Peter A. Zimmerman

CCR5 promoter polymorphism and HIV-1 disease progression

BACKGROUND The rate of progression to AIDS varies among individuals infected with HIV-1. Factors responsible include two inherited human alleles, CCR5 delta32 and CCR2-641, which alter the protein-coding regions for the HIV-1 coreceptors/chemokine receptors CCR5 and CCR2b. We tested the hypothesis that polymorphisms of the CCR5 promoter might affect the rate of progression of HIV-1 infected people to AIDS. METHODS We used directed heteroduplex analysis to identify polymorphism in the CCR5 promoter. Promoter-variants were compared in vitro with a chloramphenicol acetyltransferase reporter gene, and in vivo by genotyping HIV-1 seroconvertors discordant at polymorphous loci. FINDINGS An A/G polymorphism was identified at basepair 59029 (Genbank U95626) in the CCR5 promoter. Both promoter alleles were common (43-68% allelic frequency for 59029-A depending on race). When in-vitro promoter activity was measured, 59029-G had 45% lower activity than 59029-A (p=0.05). In a cohort of HIV-1 seroconvertors lacking both CCR5 delta32 and CCR2-641, 59029-G/G individuals progressed to AIDS on average 3.8 years more slowly than 59029-A/A individuals (p=0.004). 59029-G/A discordance did not correlate with discordant rates of infection. INTERPRETATION Our results are consistent with the hypothesis that CCR5 is important in HIV-1 pathogenesis. CCR5 59029-G/G appears to be protective relative to CCR5 59029-A/A, and about twice as protective relative to CCR5 delta32 or CCR2-641. This effect may be the result of reduced CCR5 mRNA production. These results identify the first site in the CCR5 promoter that may be a useful target for treatment of HIV-1 infection.

Plasmodium falciparum erythrocyte invasion through glycophorin C and selection for Gerbich negativity in human populations

Geographic overlap between malaria and the occurrence of mutant hemoglobin and erythrocyte surface proteins has indicated that polymorphisms in human genes have been selected by severe malaria. Deletion of exon 3 in the glycophorin C gene (called GYPCΔex3 here) has been found in Melanesians; this alteration changes the serologic phenotype of the Gerbich (Ge) blood group system, resulting in Ge negativity. The GYPCΔex3 allele reaches a high frequency (46.5%) in coastal areas of Papua New Guinea where malaria is hyperendemic. The Plasmodium falciparum erythrocyte-binding antigen 140 (EBA140, also known as BAEBL) binds with high affinity to the surface of human erythrocytes. Here we show that the receptor for EBA140 is glycophorin C (GYPC) and that this interaction mediates a principal P. falciparum invasion pathway into human erythrocytes. EBA140 does not bind to GYPC in Ge-negative erythrocytes, nor can P. falciparum invade such cells using this invasion pathway. This provides compelling evidence that Ge negativity has arisen in Melanesian populations through natural selection by severe malaria.

Plasmodium vivax clinical malaria is commonly observed in Duffy-negative Malagasy people

Malaria therapy, experimental, and epidemiological studies have shown that erythrocyte Duffy blood group-negative people, largely of African ancestry, are resistant to erythrocyte Plasmodium vivax infection. These findings established a paradigm that the Duffy antigen is required for P. vivax erythrocyte invasion. P. vivax is endemic in Madagascar, where admixture of Duffy-negative and Duffy-positive populations of diverse ethnic backgrounds has occurred over 2 millennia. There, we investigated susceptibility to P. vivax blood-stage infection and disease in association with Duffy blood group polymorphism. Duffy blood group genotyping identified 72% Duffy-negative individuals (FY*BES/*BES) in community surveys conducted at eight sentinel sites. Flow cytometry and adsorption–elution results confirmed the absence of Duffy antigen expression on Duffy-negative erythrocytes. P. vivax PCR positivity was observed in 8.8% (42/476) of asymptomatic Duffy-negative people. Clinical vivax malaria was identified in Duffy-negative subjects with nine P. vivax monoinfections and eight mixed Plasmodium species infections that included P. vivax (4.9 and 4.4% of 183 participants, respectively). Microscopy examination of blood smears confirmed blood-stage development of P. vivax, including gametocytes. Genotyping of polymorphic surface and microsatellite markers suggested that multiple P. vivax strains were infecting Duffy-negative people. In Madagascar, P. vivax has broken through its dependence on the Duffy antigen for establishing human blood-stage infection and disease. Further studies are necessary to identify the parasite and host molecules that enable this Duffy-independent P. vivax invasion of human erythrocytes.

