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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.

CD4+ T lymphocyte depletion in human immunodeficiency virus type 1 (HIV-1)–infected humans underlies the development of acquired immune deficiency syndrome. Using a model in which rhesus macaques were infected with chimeric simian–human immunodeficiency viruses (SHIVs), we show that both the level of viremia and the structure of the HIV-1 envelope glycoprotein ectodomains individually contributed to the efficiency with which CD4+ T lymphocytes were depleted. The envelope glycoproteins of recombinant SHIVs that efficiently caused loss of CD4+ T lymphocytes exhibited increased chemokine receptor binding and membrane-fusing capacity compared with those of less pathogenic viruses. These studies identify the HIV-1 envelope glycoprotein ectodomains as determinants of CD4+ T lymphocyte loss in vivo and provide a foundation for studying pathogenic mechanisms.

Human immunodeficiency virus type 2 (HIV-2) is similar in genetic organization to HIV-1 but contains a unique gene (vpx) that encodes a 16-kDa protein. A replication-competent molecular clone of HIV-2 (HIV-2sbl/isy) that infects human primary cells in vitro and rhesus monkeys was used to generate three mutations in the vpx gene. In the first mutant, the vpx open reading frame was truncated at amino acid 20; the second mutant was tailored to eliminate the proline-rich carboxyl terminus of the protein; and the third mutant was obtained by addition of four amino acids (KDEL) to the carboxyl terminus of the protein to provide a retention signal in the endoplasmic reticulum. The viral infection kinetics of the three mutant viruses and isogeneic HIV-2sbl/isy in the SupT1 cell line were similar. Slight impairment in the early phases of viral replication was observed during infection of primary human peripheral blood mononuclear cells with the vpx mutant viruses. All of the vpx mutant viruses readily infected macrophages, indicating that vpx expression is dispensable for HIV-2 infection and replication in human 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.

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.

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. © 2004 Lippincott Williams & Wilkins.

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.

A soluble form of the CD4 receptor (sCD4) can either enhance or inhibit the infection of cells by simian immunodeficiency virus (SIV) and human immunodeficiency virus. We investigated the basis for these varying effects by studying the entry of three SIV isolates into CD4-positive and CD4-negative cells expressing different chemokine receptors. Infection of CD4-negative cells depended upon the viral envelope glycoproteins and upon the chemokine receptor, with CCR5 and gpr15 being more efficient than STRL33. Likewise, enhancement of infection by sCD4 was observed when CCR5- and gpr15-expressing target cells were used but not when those expressing STRL33 were used. The sCD4-mediated enhancement of virus infection of CD4-negative, CCR5-positive cells was related to the sCD4-induced increase in binding of the viral gp120 envelope glycoprotein to CCR5. Inhibitory effects of sCD4 could largely be explained by competition for virus attachment to cellular CD4 rather than other detrimental effects on virus infectivity (e.g., disruption of the envelope glycoprotein spike). Consistent with this, the sCD4-activated SIV envelope glycoprotein intermediate on the virus was long-lived. Thus, the net effect of sCD4 on SIV infectivity appears to depend upon the degree of enhancement of chemokine receptor binding and upon the efficiency of competition for cellular CD4.

Human immunodeficiency virus type 1 (HIV-1) enters target cells by sequential binding to CD4 and specific seven-transmembrane-segment (7TMS) coreceptors. Viruses use the chemokine receptor CCR5 as a coreceptor in the early, asymptomatic stages of HIV-1 infection but can adapt to the use of other receptors such as CXCR4 and CCR3 as the infection proceeds. Here we identify one such coreceptor, Apj, which supported the efficient entry of several primary T-cell-line tropic (T-tropic) and dualtropic HIV-1 isolates and the simian immunodeficiency virus SIVmac316. Another 7TMS protein, CCR9, supported the less efficient entry of one primary T-tropic isolate. mRNAs for both receptors were present in phytohemagglutinin- and interleukin-2-activated peripheral blood mononuclear cells. Apj and CCR9 share with other coreceptors for HIV-1 and SIV an N-terminal region rich in aromatic and acidic residues. These results highlight properties common to 7TMS proteins that can function as HIV-1 coreceptors, and they may contribute to an understanding of viral evolution in infected individuals.

