Quirina Santos-Costa

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.

HIV-2 Infection and Chemokine Receptors Usage - Clues to Reduced Virulence of HIV-2

Human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) are the causative agents of Acquired Immunodeficiency Syndrome (AIDS). Without therapeutic intervention, HIV-1 or HIV-2 infections in humans are characterized by a gradual and irreversible immunologic failure that ultimately leads to the onset of a severe immunodeficiency that constitutes the hallmark of AIDS. In the last two decades AIDS has evolved into a global epidemic affecting millions of persons worldwide. Although sharing several identical properties, HIV-1 and HIV-2 have shown some important differences in vivo. In fact, a significant amount of epidemiologic, clinical and virologic data suggest that HIV-2 is in general less virulent than HIV-1. This reduced virulence is revealed by the longer asymptomatic period and the smaller transmission rate that characteristically are observed in HIV-2 infection. In this context, studies using HIV-2 as a model of a naturally less pathogenic infection could bring important new insights to HIV pathogenesis opening to new strategies to vaccines or therapeutic design. The reasons underlying the reduced pathogenicity of HIV-2 are still essentially unknown and surely are the outcome of a combination of distinct factors. In this review we will discuss the importance and the possible implications in HIV-2 pathogenesis, particularly during the asymptomatic period, of a less fitted interaction between viral envelope glycoproteins and cellular receptors that have been described in the way HIV-2 and HIV-1 use these receptors.

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.

Molecular characterization of the env gene of two CCR5/CXCR4-independent human immunodeficiency 2 primary isolates.

Human immunodeficiency virus 2 (HIV‐2) infection is characterized by a slower disease progression and lower transmission rates. The molecular features that could be assigned as directly involved in this in vivo phenotype remain essentially unknown, and the importance of HIV‐2 as a model to understand pathogenicity of HIV infection has been frequently underestimated. The early events of the HIV replication cycle involve the interaction between viral envelope glycoproteins and cellular receptors: the CD4 molecule and a chemokine receptor, usually CCR5 or CXCR4. Despite the importance of these two chemokine receptors in human immunodeficiency virus 1 (HIV‐1) entry into cells, we have previously shown that in some HIV‐2 asymptomatic individuals, a viral population exists that is unable to use both CCR5 and CXCR4. The goal of the present study was to investigate whether possible regions in the env gene of these viruses might account for this phenotype. From the molecular characterization of these env genes we could not detect any correlation between V3 loop sequence and viral phenotype. In contrast, it reveals the existence of remarkable differences in the V1/V2 and C5 regions of the surface glycoprotein, including the loss of a putative glycosilation site. Moreover, in the transmembrane glycoprotein some unique sequence signatures could be detected in the central ectodomain and second heptad repeat (HR2). Some of the mutations affect well‐conserved residues, and may affect the conformation and/or the dynamics of envelope glycoproteins complex, including the SU–TM association and the modulation of viral entry function. J. Med. Virol. 81:1869–1881, 2009.

Development of synthetic light-chain antibodies as novel and potent HIV fusion inhibitors.

Objective:To develop a novel and potent fusion inhibitor of HIV infection based on a rational strategy for synthetic antibody library construction. Design:The reduced molecular weight of single-domain antibodies (sdAbs) allows targeting of cryptic epitopes, the most conserved and critical ones in the context of HIV entry. Heavy-chain sdAbs from camelids are particularly suited for this type of epitope recognition because of the presence of long and flexible antigen-binding regions [complementary-determining regions (CDRs)]. Methods:We translated camelid CDR features to a rabbit light-chain variable domain (VL) and constructed a library of minimal antibody fragments with elongated CDRs. Additionally to elongation, CDRs’ variability was restricted to binding favorable amino acids to potentiate the selection of high-affinity sdAbs. The synthetic library was screened against a conserved, hidden, and crucial-to-fusion sequence on the heptad-repeat 1 (HR1) region of the HIV-1 envelope glycoprotein. Results:Two anti-HR1 VLs, named F63 and D104, strongly inhibited laboratory-adapted HIV-1 infectivity. F63 also inhibited infectivity of HIV-1 and HIV-2 primary isolates similarly to the Food and Drug Administration-approved fusion inhibitor T-20 and HIV-1 strains resistant to T-20. Moreover, epitope mapping of F63 revealed a novel target sequence within the highly conserved hydrophobic pocket of HR1. F63 was also capable of interacting with viral and cell lipid membrane models, a property previously associated with T-20s inhibitory mechanism. Conclusion:In summary, to our best knowledge, we developed the first potent and broad VL sdAb fusion inhibitor of HIV infection. Our study also gives insights into engineering strategies that could be explored to enhance the development of antiviral drugs.