Chemokine RANTES promoter polymorphism affects risk of both HIV infection and disease progression in the Multicenter AIDS Cohort Study.

ObjectiveTo examine whether polymorphism in the RANTES gene is associated with HIV disease outcome. DesignRANTES, a ligand of the major HIV co-receptor, CCR5, is known to block HIV-CCR5 interactions. Recently, two single nucleotide polymorphisms in the RANTES gene promoter region, designated −403G/A and −28C/G, have been described. Both polymorphisms can affect in-vitro promoter activity, and the RANTES −403A, −28G haplotype has been associated with a slower CD4 cell count decline rate in a Japanese cohort. MethodsWe compared RANTES compound genotype frequencies between HIV-positive and exposed-uninfected participants of the Multicenter AIDS Cohort Study (MACS) and rates of progression to AIDS for MACS seroconverters. ResultsWe found that the two most common RANTES promoter compound genotypes, G1 (−403G/G, −28C/C) found in 67% of Caucasians, and G4 (−403G/A, −28C/C) found in 23% of Caucasians, were associated with altered risk of HIV transmission and progression, particularly in individuals who lacked the protective CCR5 mutation, CCR5Δ32. In this study, individuals with a G4 compound genotype were more likely to acquire HIV than individuals with a G1 compound genotype (OR 1.72, P = 0.016) and the risk increased when individuals possessing CCR5Δ32 were omitted from consideration (OR 2.13, P = 0.005). Among seroconverters lacking CCR5Δ32, those who had the G4 compound genotype progressed significantly slower to AIDS-1993 than those with the G1 compound genotype (median time to AIDS 7.6 versus 5.4 years; RH 0.65;P = 0.007). ConclusionsThese data implicate the RANTES-403A allele as a risk factor for HIV transmission and as a protective factor for HIV progression.

Epidemiologic and biologic characterization of a cohort of human immunodeficiency virus type 1 highly exposed, persistently seronegative female sex workers in northern Thailand

Characterization of persons highly exposed to human immunodeficiency virus (HIV)-1 who remain uninfected may help define protective immunity. Seventeen HIV-1-seronegative Thai female sex workers (CSWs) with epidemiologic evidence of exposure to HIV-1 were studied for humoral immune responses and phenotypic and genotypic analyses of HLA class I and CCR5 allelic profiles. Infected CSWs and low-risk HIV-1-seronegative Thai women were controls. Highly exposed, persistently seronegative (HEPS) CSWs did not differ from HIV-infected CSWs in HIV risks, condom use, or sexually transmitted diseases. Significant differences were seen in humoral immune responses: gp160-specific IgA responses were detected in cervicovaginal lavage fluids in 6 of 13 HEPS CSWs but 0 of 21 seronegative subjects. All women had wild-type CCR5. HEPS CSWs were more likely to have the HLA-B18 phenotype and genotype than were matched controls (corrected P=.018). Epidemiologic exposure to HIV-1 without apparent infection, an unusual distribution of HLA class I alleles, and HIV-1 gp160-specific IgA responses suggest a biologic basis for this phenomenon.

Comparing the ex vivo fitness of CCR5-tropic human immunodeficiency virus type 1 isolates of subtypes B and C.