CCR5 and CD4 are coreceptors for immunodeficiency virus entry into target cells. The gp120 envelope glycoprotein from human immunodeficiency virus strain HIV-1(YU2) bound human CCR5 (CCR5hu) or rhesus macaque CCR5 (CCR5rh) only in the presence of CD4. The gp120 from simian immunodeficiency virus strain SIVmac239 bound CCR5rhwithout CD4, but CCR5huremained CD4-dependent. The CD4-independent binding of SIVmac239 gp120 depended on a single amino acid, Asp13, in the CCR5rhamino-terminus. Thus, CCR5-binding moieties on the immunodeficiency virus envelope glycoprotein can be generated by interaction with CD4 or by direct interaction with the CCR5 amino-terminus. These results may have implications for the evolution of receptor use among lentiviruses as well as utility in the development of effective intervention.

Clinical isolates of primate immunodeficiency viruses, including human immunodeficiency virus type 1 (HIV-1), enter target cells by sequential binding to CD4 and the chemokine receptor CCR5, a member of the seven-transmembrane receptor family. HIV-1 variants which use additional chemokine receptors are present in the central nervous system or emerge during the course of infection. Simian immunodeficiency viruses (SIV) have been shown to use CCR5 as a coreceptor, but no other receptors for these viruses have been identified. Here we show that two orphan seven-transmembrane segment receptors, gpr1 and gpr15, serve as coreceptors for SIV, and are expressed in human alveolar macrophages. The more efficient of these, gpr15, is also expressed in human CD4+ T lymphocytes and activated rhesus macaque peripheral blood mononuclear cells. The gpr15 and gpr1 proteins lack several hallmarks of chemokine receptors, but share with CCR5 an amino-terminal motif rich in tyrosine residues. These results underscore the potential diversity of seven-transmembrane segment receptors used as entry cofactors by primate immunodeficiency viruses, and may contribute to an understanding of viral variation and pathogenesis.

The infection of macaques by simian immunodeficiency virus (SIVmac) represents an attractive model to study the pathogenic determinants of primate and human immunodeficiency viruses. The utility of this model would be enhanced if genetic changes in human immunodeficiency virus (HIV-1) associated with interesting in vitro properties would, when introduced into SIVmac, result in similar phenotypes. In this study, we introduced amino acid changes into the SIVmac239 envelope glycoproteins that, in the context of the HIV-1 envelope glycoproteins, disproportionately attenuated in vitro cytopathic effects compared with the viral replication rate. Amino acid changes in the SIVmac239 gp41 ectodomain altered the noncovalent association of the gp120 and gp41 glycoproteins significantly more than did analogous changes in the HIV-1 envelope glycoproteins. Decreases in the affinity of the gp120-gp41 interaction were observed and were associated with a dramatic attenuation of virus replication not seen in the HIV-1 studies. The increased sensitivity of the SIVmac gp120-gp41 interaction to amino acid changes presents an obstacle to the direct extension of results obtained with the HIV-1 envelope glycoproteins to the SIVmacaque model.

We examined chemokine receptors for the ability to facilitate the infection of CD4-expressing cells by viruses containing the envelope glycoproteins of a pathogenic simian immunodeficiency virus, SIVmac239. Expression of either human or simian C-C chemokine receptor CCR5 allowed the SIVmac239 envelope glycoproteins to mediate virus entry and cell-to-cell fusion. Thus, distantly related immunodeficiency viruses such as SIV and the primary human immunodeficiency virus type 1 isolates can utilize CCR5 as an entry cofactor.

