots," resulting in the selection of sequences more similar to the B consensus. These included additions on V1, the N-terminal side of V4, and the outer region of C2. Complex mutational patterns resulted in convergent PNG shifts in V2 and V5. Charge changes in Env V1V2, resulting in a net acidic charge, and a proline addition in V5 occurred in several macaques. Molecular modeling of the 89.6P sequence showed that the conserved glycans lie on the silent face of Env and that many are proximal to disulfide bonds, while PNG additions and shifts are proximal to the CD4 binding site. Nonsynonymous-to-synonymous substitution ratios suggest that these changes result from selective pressure. This longitudinal and cross-sectional study of mutations in human immunodeficiency virus (HIV) env in the SHIV background provides evidence that there are more constraints on the configuration of the glycan shield than were previously appreciated.

Functionalized head-to-head hairpin polyamides: synthesis, double-stranded DNA-binding activity and affinity.

A series of 4 functionalized head-to-head-linked hairpin oligo(N-methylpyrrole) carboxamides with different linkers have been synthesized. Their ability to bind double-stranded DNA and sequence specificity were compared and the apparent Kd values of their DNA complexes were determined. These compounds, particularly those with iminodiacetic linkers, revealed a high affinity for DNA (Kd = 4.5-4.8 x 10(-9) M) and sequence specific recognition of 9-10 base pairs.

Identification of amino acid residues in the human immunodeficiency virus type-1 reverse transcriptase tryptophan-repeat motif that are required for subunit interaction using infectious virions.

The human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) functions as a heterodimer (p51/p66), which makes disruption of subunit interactions a possible target for antiviral drug design. Our understanding of subunit interface interactions has been limited by the lack of virus-based approaches for studying the heterodimer. Therefore, we developed a novel subunit-specific mutagenesis approach that enables precise molecular analysis of the heterodimer in the context of infectious HIV-1 particles. Here, we analyzed the contributions of amino acid residues comprising the Trp-motif to RT subunit interaction and function. Our results reveal important inter- and intra-subunit interactions of residues in the Trp-motif. A tryptophan cluster in p51 (W398, W402, W406, W414), proximal to the interface, was found to be important for p51/p66 interaction and stability. At the dimer interface, residues W401, Y405 and N363 in p51 and W410 in p66 mediate inter-subunit interactions. The W401 residue is critical for RT dimerization, exerting distinct effects in p51 and p66. Our analysis of the RT heterodimerization enhancing non-nucleoside RT inhibitor (NNRTI), efavirenz, indicates that the effects of drugs on RT dimer stability can be examined in human cells. Thus, we provide the first description of subunit-specific molecular interactions that affect RT heterodimer function and virus infection in vivo. Moreover, with heightened interest in novel RT inhibitors that affect dimerization, we demonstrate the ability to assess the effects of RT inhibitors on subunit interactions in a physiologically relevant context.

Localization of human T-cell lymphotropic virus-1 gag proviral sequences in dermato-immunological disorders with eosinophilia.

The mechanisms leading to the development of eosinophilia were investigated in 65 patients with immunodermatological disorders, including the role of eosinophilotactic cytokines and the possible involvement of human T-cell leukemia virus, HTLV. HTLV-1 gag proviral sequences were revealed in two cases of lymphoproliferative disorders such as angiolymphoid hyperplasia with eosinophilia (ALHE) and CD4+ cutaneous lymphoma, respectively. Increased level of GM-CSF was detected in 33% of disorders studied. Elevated level of IL-5 and eotaxin was detected in 27% and 30%, respectively, of patients with bullous diseases. Elevated level of GM-CSF and eotaxin was found in 33% and 46%, respectively, of patients with inflammatory diseases. Neither of the four cytokines, however proved to be responsible alone or together for the induction of eosinophilia. The possible indirect role of human retroviruses through induction of eosinophilic chemotactic cytokines is hypothesized.

Efficacy and mode of action of hammerhead and hairpin ribozymes against various HIV-1 target sites.

Ribozymes have been proposed as gene therapy agents against HIV-1, although many fundamental questions about their mechanism of action remain unclear. Few studies have compared directly the potential of different modified ribozyme species against a particular target. Here we compare the relative abilities of hammerhead (HhU5) and hairpin (HpU5) ribozymes directed against a well-studied target RNA that has therapeutic potential, located in the untranslated 5' region (U5), to inhibit HIV-1 replication. The two types of ribozymes showed similar antiviral efficacy after being stably transfected into HUT78 cells and subsequently challenged with HIV-1(SF2), but the HhU5 ribozyme showed faster cleavage kinetics when tested in a cell-free system. In the second part of this study, we examined whether different ribozymes were able to inhibit the integration of proviral DNA in infected HUT78 cells. We found that cell pools stably expressing HpU5 could limit the appearance of integrated provirus, indicating that they could inhibit the infecting viral RNA before reverse transcription. A preintegration effect was also found for cell pools expressing a ribozyme targeting the nef gene (HhNef) or a ribozyme targeting the LTR (HhLTR). However, no discernible preintegration effects were seen for the HhU5 ribozyme or an active ribozyme directed against an RNA target site in the pol gene (HhPol). Thus, the results suggest that the mode of ribozyme action varied between sites and is not dependent solely on inhibiting the infecting viral RNA. Evidence for a preintegration effect is extremely encouraging and indicates that "resistant" cells have some chance to repopulate the immune system through such a selective advantage. We also studied the ability of the different ribozymes to down regulate viral RNA postintegration.

