The performance of the VIKIA(®) HIV1/2 rapid test-evaluation of the reliability and sensitivity.

The use of rapid human immunodeficiency virus (HIV) antibody tests can help reduce the number of individuals positive for HIV who are unaware of their infection. Although several studies have demonstrated that the sensitivity and specificity of rapid HIV tests are comparable to those of enzyme immunoassays, none have addressed the rapidity with which these tests can yield a result and the reliability of such results. In this study, we investigated the performance of VIKIA(®) HIV1/2 rapid tests regarding early reactive results and the stability of these results after sample addition. The results showed that using HIV-1 or HIV-2 positive samples, a positive result could be observed as early as 1 min after the addition of the sample. The ability of this test to detect early HIV-1 primary infection was also assessed using seroconversion specimens. The results demonstrate the high sensitivity of this test, and its suitability for the identification of seroconversion samples in the context of primary infection with HIV-1.

[The chemokines and their receptors: characteristics and physiological functions]. / As quimiocinas e os seus receptores: características e funções fisiológicas.

Chemokines are members of a large family of small soluble proteins, which were discovered by their adhesion control, chemotaxis and leukocyte activation abilities. Nevertheless, it is now known they are involved in other equally important functions, namely, angiogenesis, haematopoiesis, embryologic development, B and T cell development, dendritic cell maturation, inflammation, infection, tumour growth and metastasis. Hence, the increasing interest on chemokines and their receptors is due not only to chemokine chemoattractant properties but also to their contribution to immune processes that do not directly involve leukocyte migration. According to the number and spacing of the first two conserved cysteine residues in the N-terminal, chemokines have been divided into four subfamilies (CXC, CC, CX3C and C) and mediate their functions by binding to G-protein coupled receptors. This interaction may result in multiple signal transduction pathways, depending on the player subunit and the effector protein activated. It triggers a cascade of intracellular events that promote from gene transcription to cytoskeleton rearrangement and chemotaxis. The pattern of migration of cell populations and of leukocytes in particular is closely related to chemokine receptors expression. One of the characteristics associated with the chemokine system is an apparent redundancy: several chemokine receptors bind multiple ligands and in turn, a single ligand binds multiple receptors. Another overall classification of chemokines is based on functional criteria that group them into homeostatic and inflammatory chemokines. Homeostatic chemokines are constitutively expressed and regulate the migration of leukocytes and their precursors. The inflammatory chemokines are inducible and regulate the leukocyte migration into tissues in response to an inflammatory stimulus, such as tissue damage, inflammation or infection. Many of the inflammatory chemokines have wide target-cell selectivity, acting both on the cells of the innate and adaptive immunity. The purpose of this review is to collect all the research that has been done so far concerning chemokines and their receptors through analysing their expression patterns, in order to define their cellular localisation with the aim of understanding their role in human physiology.

Coreceptor usage by HIV-1 and HIV-2 primary isolates: the relevance of CCR8 chemokine receptor as an alternative coreceptor.

The human immunodeficiency virus replication cycle begins by sequential interactions between viral envelope glycoproteins with CD4 molecule and a member of the seven-transmembrane, G-protein-coupled, receptors' family (coreceptor). In this report we focused on the contribution of CCR8 as alternative coreceptor for HIV-1 and HIV-2 isolates. We found that this coreceptor was efficiently used not only by HIV-2 but particularly by HIV-1 isolates. We demonstrate that CXCR4 usage, either alone or together with CCR5 and/or CCR8, was more frequently observed in HIV-1 than in HIV-2 isolates. Directly related to this is the finding that the non-usage of CXCR4 is significantly more common in HIV-2 isolates; both features could be associated with the slower disease progression generally observed in HIV-2 infected patients. The ability of some viral isolates to use alternative coreceptors besides CCR5 and CXCR4 could further impact on the efficacy of entry inhibitor therapy and possibly also in HIV pathogenesis.

Characterization of HIV-2 chimeric viruses unable to use CCR5 and CXCR4 coreceptors.

