Lokesh Agrawal

Role for CCR5Δ32 Protein in Resistance to R5, R5X4, and X4 Human Immunodeficiency Virus Type 1 in Primary CD4+ Cells

ABSTRACT CCR5Δ32 is a loss-of-function mutation that abolishes cell surface expression of the human immunodeficiency virus (HIV) coreceptor CCR5 and provides genetic resistance to HIV infection and disease progression. Since CXCR4 and other HIV coreceptors also exist, we hypothesized that CCR5Δ32-mediated resistance may be due not only to the loss of CCR5 function but also to a gain-of-function mechanism, specifically the active inhibition of alternative coreceptors by the mutant CCR5Δ32 protein. Here we demonstrate that efficient expression of the CCR5Δ32 protein in primary CD4+ cells by use of a recombinant adenovirus (Ad5/Δ32) was able to down-regulate surface expression of both wild-type CCR5 and CXCR4 and to confer broad resistance to R5, R5X4, and X4 HIV type 1 (HIV-1). This may be important clinically, since we found that CD4+ cells purified from peripheral blood mononuclear cells of individuals who were homozygous for CCR5Δ32, which expressed the mutant protein endogenously, consistently expressed lower levels of CXCR4 and showed less susceptibility to X4 HIV-1 isolates than cells from individuals lacking the mutation. Moreover, CD4+ cells from individuals who were homozygous for CCR5Δ32 expressed the mutant protein in five of five HIV-exposed, uninfected donors tested but not in either of two HIV-infected donors tested. The mechanism of inhibition may involve direct scavenging, since we were able to observe a direct interaction of CCR5 and CXCR4 with CCR5Δ32, both by genetic criteria using the yeast two-hybrid system and by biochemical criteria using the coimmunoprecipitation of heterodimers. Thus, these results suggest that at least two distinct mechanisms may account for genetic resistance to HIV conferred by CCR5Δ32: the loss of wild-type CCR5 surface expression and the generation of CCR5Δ32 protein, which functions as a scavenger of both CCR5 and CXCR4.

Anti-HIV Therapy: Current and Future Directions

Although combinations of drugs that target the HIV reverse transcriptase and protease enzymes have clearly revolutionized the treatment of HIV/AIDS, problems with these agents, such as viral escape mutants, persistence of viral reservoirs, poor patient compliance due to complicated regimens, and toxic side effects, have emphasized the need for development of new drugs with novel mechanisms of action, as well as an HIV vaccine. Recently two new classes of drugs have been identified that interfere with the membrane fusion reaction required for HIV entry of target cells. Two such agents, T-20 (enfuvirtide) and T-1249, which have been approved by the Food and Drug Administration (FDA), block the action of the fusogenic envelope glycoprotein gp41. Others target the HIV coreceptors CCR5 and CXCR4, and are now in clinical trials. Also under development are novel agents that target the HIV integrase and HIV regulatory gene products as well as immunomodulators such as IL-12 and IL-2. This article will focus on these and other novel approaches to HIV therapeutics.

Generating neutralizing antibodies, Th1 response and MHC non restricted immunogenicity of HIV-I env and gag peptides in liposomes and ISCOMs with in-built adjuvanticity

For enhancing immunogenicity and develop vaccine strategies using peptide based constructs against HIV-1, a chimeric peptide containing V3 loop and transmembrane sequence of gp41 with two glycine motifs as spacer was constructed. The V3-gp41, gp41 peptide and p17 and p24 peptides separately or in a cocktail were entrapped with or without MA729 as an immunoadjuvant in liposomes or ISCOMs. The immunogenicity, antigen induced T-cell proliferation and cytokine profiles of various formulations were studied in four different inbred strains of mice of H-2d, H-2b, H-2k and H-2q haplotypes, keeping alum as a control adjuvant. Both liposomes and ISCOM preparations elicited high titer and long lasting antibody response (60 days and above). When compared to the alum formulation, the liposomes co-entrapped with MA729 produced high antibody levels, comparable with that induced by ISCOMs. Peptide in alum, liposomes and ISCOMs enhanced both antigen specific IgG2a and IgG2b isotypes and high T-cell stimulation index. Peptide formulations also induced antibodies with high affinity and in vitro neutralizated the formation of HIV-1 syncytia. T-cell supernatants contained high levels of IFN-γ and IL-2. Thus formulation in these adjuvants induced a predominant Th1 like response with MA729 as a versatile novel delivery vehicle for stimulating the appropriate arm of the immune response that can selectively modulate MHC class I or MHC class II response. The above peptide can be of wide vaccination interest as a means to improve immune responses to several other HIV-1 antigens and may serve as candidates for vaccine development.

