Past and future of HIV infection. A document based on expert opinion.

HIV infection is now almost 40 years old. In this time, along with the catastrophe and tragedy that it has entailed, it has also represented the capacity of modern society to take on a challenge of this magnitude and to transform an almost uniformly lethal disease into a chronic illness, compatible with a practically normal personal and relationship life. This anniversary seemed an ideal moment to pause and reflect on the future of HIV infection, the challenges that remain to be addressed and the prospects for the immediate future. This reflection has to go beyond merely technical approaches, by specialized professionals, to also address social and ethical aspects. For this reason, the Health Sciences Foundation convened a group of experts in different aspects of this disease to discuss a series of questions that seemed pertinent to all those present. Each question was presented by one of the participants and discussed by the group. The document we offer is the result of this reflection.

Pharmacologic control of homeostatic and antigen-driven proliferation to target HIV-1 persistence.

The presence of latent human immunodeficiency virus 1 (HIV-1) in quiescent memory CD4 + T cells represents a major barrier to viral eradication. Proliferation of memory CD4 + T cells is the primary mechanism that leads to persistence of the latent reservoir, despite effective antiretroviral therapy (ART). Memory CD4 + T cells are long-lived and can proliferate through two mechanisms: homeostatic proliferation via γc-cytokine stimulation or antigen-driven proliferation. Therefore, therapeutic modalities that perturb homeostatic and antigen-driven proliferation, combined with ART, represent promising strategies to reduce the latent reservoir. In this study, we investigated a library of FDA-approved oncology drugs to determine their ability to inhibit homeostatic and/or antigen-driven proliferation. We confirmed potential hits by evaluating their effects on proliferation in memory CD4 + T cells from people living with HIV-1 on ART (PLWH) and interrogated downstream signaling of γc-cytokine stimulation. We found that dasatinib and ponatinib, tyrosine kinase inhibitors, and trametinib, a MEK inhibitor, reduced both homeostatic and antigen-driven proliferationby >65%, with a reduction in viability <45%, ex vivo. In memory CD4 + T cells from PLWH, only dasatinib restricted both homeostatic and antigen-driven proliferation and prevented spontaneous rebound, consistent with promoting a smaller reservoir size. We show that dasatinib restricts IL-7 induced proliferation through STAT5 phosphorylation inhibition. Our results establish that the anti-cancer agent dasatinib is an exciting candidate to be used as an anti-proliferative drug in a clinical trial, since it efficiently blocks proliferation and iswell tolerated in patients with chronic myeloid leukemia (CML).

Modifications in host cell cytoskeleton structure and function mediated by intracellular HIV-1 Tat protein are greatly dependent on the second coding exon.

The human immunodeficiency virus type 1 (HIV-1) regulator Tat is essential for viral replication because it achieves complete elongation of viral transcripts. Tat can be released to the extracellular space and taken up by adjacent cells, exerting profound cytoskeleton rearrangements that lead to apoptosis. In contrast, intracellular Tat has been described as protector from apoptosis. Tat gene is composed by two coding exons that yield a protein of 101 amino acids (aa). First exon (1-72aa) is sufficient for viral transcript elongation and second exon (73-101 aa) appears to contribute to non-transcriptional functions. We observed that Jurkat cells stably expressing intracellular Tat101 showed gene expression deregulation 4-fold higher than cells expressing Tat72. Functional experiments were performed to evaluate the effect of this deregulation. First, NF-kappaB-, NF-AT- and Sp1-dependent transcriptional activities were greatly enhanced in Jurkat-Tat101, whereas Tat72 induced milder but efficient activation. Second, cytoskeleton-related functions as cell morphology, proliferation, chemotaxis, polarization and actin polymerization were deeply altered in Jurkat-Tat101, but not in Jurkat-Tat72. Finally, expression of several cell surface receptors was dramatically impaired by intracellular Tat101 but not by Tat72. Consequently, these modifications were greatly dependent on Tat second exon and they could be related to the anergy observed in HIV-1-infected T cells.

Oligonucleotide array for simultaneous detection of respiratory viruses using a reverse-line blot hybridization assay.

