Limited development and progression of resistance of HIV-1 to the nucleoside analogue reverse transcriptase inhibitor lamivudine in human primary macrophages.

OBJECTIVES: The aim of this study was to investigate the development and progression of phenotypic resistance to the HIV-1-reverse transcriptase (RT) inhibitor lamivudine, and genotypic variations of HIV-1-RT occurring under lamivudine treatment in HIV-1-infected human primary monocytes-macrophages (M/M). METHODS: Cellular passages in the presence of lamivudine were performed every 2 weeks by transferring supernatants of infected M/M to fresh M/M. A fitness assay using wild-type virus and a lamivudine-resistant HIV-1 virus (harbouring the M184V RT mutation) was performed in peripheral blood mononuclear cells. Culture supernatants were tested for p24 antigen production and RT activity. The M184V RT mutant virus was obtained by site-directed mutagenesis on a CCR5-using HIV-1 backbone. RESULTS: The mutagenized M184V RT virus showed full resistance to lamivudine in M/M. However, no detectable phenotypic and genotypic resistance (neither virus breakthrough, nor RT resistance-related mutations) developed in M/M infected by HIV-1 and cultured for up to seven passages in vitro (i.e. 105 days). This inefficiency of M/M to develop M184V RT mutated virus is tightly related to the low 2'-deoxynucleotide (dNTP) pool in such cells, which in turn decreases the kinetics of HIV-1-RT. Despite this, the M184V RT mutant virus replicates in M/M, although with a 30% decreased efficiency compared with the wild-type. CONCLUSIONS: Our results show that the chances of development of resistance are far lower in M/M than in lymphocytes. This underlines the importance and the peculiar role of M/M as reservoirs of either wild-type or resistant strains in human organs.

Developing an epitope-driven tuberculosis (TB) vaccine.

Epitope-driven vaccines are created from selected sub-sequences of proteins, or epitopes, derived by scanning the protein sequences of pathogens for patterns of amino acids that permit binding to human MHC molecules. We developed a prototype tuberculosis (TB) vaccine that contains epitopes derived by (1) EpiMer mapping of previously published secreted proteins derived from Mycobacterium tuberculosis (Mtb), and (2) EpiMatrix mapping of selected Mtb genome open reading frames (ORFs). Each of the epitopes contains at least three distinct class II MHC binding motif matches. These Mtb epitope selections were validated by measuring T cell responses from peripheral blood mononuclear cells (PBMC) obtained from healthy, asymptomatic tuberculin skin test-positive donors. Twenty-four validated Mtb epitopes were selected for inclusion in a DNA plasmid vector. We immunized HLA-DR B*0101 transgenic mice with this vaccine prototype augmented by co-administration of rIL-15. Following administration of three immunizations at 14-day intervals in conjunction with rIL-15, epitope-specific T cell responses were observed to eight of the 24 epitopes contained in the DNA construct, one week following the last injection. The systematic application of bioinformatics tools to whole genomes, in combination with in vitro methods for screening and confirming epitopes, may lead to the development of novel vaccines for infectious diseases like TB.

HIV vaccine development by computer assisted design: the GAIA vaccine.

The design of epitope-driven vaccines that address the global variability of HIV has been significantly hampered by concerns about conservation of the vaccine epitopes across clades of HIV. We developed two computer-driven methods for improving epitope-driven HIV vaccines: the Epi-Assembler, which derives representative or "immunogenic consensus sequence" (ICS) epitopes from multiple viral variants, and VaccineCAD, which reduces junctional immunogenicity when epitopes are aligned in a string-of-beads format for insertion in a DNA expression vector. In this study, we report on 20 ICS HIV-1 peptides. The core 9-mer contained in these consensus peptides was conserved in 105-2250 individual HIV-1 strains. Nineteen of the 20 ICS epitopes (95%) evaluated in this study were confirmed in ELISpot assays using peripheral blood monocytes obtained from 13 healthy HIV-1 infected subjects. Twenty-five ICS peptides (all 20 of the peptides evaluated in this study and 5 additional ICS epitopes) were then aligned in a pseudoprotein string using "VaccineCAD", an epitope alignment tool that eliminates immunogenicity created by the junctions between the epitopes. Reordering the construct reduced the immunogenicity of the junctions between epitopes as measured by EpiMatrix, an epitope mapping algorithm. The reordered construct was also a more effective immunogen in vivo when tested in HLA-DR transgenic mice. These data confirm the utility of bioinformatics tools to design novel vaccines containing "immunogenic consensus sequence" T cell epitopes for a globally relevant vaccine against HIV.

Engineering immunogenic consensus T helper epitopes for a cross-clade HIV vaccine.

