Gustavo del Real

PATHOGENS: RAFT HIJACKERS

Throughout evolution, organisms have developed immune-surveillance networks to protect themselves from potential pathogens. At the cellular level, the signalling events that regulate these defensive responses take place in membrane rafts — dynamic microdomains that are enriched in cholesterol and glycosphingolipids — that facilitate many protein–protein and lipid–protein interactions at the cell surface. Pathogens have evolved many strategies to ensure their own survival and to evade the host immune system, in some cases by hijacking rafts. However, understanding the means by which pathogens exploit rafts might lead to new therapeutic strategies to prevent or alleviate certain infectious diseases, such as those caused by HIV-1 or Ebola virus.

Membrane raft microdomains mediate lateral assemblies required for HIV-1 infection

HIV‐1 infection triggers lateral membrane diffusion following interaction of the viral envelope with cell surface receptors. We show that these membrane changes are necessary for infection, as initial gp120–CD4 engagement leads to redistribution and clustering of membrane microdomains, enabling subsequent interaction of this complex with HIV‐1 co‐receptors. Disruption of cell membrane rafts by cholesterol depletion before viral exposure inhibits entry by both X4 and R5 strains of HIV‐1, although viral replication in infected cells is unaffected by this treatment. This inhibitory effect is fully reversed by cholesterol replenishment of the cell membrane. These results indicate a general mechanism for HIV‐1 envelope glycoprotein‐mediated fusion by reorganization of membrane microdomains in the target cell, and offer new strategies for preventing HIV‐1 infection.

Statins Inhibit HIV-1 infection by down-regulating rho activity

Human immunodeficiency virus (HIV)-1 infectivity requires actin-dependent clustering of host lipid raft–associated receptors, a process that might be linked to Rho guanosine triphosphatase (GTPase) activation. Rho GTPase activity can be negatively regulated by statins, a family of drugs used to treat hypercholesterolemia in man. Statins mediate inhibition of Rho GTPases by impeding prenylation of small G proteins through blockade of 3-hydroxy-3-methylglutaryl coenzyme A reductase. We show that statins decreased viral load and increased CD4+ cell counts in acute infection models and in chronically HIV-1–infected patients. Viral entry and exit was reduced in statin-treated cells, and inhibition was blocked by the addition of l-mevalonate or of geranylgeranylpyrophosphate, but not by cholesterol. Cell treatment with a geranylgeranyl transferase inhibitor, but not a farnesyl transferase inhibitor, specifically inhibited entry of HIV-1–pseudotyped viruses. Statins blocked Rho-A activation induced by HIV-1 binding to target cells, and expression of the dominant negative mutant RhoN19 inhibited HIV-1 envelope fusion with target cell membranes, reducing cell infection rates. We suggest that statins have direct anti–HIV-1 effects by targeting Rho.

A heterologous prime-boost regime using DNA and recombinant vaccinia virus expressing the Leishmania infantum P36/LACK antigen protects BALB/c mice from cutaneous leishmaniasis.

A heterologous prime-boost vaccination with DNA vectors and vaccinia virus recombinants (VVr) has been shown to enhance specific cellular immune responses and to elicit significant protection against pathogens in animal models. In this study, we have analyzed, in the leishmaniasis cutaneous murine model, the effectiveness of this prime-boost strategy by immunizing with a DNA vector followed by boost with a VVr expressing the same Leishmania infantum P36/LACK antigen. After DNA priming and VVr boost, we challenged susceptible BALB/c mice with live L. major promastigotes, and examined the increase in footpad lesion size and parasite load in draining lymph nodes. Compared to controls, we observed reduction of up to 70% in lesion size and 1000-fold in parasite load. DNA prime-VVr boost before challenge elicited a Th1 type immune response in spleen cells from immunized animals. This DNA/VVr vaccination approach could be of utility in the prophylaxis against leishmaniasis.

Blocking of HIV-1 Infection by Targeting CD4 to Nonraft Membrane Domains

Human immunodeficiency virus (HIV)-1 infection depends on multiple lateral interactions between the viral envelope and host cell receptors. Previous studies have suggested that these interactions are possible because HIV-1 receptors CD4, CXCR4, and CCR5 partition in cholesterol-enriched membrane raft domains. We generated CD4 partitioning mutants by substituting or deleting CD4 transmembrane and cytoplasmic domains and the CD4 ectodomain was unaltered. We report that all CD4 mutants that retain raft partitioning mediate HIV-1 entry and CD4-induced Lck activation independently of their transmembrane and cytoplasmic domains. Conversely, CD4 ectodomain targeting to a nonraft membrane fraction results in a CD4 receptor with severely diminished capacity to mediate Lck activation or HIV-1 entry, although this mutant binds gp120 as well as CD4wt. In addition, the nonraft CD4 mutant inhibits HIV-1 X4 and R5 entry in a CD4+ cell line. These results not only indicate that HIV-1 exploits host membrane raft domains as cell entry sites, but also suggest new strategies for preventing HIV-1 infection.