HIV-2 interaction with cell coreceptors: amino acids within the V1/V2 region of viral envelope are determinant for CCR8, CCR5 and CXCR4 usage

BackgroundHuman immunodeficiency virus 1 and 2 (HIV-1 and HIV-2) use cellular receptors in distinct ways. Besides a more promiscuous usage of coreceptors by HIV-2 and a more frequent detection of CD4-independent HIV-2 isolates, we have previously identified two HIV-2 isolates (HIV-2MIC97 and HIV-2MJC97) that do not use the two major HIV coreceptors: CCR5 and CXCR4. All these features suggest that in HIV-2 the Env glycoprotein subunits may have a different structural organization enabling distinct - although probably less efficient - interactions with cellular receptors.ResultsBy infectivity assays using GHOST cell line expressing CD4 and CCR8 and blocking experiments using CCR8-specific ligand, I-309, we show that efficient replication of HIV-2MIC97 and HIV-2MJC97 requires the presence of CCR8 at plasma cell membrane. Additionally, we disclosed the determinants of chemokine receptor usage at the molecular level, and deciphered the amino acids involved in the usage of CCR8 (R8 phenotype) and in the switch from CCR8 to CCR5 or to CCR5/CXCR4 usage (R5 or R5X4 phenotype). The data obtained from site-directed mutagenesis clearly indicates that the main genetic determinants of coreceptor tropism are located within the V1/V2 region of Env surface glycoprotein of these two viruses.ConclusionsWe conclude that a viral population able to use CCR8 and unable to infect CCR5 or CXCR4-positive cells, may exist in some HIV-2 infected individuals during an undefined time period, in the course of the asymptomatic stage of infection. This suggests that in vivo alternate molecules might contribute to HIV infection of natural target cells, at least under certain circumstances. Furthermore we provide direct and unequivocal evidence that the usage of CCR8 and the switch from R8 to R5 or R5X4 phenotype is determined by amino acids located in the base and tip of V1 and V2 loops of HIV-2 Env surface glycoprotein.

Characterization of Env surface glycoprotein from HIV-2 primary isolates with different coreceptor usage profile

The main goal of this work was to identify molecular signatures in envelope surface glycoprotein that may be correlated with coreceptor usage by different human immunodeficiency virus (HIV)-2 strains. From inspection of aligned HIV-2 sequences, we verified that V1/V2 region showed the highest degree of amino acid sequence heterogeneity, including polymorphisms in N-linked glycosylation sites, sequence, and length. Furthermore, we did not find any correlation between the net charge and specific amino acid positions in V3 region with any particular coreceptor usage pattern. In conclusion, we showed that for HIV-2, the genetic determinants for coreceptor usage are distinct from those of HIV-1. More specifically, we did not identify any molecular signature, based on discrete amino acid positions either in V1/V2 or in V3 regions, which could be assigned to the preferential usage of a specific coreceptor.

The HIV-2 SU Glycoprotein Influence Proviral Integration Dynamics into Human CD4+ T-Lymphocytes

*Corresponding author Quirina Santos-Costa, PhD, MsD, PharmD Director of Biossafety Level 3 Facility (BSL3/P3); Assistant Professor, Departamento de Microbiologia e Imunologia da Faculdade de Farmácia da Universidade de Lisboa (DMI-FFULisboa); Investigator at Host-Pathogen Interaction Unit Research Institute for Medicines and Pharmaceutical Sciences Instituto de Investigação do Medicamento (iMed.ULisboa) and Instituto de Medicina Molecular (IMM), Faculdade de Medicina da Universidade de Lisboa, Portugal Tel. +351 217946400; 00351-919700131 Fax: +351 217934212 E-mail: quirina.c@ff.ul.pt