Developing a vaccine that will stimulate broad HIV-specific T cell responses is difficult because of the variability in HIV T cell epitope sequences, which is in turn due to the high mutation rate and consequent strain diversity of HIV-1. We used a new Class II version of the EpiMatrix T cell epitope-mapping tool and Conservatrix to select highly conserved and promiscuous Class II HLA-restricted T cell epitopes from a database of 18,313 HIV-1 env sequences. Criteria for selection were: (1) number of HIV-1 strains represented as measured by Conservatrix; (2) EpiMatrix score; and (3) promiscuity (number of unique MHC motifs contained in the peptide). Using another vaccine design tool called the EpiAssembler, a new set of overlapping, conserved and immunogenic HIV-1 peptides were engineered creating extended "immunogenic consensus" sequences. Each overlapping 9-mer of the 20-23 amino acid long immunogenic consensus peptides was conserved in a large number (range 893-2254) of individual HIV-1 strains, although the novel peptides were not representative of any single strain of HIV. We synthesized nine representative peptides. T helper cell responses to the peptides were evaluated by ELISpot (gamma-interferon) assay, using peripheral blood monocytes (PBMC) obtained from 34 healthy long term non-progressor (LT) or moderate-progressor (MP) donors (median years infected = 8.88, median CD4 T cells = 595, median VL = 1044). Nine peptides were tested, of which eight were confirmed in ELISpot assays using PBMC from the LT/MP subjects. These epitopes were ranked by Conservation and EpiMatrix score 1, 2, 3, 5, 7, 11, and 14 out of the set of 9 original peptides. Five of these peptides were selected for inclusion in an epitope-driven cross-clade HIV-1 vaccine (the GAIA vaccine). These data confirm the utility of bioinformatics tools to select and construct novel "immunogenic consensus sequence" T cell epitopes for a globally relevant vaccine against HIV.

Identification of the minimal conserved structure of HIV-1 protease in the presence and absence of drug pressure.

OBJECTIVE: To define the extent of amino acid protease (PR) conservation in vivo in the absence and presence of pharmacological pressure in a large patient cohort. METHODS: Plasma-derived complete protein PR sequences from a well-defined cohort of 1096 HIV-1 infected individuals (457 drug-naive and 639 under antiretroviral therapy including PR-inhibitors) were obtained and analysed, and are discussed in a structural context. RESULTS: In naive patients, the PR sequence showed conservation (< 1% variability) in 68 out of 99 (69%) residues. Five large conserved regions were observed, one (P1-P9) at the N-terminal site, another (E21-V32) comprised the catalytic active-site, a third (P44-V56) contained the flap, a fourth contained the region G78-N88, and another (G94-F99) contained the C-terminal site. In PR-inhibitor treated patients, the appearance of mutations primarily associated with drug resistance determined a decrease of amino acid invariance to 45 out of 99 residues (45% conservation). The overall degree of enzyme conservation, when compared to the PR sequences in drug-naive patients, was preserved at the N- and C-terminal regions, whereas the other large conserved areas decreased to smaller domains containing, respectively, the active-site residues D25-D29, the tip of the flap G49-G52, and the G78-P81 and G86-R87 turns. CONCLUSIONS: Amino acid conservation in HIV PR can be minimally present in 45 residues out of 99. Identification of these invariable residues, with crucial roles in dimer stability, protein flexibility and catalytic activity, and their mapping on the three-dimensional structure of the enzyme will help guide the design of novel resistance-evading drugs.

HIV fitness and resistance as covariates associated with the appearance of mutations under antiretroviral treatment.

The ability of human immunodeficiency virus (HIV) strains to replicate in human target cells represents a major driving force of the progression of the disease. Despite antiretroviral treatment, HIV overcomes drug pressure by adding new (compensatory) mutations, appearing in a specific and sequential order, that modulate its replication capacity and favour viral escape. In the case of M184V (a mutation involving the catalytic site of HIV reverse transcriptase), no pathways of viral escape have been defined so far; it is thus conceivable that the mutated virus maintains a relatively low replicative capacity. At the time of interruption of specific viral pressure (lamivudine in the case of M184V), wild-type virus easily overgrows mutated strains. A deep molecular analysis (90 clones) conducted on proviral DNA of lymphocytes demonstrates that M184V strains are no longer detected in plasma and proviral DNA shortly after interruption of therapeutic regimens including lamivudine (even if a new therapeutic regimen has been started). This supports the concept that the low fitness of M184V strains is not easily compensated by additional mutations. Taken together, the results suggest that the assessment of viral fitness, either direct (through biological methods) or indirect (through the identification of specific mutations that affect the replicative capacity), may provide substantial advancements in the definition of the long-term efficacy of antiretroviral therapy.