ABSTRACT Continual human immunodeficiency virus type 1 (HIV-1) evolution and expansion within the human population have led to unequal distribution of HIV-1 group M subtypes. In particular, recent outgrowth of subtype C in southern Africa, India, and China has fueled speculation that subtype C isolates may be more fit in vivo. In this study, nine subtype B and six subtype C HIV-1 isolates were added to peripheral blood mononuclear cell cultures for a complete pairwise competition experiment. All subtype C HIV-1 isolates were less fit than subtype B isolates (P < 0.0001), but intrasubtype variations in HIV-1 fitness were not significant. Increased fitness of subtype B over subtype C was also observed in primary CD4+ T cells and macrophages from different human donors but not in skin-derived human Langerhans cells. Detailed analysis of the retroviral life cycle during several B and C virus competitions indicated that the efficiency of host cell entry may have a significant impact on relative fitness. Furthermore, phyletic analyses of fitness differences suggested that, for a recombined subtype B/C HIV-1 isolate, higher fitness mapped to the subtype B env gene rather than the subtype C gag and pol genes. These results suggest that subtype B and C HIV-1 may be transmitted with equal efficiency (Langerhans cell data) but that subtype C isolates may be less fit following initial infection (T-cell and macrophage data) and may lead to slower disease progression.

R5 HIV productively infects Langerhans cells, and infection levels are regulated by compound CCR5 polymorphisms

Langerhans cells (LCs) are suspected to be initial targets for HIV after sexual exposure (by becoming infected or by capturing virus). Here, productive R5 HIV infection of LC ex vivo and LC-mediated transmission of virus to CD4+ T cells were both found to depend on CCR5. By contrast, infection of monocyte-derived dendritic cells and transfer of infection from monocyte-derived dendritic cells to CD4+ T cells were mediated by CCR5-dependent as well as DC-specific ICAM-3-grabbing nonintegrin-dependent pathways. Furthermore, in 62 healthy individuals, R5 HIV infection levels in LCs ex vivo were associated with CCR5 genotype. Specifically, genotyping for ORFΔ32 revealed that LCs isolated from ORFΔ32/wt individuals were significantly less susceptible to HIV when compared with LCs isolated from ORFwt/wt individuals (P = 0.016). Strikingly, further genetic analyses of the A-2459G CCR5 promoter polymorphism in ORFΔ32/wt heterozygous individuals revealed that LCs isolated from -2459A/G + ORFΔ32/wt individuals were markedly less susceptible to HIV than were LCs from -2459A/A + ORFΔ32/wt individuals (P = 0.012). Interestingly, these genetic susceptibility data in LCs parallel those of genetic susceptibility studies performed in cohorts of HIV-infected individuals. Thus, we suggest that CCR5-mediated infection of LCs, and not capture of virus by LCs, provides a biologic basis for understanding certain aspects of host genetic susceptibility to initial HIV infection.

Evolution of a unique Plasmodium falciparum chloroquine-resistance phenotype in association with pfcrt polymorphism in Papua New Guinea and South America

The mechanistic basis for chloroquine resistance (CQR) in Plasmodium falciparum recently has been linked to the polymorphic gene pfcrt. Alleles associated with CQR in natural parasite isolates harbor threonine (T), as opposed to lysine (K) at amino acid 76. P. falciparum CQR strains of African and Southeast Asian origin carry pfcrt alleles encoding an amino acid haplotype of CVIET (residues 72–76), whereas most South American CQR strains studied carry an allele encoding an SVMNT haplotype; chloroquine-sensitive strains from malarious regions around the world carry a CVMNK haplotype. Upon investigating the origin of pfcrt alleles in Papua New Guinean (PNG) P. falciparum we found either the chloroquine-sensitive-associated CVMNK or CQR-associated SVMNT haplotypes previously seen in Brazilian isolates. Remarkably we did not find the CVIET haplotype observed in CQR strains from Southeast Asian regions more proximal to PNG. Further we found a previously undescribed CQR phenotype to be associated with the SVMNT haplotype from PNG and South America. This CQR phenotype is significantly less responsive to verapamil chemosensitization compared with the effect associated with the CVIET haplotype. Consistent with this, we observed that verapamil treatment of P. falciparum isolates carrying pfcrt SVMNT is associated with an attenuated increase in digestive vacuole pH relative to CVIET pfcrt-carrying isolates. These data suggest a key role for pH-dependent changes in hematin receptor concentration in the P. falciparum CQR mechanism. Our findings also suggest that P. falciparum CQR has arisen through multiple evolutionary pathways associated with pfcrt K76T.