Provirus variants of bovine leukemia virus in naturally infected cattle from Argentina and Japan.

A serologic subgroup of bovine leukemia virus (BLV) has not been identified, whereas genetic diversity among BLVs has been reported by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP). To investigate the distribution of BLV provirus variants, 42 isolates from Argentina and Japan were examined by nested PCR for a segment of the env gene, followed by DNA sequencing. The nucleotide sequences were compared with other previously characterized BLV variants from different geographical areas (Belgium, France, Italy, North America, Australia, Japan and Argentina). The majority of analyzed segments had a tendency for nucleotide substitution without changing the amino acid. The constructed phylogenetic tree showed the relations and differences between proviruses and within each one. Most of the samples in Argentina formed one cluster. The samples in Japan, except one, also formed one cluster and some of them showed high homology with the isolates from Australia and the USA. Considering the sequence analysis of env PCR products of all Japanese and Argentine samples and comparing them with the other previously isolated sequences, the variation was up to 3.5% and was characterized geographically in each area.

[Defective proviruses of the human T-cell leukemia virus: structure and classification]. / Defektnye provirusy virusa T-kletochnogo leikoza cheloveka: struktura i klassifikatsiia.

Peripheral blood mononuclear cells of 24-70% individuals infected with HTLV-1 contain defective proviruses (dp) in addition to the full size ones. Most of them remain silent lacking regions sufficient for viral genes transcription except those activated under cell promoter or retaining viral open reading frames (orfs). It is still unclear whether these proviruses are associated with the development of T-cell leukemia in adults, tropic spastic paresis, or myelopathy. Classification of previously reported dp is presented, their origin and possible function in human HTLV-1 associated diseases are discussed.

The protelomerase of the phage-plasmid N15 is responsible for its maintenance in linear form.

The prophage of coliphage N15 is not integrated into the bacterial chromosome but exists as a linear plasmid molecule with covalently closed ends. Upon infection of an Escherichia coli cell, the phage DNA circularises via cohesive ends. A phage-encoded enzyme, protelomerase, then cuts at another site, telRL, and forms hairpin ends (telomeres). We demonstrate that this enzyme acts in vivo on specific substrates, and show that it is necessary for replication of the linear prophage. We show that protelomerase is an end-resolving enzyme responsible for processing of replicative intermediates. Removal of protelomerase activity resulted in accumulation of replicative intermediates that were found to be circular head-to-head dimers. N15 protelomerase and its target site constitute a functional unit acting on other replicons independently of other phage genes; a mini-F or mini-P1 plasmid carrying this unit replicates as a linear plasmid with covalently closed ends. Our results suggest the following model of N15 prophage DNA replication. Replication is initiated at an internal ori site located close to the left end of plasmid DNA and proceeds bidirectionally. After replication of the left telomere, protelomerase cuts this sequence and forms two hairpin loops telL. After duplication of the right telomere (telR) the same enzyme resolves this sequence producing two linear plasmids. Alternatively, full replication of the linear prophage to form a circular head-to-head dimer may precede protelomerase-mediated formation of hairpin ends.

Initiation of therapy during primary HIV type 1 infection results in a continuous decay of proviral DNA and a highly restricted viral evolution.

A latent pool of HIV-1 is established early in memory CD4+ T lymphocytes and persists during antiretroviral therapy. Also, viral replication may continue in subjects despite undetectable viremia. However, it remains unclear whether this residual replication results in any significant sequence evolution. We were therefore interested in studying the viral evolution and HIV-1 DNA dynamics in subjects with primary infection receiving or not receiving early potent antiretroviral therapy. In 16 subjects, HIV-1 DNA load was monitored from 1 to 23 days, up to 1253 days, after onset of symptoms. Extensive sequential cloning and sequence analysis of the V3 region was performed in four subjects. In the treated subjects a continuous decline in the proviral load was found, corresponding to a half-life of about 6 months. As expected in newly infected individuals the founder virus populations showed high intrasubject sequence similarity. Also, a limited increase in the viral divergence was detected during the first 6 months in three treated subjects. Thereafter, no significant sequence changes were found despite analysis of a large number of clones. Our data thus suggest that early and successful therapy in compliant subjects with primary HIV-1 infection results in a highly restricted viral evolution and a decline in the proviral load close to the decay rate of human memory T lymphocytes.

Low copy numbers of human T-cell lymphotropic virus type I (HTLV-I) tax-like DNA detected in the salivary gland of seronegative patients with Sjögren's syndrome in an HTLV-I endemic area.