In a natural context, membrane fusion mediated by the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins involves both the exterior envelope glycoprotein (gp120) and the transmembrane glycoprotein (gp41). Perez et al. (J. Virol. 66:4134-4143, 1992) reported that a mutant HIV-1 envelope glycoprotein containing only the signal peptide and carboxyl terminus of the gp120 exterior glycoprotein fused to the complete gp41 glycoprotein was properly cleaved and that the resultant gp41 glycoprotein was able to induce the fusion of even CD4-negative cells. In the studies reported herein, mutant proteins identical or similar to those studied by Perez et al. lacked detectable cell fusion activity. The proteolytic processing of these proteins was very inefficient, and one processed product identified by Perez et al. as the authentic gp41 glycoprotein was shown to contain carboxyl-terminal gp120 sequences. Furthermore, no fusion activity was observed for gp41 glycoproteins exposed after shedding of the gp120 glycoprotein by soluble CD4. Thus, evidence supporting a gp120-independent cell fusion activity for the HIV-1 gp41 glycoprotein is currently lacking.

The human immunodeficiency virus (HIV) genetic determinant(s) responsible for tropism in human T cells or macrophages are not well defined. We studied the role of the HIV type 2 (HIV-2) nef and vpr genes in viral tropism. HIV-2 mutants, lacking either vpr or nef genes, or both vpr and nef, were obtained by site-specific mutagenesis of a biologically active HIV-2 proviral clone (HIV-2sbl/isy), which is infectious in both human T cells and macrophages. Viral progeny carrying mutations of nef, vpr, or of both nef and vpr genes replicated more efficiently than the parental virus in primary human peripheral blood cells and in the human Hut 78 T-cell line. In contrast, the HIV-2 nef- mutant infected human macrophages as efficiently as the parental virus, whereas viruses lacking the vpr gene either alone or in conjunction with the lack of the nef gene did not replicate in macrophages. Thus, some lack of nef in HIV-2 enhances viral replication in T cells and does not interfere with viral replication in primary macrophages, whereas vpr is essential for replication of HIV-2 in human macrophages. Because the parental HIV-2sbl/isy cloned virus also infects rhesus macaques, the use in animal studies of these HIV-2 mutants with differences in cell tropism and rates of replication will be highly useful in understanding the mechanism of viral infectivity and possibly pathogenicity in vivo.

Activated T cells synthesize and express a cell membrane-bound receptor for interleukin-2 (IL-2) and have recently been shown to secrete a soluble form of the same receptor. Hairy cell leukemia is a chronic disorder caused by expansion of a clonal population of an unusual mononuclear cell of B cell origin. These cells have previously been shown to express an IL-2 receptor on the cell membrane. The sera of 26 patients with hairy cell leukemia were examined for the presence of a soluble IL-2 receptor before and during therapy with either recombinant interferon alpha-2a or 2′-deoxycoformycin. Before therapy, all patients had markedly elevated levels of this soluble IL-2 receptor ranging from five to 60 times the highest level observed in normal control sera. In individual patients changes in the level during therapy correlated well with clinical assessments of tumor response; levels fell to near the normal range in patients responding to therapy. Patients not responding to interferon alpha had no significant change in the soluble IL-2 receptor level. These results suggest that hairy cells secrete a soluble IL-2 receptor and that serial measurements of the level of this receptor in the serum can be used as a noninvasive means to assess disease response to therapy.

A solid-phase, competition enzyme-linked immunosorbent assay (ELISA) was established for the quantitative measurement of soluble (human) interleukin-2 receptors (IL-2R). The ladder of reagents from the solid phase up consisted of: (1) recombinant DNA-derived, purified IL-2R, (2) sample-containing soluble IL-2R and fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody, 7G7/B6, directed against the IL-2R, (3) alkaline phosphatase-conjugated rabbit anti-FITC, and (4) substrate. This ELISA was compared with a 'sandwich' ELISA for soluble IL-2R. The competitive ELISA was less sensitive than the 'sandwich' assay, being capable of measuring 5000 versus 31 U/ml, respectively. While both anti-Tac and 7G7/B6 in the IL-2R-containing sample inhibited the 'sandwich' assay, only 7G7/B6 inhibited the competition assay. Anti-mouse immunoglobulin enhanced the 'sandwich' assay and inhibited the competitive assay; both effects could be overcome by the addition of normal mouse immunoglobulin in the sample buffer. Studies of a patient's serum receiving anti-Tac as therapy for the adult T cell leukemia demonstrated that rises in the level of IL-2R occurring with anti-Tac therapy, as measured with the competition assay, were masked in the 'sandwich' assay. This competition ELISA will be useful for measuring soluble IL-2R levels in patients receiving anti-Tac as therapy for various immunologic disorders.