Plasmodium vivax Invasion of Human Erythrocytes Inhibited by Antibodies Directed against the Duffy Binding Protein

Background Plasmodium vivax invasion requires interaction between the human Duffy antigen on the surface of erythrocytes and the P. vivax Duffy binding protein (PvDBP) expressed by the parasite. Given that Duffy-negative individuals are resistant and that Duffy-negative heterozygotes show reduced susceptibility to blood-stage infection, we hypothesized that antibodies directed against region two of P. vivax Duffy binding protein (PvDBPII) would inhibit P. vivax invasion of human erythrocytes. Methods and Findings Using a recombinant region two of the P. vivax Duffy binding protein (rPvDBPII), polyclonal antibodies were generated from immunized rabbits and affinity purified from the pooled sera of 14 P. vivax–exposed Papua New Guineans. It was determined by ELISA and by flow cytometry, respectively, that both rabbit and human antibodies inhibited binding of rPvDBPII to the Duffy antigen N-terminal region and to Duffy-positive human erythrocytes. Additionally, using immunofluorescent microscopy, the antibodies were shown to attach to native PvDBP on the apical end of the P. vivax merozoite. In vitro invasion assays, using blood isolates from individuals in the Mae Sot district of Thailand, showed that addition of rabbit anti-PvDBPII Ab or serum (antibodies against, or serum containing antibodies against, region two of the Plasmodium vivax Duffy binding protein) (1:100) reduced the number of parasite invasions by up to 64%, while pooled PvDBPII antisera from P. vivax–exposed people reduced P. vivax invasion by up to 54%. Conclusions These results show, for what we believe to be the first time, that both rabbit and human antibodies directed against PvDBPII reduce invasion efficiency of wild P. vivax isolated from infected patients, and suggest that a PvDBP-based vaccine may reduce human blood-stage P. vivax infection.

Genetic polymorphisms in molecules of innate immunity and susceptibility to infection with Wuchereria bancrofti in South India.

A pilot study was conducted to determine if host genetic factors influence susceptibility and outcomes in human filariasis. Using the candidate gene approach, a well-characterized population in South India was studied using common polymorphisms in six genes (CHIT1, MPO, NRAMP, CYBA, NCF2, and MBL2). A total of 216 individuals from South India were genotyped; 67 normal (N), 63 asymptomatic microfilaria positive (MF+), 50 with chronic lymphatic dysfunction/elephantiasis (CP), and 36 tropical pulmonary eosinophilia (TPE). An association was observed between the HH variant CHIT1 genotype, which correlates with decreased activity and levels of chitotriosidase and susceptibility to filarial infection (MF+ and CP; P = 0.013). The heterozygosity of CHIT1 gene was over-represented in the normal individuals (P = 0.034). The XX genotype of the promoter region in MBL2 was associated with susceptibility to filariasis (P = 0.0093). Since analysis for MBL-sufficient vs insufficient haplotypes was not informative, it is possible the MBL2 promoter association results from linkage disequilibrium with neighboring loci. We have identified two polymorphisms, CHIT1 and MBL2 that are associated with susceptibility to human filarial infection, findings that merit further follow-up in a larger study.