To evaluate the hypothesis, proposed in previous reports from HTLV-I non-endemic areas, that HTLV-I is involved in a significant proportion, about a quarter, of Sjögren's syndrome patients who lack serum antibodies to the virus, we examined for the presence or absence of HTLV-I in DNA samples isolated from salivary gland tissues of 17 seronegative as well as 7 seropositive patients with Sjögren's syndrome in Nagasaki, Japan, where the virus is highly endemic. The nested two-step polymerase chain reaction (PCR), with a sensitivity capable of detecting a single DNA molecule, failed to amplify the HTLV-I tax sequence from DNA of 14 of the 17 seronegative patients. The tax was only amplifiable from the tissue DNA of the remaining three seronegative patients. The detection rate, 3/17 (18%), was, unexpectedly, less than those previously reported from the HTLV-I non-endemic areas. Moreover, in contrast to high viral loads (10(-1) to 10(-3) per cell) in the salivary gland of the seropositive patients, a semiquantitative PCR revealed that the copy number of the HTLV-I tax in the gland tissue of these seronegative patients was very low, 10(-5) per cell. This level is unlikely to be sufficient to promote an inflammatory reaction in the tissue. Our findings might argue against the involvement of "prototype" HTLV-I in the pathogenesis of Sjögren's syndrome in seronegative patients.

A rapid method for comparative quantitative polymerase chain reaction of HIV-1 proviral DNA extracted from cryopreserved brain tissues.

The quantitative polymerase chain reaction (PCR) method devised by Fujimura and Bockstahler (1995) was modified for rapid determination of distribution of HIV-1 proviral DNA load in AIDS brains. It was used for analysis of an association with HIV-1 associated dementia and HIV-1 encephalitis (Fujimura et al., 1997). The method has wider applicability for comparative studies of viral DNA load based on PCR amplification. The method is applicable under conditions where target DNA and its PCR-amplified product increase proportionally. An equation was derived to obtain the number of copies of HIV-1 DNA per cellular genome from the amount of PCR amplified product of a tissue specimen DNA. The equalizing constant is the reciprocal of the slope of the amplification of the HIV-1 proviral DNA sequence of the standard cellular DNA included in each experiment. The intercept of the equation is zero.

Contribution of virion ICAM-1 to human immunodeficiency virus infectivity and sensitivity to neutralization.

Incorporation of the intercellular adhesion molecule ICAM-1 into human immunodeficiency virus type 1 (HIV-1) particles increased virus infectivity on peripheral blood mononuclear cells (PBMCs) by two- to sevenfold. The degree of ICAM-1-mediated enhancement was greater for viruses bearing envelope glycoproteins derived from primary HIV-1 isolates than for those bearing envelope glycoproteins from laboratory-adapted strains. Treatment of target PBMCs with an antibody against LFA-1, a principal ICAM-1 receptor, was able to nullify the ICAM-1-mediated enhancement. The incorporation of ICAM-1 rendered HIV-1 virions less susceptible to neutralization by a monoclonal antibody directed against the viral envelope glycoproteins. Surprisingly, an antibody against ICAM-1 completely neutralized infection by ICAM-1-containing viruses, reducing the efficiency of virus entry by almost 100-fold. Thus, HIV-1 neutralization by an ICAM-1-directed antibody involves more than an inhibition of the contribution of ICAM-1 to virus entry.

Live attenuated simian immunodeficiency virus prevents super-infection by cloned SIVmac251 in cynomolgus monkeys.

The ability of a live attenuated simian immunodeficiency virus (SIV) to protect against challenge with cloned SIVmac251/BK28 was evaluated in four cynomolgus macaques. The intravenous infection of the C8 variant of the SIVmac251/32H virus, carrying an in-frame 12 bp deletion in the nef gene, did not affect the CD4+ and CD8+ cell counts, and a persistent infection associated with an extremely low virus burden in peripheral blood mononuclear cells (PBMCs) was established. After 40 weeks, these monkeys were challenged intravenously with a 50 MID50 dose of SIVmac251/BK28 virus grown on macaque cells. Four naive monkeys were infected as controls. Monkeys were monitored for 62 weeks following challenge. Attempts to rescue virus from either PBMCs or bone marrow from the C8-vaccinated monkeys were unsuccessful, but in two cases virus was re-isolated from lymph node cells. The presence of the SIV provirus with the C8 variant genotype maintaining its original nef deletion was shown by differential PCR in PBMCs, lymph nodes and bone marrow. Furthermore, in contrast to the control monkeys, the vaccinated monkeys showed normal levels for CD4+ and CD8+ cells, minimal lymphoid hyperplasia and no clinical signs of infection. Our results confirm that vaccination with live attenuated virus can confer protection. This appears to be dependent on the ability of the C8 variant to establish a persistent but attenuated infection which is necessary for inducing an immune response, as suggested by the persistence of a strong immune B cell memory and by the over-expression of interleukin (IL)-2, interferon-gamma and IL-15 mRNAs in PBMCs of C8-vaccinated monkeys but not in those of control monkeys.