We have previously shown the existence of primary human immunodeficiency virus type 2 isolates (MIC97 and MJC97) unable to use major coreceptors to entry into peripheral blood mononuclear cells, including CCR5 and CXCR4. We have now created a set of chimeric viruses derived from HIV-2(ROD), to study the contribution of env gene products in chemokine receptors usage and replication kinetics phenotype. The results obtained indicate that an env gene fragment, corresponding to the C1-C4 region of SU glycoprotein of both MIC97 and MJC97, impair efficient utilization of the major HIV coreceptors CCR5 and CXCR4 in phytohemagglutinin-stimulated peripheral blood mononuclear cells by ROD-MIC97 and ROD-MJC97 chimeric viruses. It also disrupts the ability to utilize established coreceptors for viral entry into GHOST-CD4 coreceptor-expressing cell lines. Resistance to CCR5 and CXCR4 inhibitors, as well as the ability to infect Delta32/Delta32ccr5 PBMC, was also observed in recombinant viruses containing the C1-C4 region from either MIC97 or MJC97. We also show that the presence of the TM region of env gene from MIC97 and MJC97 is sufficient to reduce viral replicative kinetics of ROD strain, indicating that this region, despite the presence and contribution of ROD genetic backbone, has an important role in viral progeny production efficiency.

[Chemokine receptors and its importance in the replication cycle of human immunodeficiency virus: clinical and therapeutic implications]. / Os receptores das quimiocinas e a sua importância no ciclo replicativo do vírus da imunodeficiência humana: implicações clínicas e terapéuticas.

Human immunodeficiency virus (HIV) infection of host cells begins with binding of viral envelope (Env) surface (SU) glycoprotein to specific receptors present at plasma cell membrane. One of these receptors is the CD4 molecule which can be found namely in T-helper lymphocytes (T-CD4+), macrophages and dendritic cells. Binding of SU glycoprotein to CD4 molecule, enables HIV to adsorb to target cell membrane and also initiates conformational changes in SU glycoprotein that enable it to bind to a second receptor (coreceptor). This coreceptor belongs to a family of plasma cell membrane molecules that acts, in vivo, as chemokine receptors. The SU glycoprotein binding to coreceptor molecule triggers further conformational changes and consequently the exposure of a hydrophobic region of viral envelope transmembrane glycoprotein (TM), named fusion peptide that ultimately leads to viral envelope fusion with target cell membrane. Nowadays, 19 of these chemokine receptors have been thus considered, in vitro, as coreceptors for HIV. Interestingly, despite this extensive range of potential coreceptors, only CCR5 and CXCR4 seem to be relevant in HIV transmission and in the pathogenesis of HIV infection. Identification of cell surface coreceptors, specific for HIV envelope SU glycoprotein, has provided an elucidative explanation for molecular mechanisms involved in viral cell tropism and pathogenesis. Furthermore, the recognition of a coreceptor-mediated HIV's entry has also provided novel viral and cellular targets for antiretroviral intervention. During the last few years, the inhibition of HIV entry has become an incontestable target for anti-HIV drug discovery. Enfuvirtide is one example of these new antiretroviral molecules. It is the only member of fusion inhibitors targeting fusion peptide region, which prevents HIV entry by blocking the TM-mediated fusion between viral envelope and plasma cell membrane. More recently, CCR5-specific antagonists have been described, including monoclonal antibodies, modified chemokines and more importantly small-molecules inhibitors, such as maraviroc and vicriviroc. These drugs prevent SU glycoprotein binding to CCR5 coreceptor, and thus inhibiting HIV entry into target cell. This review will focus on the influence of coreceptor engagement, by HIV Env glycoproteins, in viral replication cycle and the importance of targeting its coreceptor function, by specific inhibitors, as a new and promising class of antiretrovirals.

Construction and characterization of CD4-independent infectious recombinant HIV-2 molecular clones.

Human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) differ in their pathogenic mechanisms as evidenced by lower rate of disease progression, lower transmission rates and lower viral load in peripheral blood for HIV-2. One of the many factors that are involved in these characteristics is the interaction between viral glycoproteins and cellular receptors. The study of these interactions in an HIV-2 model could lead to important conclusions regarding pathogenesis and transmission mechanisms of HIV-2 infection. Here we report the design of a method enabling the construction of recombinant proviral HIV-2 DNAs in a moderate copy number plasmid that allows the analysis of env gene structure and functionality. This method constitutes an important tool for the study of HIV-2 env glycoproteins and for the mappings of genetic determinants of HIV-2 coreceptor usage and CD4-independent interaction. Furthermore, this knowledge will help towards the understanding of the different pathogenic mechanisms of HIV-1 and HIV-2.