In vivo T cell preactivation in chronic uremic hemodialyzed and non-hemodialyzed patients. The production and targeting of a major T cell derived lymphokine, Interleukin 2 (IL-2), were studied in 23 uremic patients undergoing regular hemodialysis treatment and 20 uremic patients prior to the onset of renal replacement therapy. In hemodialyzed patients, abnormally increased proportions of circulating T cells spontaneously expressing high affinity IL-2 receptors (IL-2 Rec) were detected; they bound a monoclonal antibody specifically directed to the IL-2 Rec 55kDa chain (Tac antigen) (mean ± SEM: 7.12 ± 0.81% in patients vs. 2.15 ±0.39% in normal controls, P < 0.0001) and significantly proliferated in presence of human recombinant IL-2 alone (mean ± SEM: 5438 ± 729cpm in patients vs. 1647 ± 244cpm in normal controls). Hemodialyzed patients also exhibited significantly increased serum levels of soluble IL-2 receptor (mean ± SEM: 4036 ± 947U/ml in patients vs. 253 ± 29 U/ml in normal controls, P < 0.001). Moreover, a significantly decreased IL-2 activity was detected in the supernatants of stimulated T cells from hemodialyzed patients (mean ± SEM: 0.93 ± 0.12 U/ml in patients vs. 2.49 ± 0.22 U/ml in normal controls, P < 0.0001). In nine hemodialyzed patients who were analyzed before and immediately after the hemodialysis session no acute modifications of the various parameters analyzed were detected. Although less profound, a similar pattern of T cell abnormalities was observed in the uremic non-hemodialyzed patients studied. These data suggest that the deficient IL-2 activity in uremic patients may be at least in part related to increased absorption by IL-2 receptor present in abnormally high amounts on T cells and in the serum. Furthermore, they underline that although exacerbated by hemodialysis treatment, a latent state of T cell preactivation is associated with end-stage chronic renal failure.

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.

Adult T-cell leukemia associated with human T-cell leukemia virus type I (HTLV-I) is characterized by a clonal expansion of a CD4-positive subset of T lymphocytes that constitutively express high numbers of interleukin-2 receptors and that frequently infiltrate the skin; osteolytic bone lesions, and hypercalcemia. Using an enzyme-linked immunosorbent assay (ELISA) test, we measured the level of soluble Tac peptide, one chain of the human interleukin-2 receptor, in the serum of 50 patients with adult T-cell leukemia (38 Japanese and 12 American patients), 8 patients with other hematologic malignancies, 8 asymptomatic HTLV-I-antibody-positive carriers, and 17 normal controls. The serum level of soluble Tac peptide (geometric mean U/mL, 95% CI) was elevated at presentation in all patients with adult T-cell leukemia (16,461; 819 to 330,896) when compared with normal controls (238; 112 to 502), patients with other hematologic malignancies (1302; 475 to 3569), and healthy HTLV-I antibody-positive carriers (490; 115 to 2086). The highest levels were seen in patients (n = 33) with acute (32,154; 2587 to 399,598) compared with chronic (5464; 661 to 45,156) disease (n = 14). Serum levels of Tac peptide also tended to be more elevated in patients with adult T-cell leukemia with hypercalcemia (32,072; 2461 to 417,908) compared with normocalcemic patients (13,885; 496 to 388,436). Serial measurements of soluble Tac peptide levels in serum were done in four patients with adult T-cell leukemia during chemotherapy and the levels reflected disease activity. These observations suggest that the measurement of soluble Tac peptide levels in patients with adult T-cell leukemia is useful as a noninvasive measure of tumor burden and will help in the diagnosis of the disease and management of these patients.