The interest in developing new diagnostic methods based on arrays of multiple probes to detect and type simultaneously a wide range of different infectious agents is increasing. This becomes a necessity in the case of infectious agents such as respiratory viruses that cause diseases with very similar signs and symptoms. Such tools will permit rapid and accurate diagnosis of different agents causing respiratory infection leading to the most adequate prevention and/or treatment measures. In this article a reverse-line blot hybridization (RLB) assay for the detection of a wide range of respiratory viruses is presented and evaluated for its usefulness in routine diagnosis. This assay employs an array of 18 oligonucleotide probes immobilized on a nylon membrane. Biotin-labeled PCR products obtained with two multiplex reverse transcription (RT)-polymerase chain reaction (PCR) assays described previously, which allow for the detection of fourteen different groups of respiratory viruses, were hybridized to the oligonucleotide array. Detection was performed using a chemiluminescent method. The standardization of the method showed that the RLB assay could be an alternative to the nested PCR assay for enhancing the sensitivity in the detection of the amplified products, avoiding the problem of cross-over contamination, increasing the specificity, and therefore simplifying the method. This is of main interest in laboratories with few facilities. The feasibility and accuracy of the RT-PCR-RLB assay for detecting respiratory viruses proves that such approach could be a first stage to develop a microarray assay for routine diagnosis of infectious diseases.

Simultaneous detection of fourteen respiratory viruses in clinical specimens by two multiplex reverse transcription nested-PCR assays.

There is a need for rapid, sensitive, and accurate diagnosis of lower respiratory tract infections in children, elderly, and immunocompromised patients, who are susceptible to serious complications. The multiplex RT-nested PCR assay has been used widely for simultaneous detection of non-related viruses involved in infectious diseases because of its high specificity and sensitivity. A new multiplex RT-PCR assay is described in this report. This approach includes nested primer sets targeted to conserve regions of human parainfluenza virus haemagglutinin, human coronavirus spike protein, and human enterovirus and rhinovirus polyprotein genes. It permits rapid, sensitive, and simultaneous detection and typing of the four types of parainfluenza viruses (1, 2, 3, 4AB), human coronavirus 229E and OC43, and the generic detection of enteroviruses and rhinoviruses. The testing of 201 clinical specimens with this multiplex assay along with other one formerly described by our group to simultaneously detect and type the influenza viruses, respiratory syncytial viruses, and a generic detection of all serotypes of adenovirus, covers the detection of most viruses causing respiratory infectious disease in humans. The results obtained were compared with conventional viral culture, immunofluorescence assay, and a third multiplex RT-PCR assay for all human parainfluenza viruses types described previously. In conclusion, both multiplex RT-PCR assays provide a system capable of detecting and identifying simultaneously 14 different respiratory viruses in clinical specimens with high sensitivity and specificity, being useful for routine diagnosis and survey of these viruses within the population.

Simultaneous detection of influenza A, B, and C viruses, respiratory syncytial virus, and adenoviruses in clinical samples by multiplex reverse transcription nested-PCR assay.

The clinical presentation of infections caused by the heterogeneous group of the respiratory viruses can be very similar. Thus, the implementation of virological assays that rapidly identify the most important viruses involved is of great interest. A new multiplex reverse transcription nested-polymerase chain reaction (RT-PCR) assay that is able to detect and type different respiratory viruses simultaneously is described. Primer sets were targeted to conserved regions of nucleoprotein genes of the influenza viruses, fusion protein genes of respiratory syncytial viruses (RSV), and hexon protein genes of adenoviruses. Individual influenza A, B, and C viruses, RSV (A and B), and a generic detection of the 48 serotypes of adenoviruses were identified and differentiated by the size of the PCR products. An internal amplification control was included in the reaction mixture to exclude false-negative results due to sample inhibitors and/or extraction failure. Detection levels of 0.1 and 0.01 TCID50 of influenza A and B viruses and 1-10 molecules of cloned amplified products of influenza C virus, RSV A and B, and adenovirus serotype 1 were achieved. The specificity was checked using specimens containing other respiratory viruses and no amplified products were detected in any case. A panel of 290 respiratory specimens from the 1999-2000 and 2000-2001 seasons was used to validate the assay. Accurately amplifying RNA from influenza and RSV prototype strains and DNA from all adenovirus serotypes demonstrates the use of this method for both laboratory routine diagnosis and surveillance of all these viruses.

Evidence for discoordinate regulation of the HLA-DPB1 gene.