CCR5Δ32 59537-G/A Promoter Polymorphism Is Associated with Low Translational Efficiency and the Loss of CCR5Δ32 Protective Effects

ABSTRACT We have recently demonstrated that the CCR5Δ32 protein interacts with CCR5 and CXCR4 and down-modulates their cell surface expression. We have also reported the absence of detectable expression of the truncated CCR5Δ32 protein in four out of six human immunodeficiency virus-infected (HIV+) CCR5−/− individuals. To explain the defect in protein expression in these samples, we cloned and sequenced the promoter regions of the six HIV+ individuals. We have identified several polymorphisms in the CCR5Δ32 promoter region, but these polymorphisms were not associated with significant differences in mRNA levels. Coupled in vitro transcription/translation and polyribosome analysis demonstrated a strong association between a variant genotype designated CCR5Δ32 59537-A/A and a low translation efficiency. Protein analysis indicated that the peripheral blood mononuclear cells from two of the HIV+CCR5−/− individuals carrying the CCR5Δ32 59537-A/A variant expressed trace amounts of CCR5Δ32 protein compared to the individuals carrying the CCR5Δ32 59537-G/G genotype. The results imply that the absence of CCR5Δ32 protein in two HIV+ individuals is due to a genetic defect in the translation of the protein. Together, these results highlight the importance of the CCR5Δ32 protein as an HIV suppressive factor and provide further insight into the mechanism of the protective effect of the CCR5Δ32 mutation.

CCR5Δ32 Protein Expression and Stability Are Critical for Resistance to Human Immunodeficiency Virus Type 1 In Vivo

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) infection of individuals carrying the two alleles of the CCR5Δ32 mutation (CCR5−/−) has rarely been reported, but how the virus overcomes the CCR5Δ32 protective effect in these cases has not been delineated. We have investigated this in 6 infected (HIV+) and 25 HIV− CCR5−/− individuals. CD4+ T lymphocytes isolated from HIV− CCR5−/− peripheral blood mononuclear cells (PBMCs) showed lower levels of CXCR4 expression that correlated with lower X4 Env-mediated fusion. Endogenous CCR5Δ32 protein was detected in all HIV− CCR5−/− PBMC samples (n = 25) but not in four of six unrelated HIV+ CCR5−/− PBMC samples. Low levels were detected in another two HIV+ CCR5−/− PBMC samples. The expression of adenovirus 5 (Ad5)-encoded CCR5Δ32 protein restored the protective effect in PBMCs from three HIV+ CCR5−/− individuals but failed to restore the protective effect in PBMCs isolated from another three HIV+ CCR5−/− individuals. In the latter samples, pulse-chase analyses demonstrated the disappearance of endogenous Ad5-encoded CCR5Δ32 protein and the accumulation of Ad5-encoded CCR5 during the chase periods. PBMCs isolated from CCR5−/− individuals showed resistance to primary X4 but were readily infected by a lab-adapted X4 strain. Low levels of Ad5-encoded CCR5Δ32 protein conferred resistance to primary X4 but not to lab-adapted X4 virus. These data provide strong support for the hypothesis that the CCR5Δ32 protein actively confers resistance to HIV-1 in vivo and suggest that the loss or reduction of CCR5Δ32 protein expression may account for HIV-1 infection of CCR5−/− individuals. The results also suggest that other cellular or virally induced factors may be involved in the stability of CCR5Δ32 protein.

Chemokine receptors: emerging opportunities for new anti-HIV therapies.

The chemokine receptors CCR5 and CXCR4 are G-protein coupled receptors (GPCRs) of the immune system and the major co-receptors required for entry of HIV into CD4+ target cells. CCR5 is critical for both human immunodeficiency virus (HIV) disease transmission and progression, whereas CXCR4 may be very important in late stages of disease. Additional co-receptors have been shown to function under certain conditions in vitro but evidence of supporting roles in HIV disease is currently lacking. The sheer number of co-receptors potentially used by HIV and the complexity of co-receptors usage are major challenges confronting usage of these molecules as drug development targets. Balanced against this, is a long history of success by the pharmaceutical industry in developing small molecule antagonists for many other classes of GPCRs. In this review, we discuss the current state of understanding of the co-receptor-based antiviral agents designed to block viral entry. The therapeutic potential of this field will be judged from future studies on the efficacy of these novel inhibitors in clinical trials. The data so far obtained from a number of studies point to the potential clinical use of this emerging class of therapeutic agents. Here we review current progress in co-receptor-based antiretroviral drug development and discuss the potential advantages and disadvantages of this approach.