Identification and characterization of HIV-2 strains obtained from asymptomatic patients that do not use CCR5 or CXCR4 coreceptors.

In vivo, human immunodeficiency virus type 2 (HIV-2) infection reveals several unique characteristics when compared to HIV-1 infection, the most remarkable of which is the extraordinarily long asymptomatic period. Here we describe two HIV-2 primary isolates, obtained from asymptomatic individuals, which do not infect any coreceptor-expressing cell lines tested. In those cells, we show that the absence of replication is directly related to cell entry events. Furthermore, productive infection observed in peripheral blood mononuclear cells (PBMC) was not inhibited by natural ligands and monoclonal antibodies directed to CCR5 and CXCR4. Finally, viral entry efficiency and viral progeny production of these viruses are markedly impaired in PBMC, indicating a reduced replicative fitness of both viruses. In conclusion, our data suggest that in some HIV-2 asymptomatic individuals, the circulating viruses are unable to use the major coreceptors to infect PBMC. This fact should have important implications in HIV-2 pathogenesis and transmission.

Primary human immunodeficiency virus type 2 (HIV-2) isolates infect CD4-negative cells via CCR5 and CXCR4: comparison with HIV-1 and simian immunodeficiency virus and relevance to cell tropism in vivo.

Cell surface receptors exploited by human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) for infection are major determinants of tropism. HIV-1 usually requires two receptors to infect cells. Gp120 on HIV-1 virions binds CD4 on the cell surface, triggering conformational rearrangements that create or expose a binding site for a seven-transmembrane (7TM) coreceptor. Although HIV-2 and SIV strains also use CD4, several laboratory-adapted HIV-2 strains infect cells without CD4, via an interaction with the coreceptor CXCR4. Moreover, the envelope glycoproteins of SIV of macaques (SIV(MAC)) can bind to and initiate infection of CD4(-) cells via CCR5. Here, we show that most primary HIV-2 isolates can infect either CCR5(+) or CXCR4(+) cells without CD4. The efficiency of CD4-independent infection by HIV-2 was comparable to that of SIV, but markedly higher than that of HIV-1. CD4-independent HIV-2 strains that could use both CCR5 and CXCR4 to infect CD4(+) cells were only able to use one of these receptors in the absence of CD4. Our observations therefore indicate (i) that HIV-2 and SIV envelope glycoproteins form a distinct conformation that enables contact with a 7TM receptor without CD4, and (ii) the use of CD4 enables a wider range of 7TM receptors to be exploited for infection and may assist adaptation or switching to new coreceptors in vivo. Primary CD4(-) fetal astrocyte cultures expressed CXCR4 and supported replication by the T-cell-line-adapted ROD/B strain. Productive infection by primary X4 strains was only triggered upon treatment of virus with soluble CD4. Thus, many primary HIV-2 strains infect CCR5(+) or CXCR4(+) cell lines without CD4 in vitro. CD4(-) cells that express these coreceptors in vivo, however, may still resist HIV-2 entry due to insufficient coreceptor concentration on the cell surface to trigger fusion or their expression in a conformation nonfunctional as a coreceptor. Our study, however, emphasizes that primary HIV-2 strains carry the potential to infect CD4(-) cells expressing CCR5 or CXCR4 in vivo.

Multicenter evaluation of a fully automated screening test, VIDAS HIV 1 + 2, for antibodies to human immunodeficiency virus types 1 and 2.

A multicenter study was done to evaluate the sensitivity, specificity, and efficiency of a new screening test for the simultaneous detection of human immunodeficiency virus type 1 and 2 (HIV-1 and HIV-2) antibodies. The VIDAS HIV 1 + 2 (bioMérieux, Marcy l'Etoile, France) is a fully automated enzyme-linked fluorescent immunoassay that uses synthetic peptides from immunodominant regions of gp41 of HIV-1 and gp36 of HIV-2 as antigens. A total of 2,984 samples were evaluated with this system in six different laboratories, and the results were compared to those obtained with other enzyme-linked immunosorbent assays. The VIDAS HIV 1 + 2 assay showed a very good performance in terms of sensitivity (100%) and specificity (99.6%), requiring minimal manipulation and short incubation time (32 min) to give results similar to or better than those of the other enzyme-linked immunosorbent assays used for screening.