Characterization of cell lines derived from patients with type II bare lymphocyte syndrome, a pathological state in which the constitutive and inducible expression of HLA class II antigens is lacking, has permitted the identification of several trans-acting factors involved in the coordinated regulation of HLA class II genes. Although an increasing body of evidence has pointed to the existence of a discoordinate regulation of HLA class II loci, the mechanisms underlying such regulation are essentially unknown. In the present study, 45.EM2, a mutant lymphoblastoid cell line with a new pattern of HLA discoordinate expression is characterized. 45.EM2 expresses HLA-DR and -DQ but fails to express HLA-DP. The absence of HLA-DP expression in 45.EM2 is the result of a transcriptional defect, leading to a lack of DPB1 mRNA. By contrast, DPA1 transcription in this LCL is not impaired. The characteristics of 45.EM2 described here suggest the existence of a specific trans-acting factor involved in the control of DPB1 gene expression.

Rapid molecular analysis of the haemagglutinin gene of human influenza A H3N2 viruses isolated in spain from 1996 to 2000.

A simple molecular technique was used for the rapid preliminary genetic characterization of human influenza A H3N2 viruses isolated in Spain from 1996 to 2000. Subtyping, based on RT-PCR, was followed by subtype-specific restriction enzyme fragment length polymorphism (RFLP) analyses of an amplified region of the HA1 domain of the H3 haemagglutinin (HA) gene to distinguish variants differentiated by common amino acid substitutions in HA1. The approach was tested using 135 Spanish H3N2 isolates and included nucleotide sequencing and phylogenetic analyses of a region of the HA1 domain of 41 representative isolates. The viruses were distinguished by haemagglutination inhibition (HI) assays into two antigenically discernible groups, the A/Wuhan/359/95-like and A/Sydney/5/97-like viruses. The results of PCR-RFLP analysis allowed a finer classification into five genetic variant subgroups, corresponding to those distinguished by phylogenetic analyses. This rapid, simple and variant-specific procedure could, therefore, be used to rapidly screen clinical specimens prior to more detailed antigenic and genetic analyses.

HLA-DPbeta residue 69 plays a crucial role in allorecognition.

To investigate the contribution to allorecognition of the individual polymorphic positions Glu 69 and Val 36 from the DPB1*02012 allele, DPB1*02012 cDNA was subjected to site-directed mutagenesis and alleles expressing Lys at 69 and Ala at 36 were generated. The lymphoblastoid cell line (LCL) 45.EM1, a previously generated mutant B-LCL which expresses normal levels of DPA mRNA but is not able to transcribe DPB, was transfected with wild-type or mutant DPB1*02012 cDNAs. The ability of two HLA-DPw2 alloreactive CD4+ cytotoxic T-lymphocyte (CTL) clones to lyse the panel of DPB1*02012 wild-type and site-directed mutant B-cell lines was tested. Both CTL clones (8.3 and 8.9) lysed the B-LCL 45.1, which is haploid for HLA and expresses wild-type DPB1*02012, and transfectants expressing Ala at 36 instead of Val, indicating that this polymorphic residue is not critical for T-cell recognition. However, the change of Glu to Lys at 69 prevented recognition by clones 8.3 and 8.9. These data demonstrate that the residue at peptide-binding position 69 is crucial for T-cell receptor recognition and suggest the requirement for a negatively charged residue at this position for allostimulation of these T-cell clones. The side chain of DPbeta-69 is predicted to point into the peptide-binding groove, and the existence of positive(Lys) or negative (Glu) residues probably leads to substantial differences in the allo- or auto-DP-bound peptides or to differences in the conformation of the peptide-MHC complex, which would therefore be responsible for specific DPw2 allorecognition. The binding of a panel of monomorphic and polymorphic anti-HLA-DP monoclonal antibodies (mAbs) to these transfectants was also tested by flow cytometry. The changes at Glu 69 and Val 36 did not affect recognition by any of the monomorphic antibodies tested. However, the binding pattern of some of the polymorphic mAbs was clearly modified. Therefore, even though it is not crucial for T-cell allorecognition, polymorphic residue 36 must be involved in epitopes recognized by some polymorphic anti-DP antibodies, while residue 69 of the DPB molecule is crucial both for T-cell allorecognition and recognition by some mAbs.