Blocking HIV-1 infection via CCR5 and CXCR4 receptors by acting in trans on the CCR2 chemokine receptor

The identification of chemokine receptors as HIV‐1 coreceptors has focused research on developing strategies to prevent HIV‐1 infection. We generated CCR2‐01, a CCR2 receptor‐specific monoclonal antibody that neither competes with the chemokine CCL2 for binding nor triggers signaling, but nonetheless blocks replication of monotropic (R5) and T‐tropic (X4) HIV‐1 strains. This effect is explained by the ability of CCR2‐01 to induce oligomerization of CCR2 with the CCR5 or CXCR4 viral coreceptors. HIV‐1 infection through CCR5 and CXCR4 receptors can thus be prevented in the absence of steric hindrance or receptor downregulation by acting in trans on a receptor that is rarely used by the virus to infect cells.

HIV-1 envelope protein gp120 triggers a Th2 response in mice that shifts to Th1 in the presence of human growth hormone.

Immunization of mice with HIV-1-gp120 results in predominant activation of the Th2 lymphocyte subset, leading to enhanced IL-4 production. Administration of human growth hormone at the time of gp120 immunization provokes a change in the cytokine production pattern, with lower IL-4 and higher gamma-IFN and IL-2 synthesis levels, indicating a preferential switch in stimulation from Th2 to Th1 cells. A growth hormone would thus be of great use for pharmacological intervention in those cases in which an infectious microorganism evades immune defenses by provoking a Th2 response. In addition, the ability of growth hormone to induce a Th1-type response upon vaccination with an HIV-antigen should be examined in the development of new therapeutic strategies or in the design of novel vaccines against HIV infection.

Novel interfering bifunctional molecules against the CCR5 coreceptor are efficient inhibitors of HIV-1 infection

CCR5 is the major coreceptor for the HIV-1 strains responsible for primary infection. Individuals homozygous for a 32-bp deletion in the CCR5 coding region are resistant to HIV-1 infection. Strategies to delete CCR5 functionally could thus be of substantial benefit in preventing HIV-1 infection or delaying disease. We evaluated new molecules for their ability to inhibit cell membrane CCR5 expression and to prevent HIV-1 infection. These inhibitors include several truncated forms of CCR5 that may act as negative transdominants, as well as bifunctional molecules resulting from the combination of a previously described anti-CCR5 ribozyme or a truncated CCR5 variant with an intracellular chemokine (RANTES-KDEL). These constructs efficiently blocked membrane CCR5 expression when cotransfected into HEK 293 cells. When expressed by retroviral transduction, some of these molecules significantly inhibited CCR5-dependent chemotaxis in the MCF-7 cell line and reduced CCR5 expression and HIV-1 infection in human T cells. Analysis of inhibitors with different efficiencies showed a strong linear correlation between CCR5 expression inhibition and prevention of HIV-1 infection. This study indicates the potential clinical application of several new CCR5 inhibitory molecules for HIV-1 gene therapy.

Molecular analysis of HIV-1 gp120 antibody response using isotype IgM and IgG phage display libraries from a long-term non-progressor HIV-1- infected individual

To characterize the variable heavy chain (VH)3 antibody response to HIV‐1 gp120, we analyzed a panel of IgM and IgG1 Fab fragments from phage display isotype libraries from a long‐term, non‐progressor HIV‐1‐infected individual. The IgM Fab antibodies isolated had low affinity for gp120, were not restricted to a particular VH3 germ‐line gene, and consisted mainly of unmutated VH genes. In contrast, IgG Fab fragments were gp120 specific, with high affinity and extensive somatic mutation; all were clonally related and were derived from a single VH3 germ‐line gene (DP50). One IgG Fab (S8) has DP50 VH region nucleotide substitutions identical to those of IgM Fab M025 and uses similar DH and JH segments, suggesting that S8 arose from M025 by isotype switching. In addition, somatic mutation in the IgG heavy chain third complementarity‐determining region results in a 100‐fold affinity increase for gp120, which correlates with a similar increase in neutralization capacity. These results imply that in vivo IgM to IgG isotype switch and affinity maturation may be important for protection and long‐term survival in certain HIV‐1‐infected individuals.

Improvement in affinity and HIV-1 neutralization by somatic mutation in the heavy chain first complementarity-determining region of antibodies triggered by HIV-1 infection

We assessed the impact of somatic hypermutation in the framework region 1 (FR1) and complementarity‐determining region 1 (CDR1) of three clonally‐related heavy chains from the human monovalent antigen‐binding fragments Fab S19, S8 and S20 on gp120 binding and HIV‐1 neutralization capacity. Nucleotide changes were introduced in the heavy chains to revert single and multiple amino acid residues, and two Fab libraries were constructed with the same light chain to express equivalent amounts of parental and reverted phage Fab. We studied the contribution of each amino acid replacement to antigen binding by calculating the frequency of phage Fab retrieval after competitive library selection on gp120. Whereas mutations in FR1 had no effect on antigen binding, somatic replacements in the CDR1 of the heavy chain (HCDR1) appeared to produce significant changes. In S19 HCDR1, somatic mutation of residue 32 reduced gp120 binding. In Fab S20, the Arg30 and Asp31 somatically replaced residues in HCDR1 improved antigen binding. Both of these residues are necessary to increase Fab binding to gp120; reversion of either residue alone results in a decrease in binding. The impact of these two replacements was confirmed by the greater neutralization capacity of S20 compared to the other Fab. Molecular modeling of S20 HCDR1 suggests that Arg30 and Asp31 are the main interaction sites for gp120, increasing antibody affinity and promoting the enhanced neutralization ability of S20. These findings are consistent with a gp120‐driven process, supporting a role for affinity maturation and intraclonal evolution of HIV‐1 neutralizing antibodies.