Human immunodeficiency virus type 1 biological variation and coreceptor use: from concept to clinical significance.

There is ample evidence for intra-patient evolution of the human immunodeficiency virus type 1 (HIV-1) biological phenotype during the pathogenic process. Evolution often involves switch of coreceptor use from CCR5 to CXCR4, but change to more flexible use of CCR5 occurs over time even in patients with maintained CCR5 use. The increasing use of entry inhibitors in the clinic, often specific for one or the other HIV-1 coreceptor or with different binding properties to CCR5, calls for virus testing in patients prior to treatment initiation. Cell lines expressing CCR5/CXCR4 chimeric receptors are tools for testing viruses for mode of CCR5 use. It is conceivable that small-molecule entry inhibitors that differentially bind to CCR5 can be matched for best effect against HIV-1 with different modes of CCR5 use, thereby allowing an individualized drug choice specifically tailored for each patient.

High prevalence of HIV-1, HIV-2 and other sexually transmitted infections among women attending two sexual health clinics in Bissau, Guinea-Bissau, West Africa.

The objective was to examine the prevalence of HIV-1, HIV-2 and 10 other sexually transmitted infections (STIs), and to explore the relationship between HIV and those STIs in women attending two sexual health clinics in Bissau, Guinea-Bissau. In all, 711 women with urogenital problems were included. Clinical examination was performed and HIV-1, HIV-2, human T-cell lymphotropic virus (HTLV)-1, HTLV-2 and syphilis were diagnosed by serology. Trichomonas vaginalis was examined using wet mount microscopy. Cervical samples (and swabs from visible ulcers, if present) were used for polymerase chain reaction (PCR) diagnosis of Chlamydia trachomatis, Mycoplasma genitalium, Haemophilus ducreyi, herpes simplex virus (HSV)-1 and HSV-2, and culture diagnosis of Neisseria gonorrhoeae. The prevalence of HIV-1, HIV-2, and HIV-1 and HIV-2 (dual infection) was 9.5%, 1.8% and 1.1%, respectively. The prevalence of HTLV-1 was 2.8%, HTLV-2 0%, HSV-1 1.4%, HSV-2 7.7%, T. vaginalis 20.4%, syphilis 1.0%, N. gonorrhoeae 1.3%, H. ducreyi 2.7%, M. genitalium 7.7% and C. trachomatis 12.6%. HIV-1 and/or HIV-2 infection was significantly associated with active HSV-2 and HIV-1 was significantly associated with M. genitalium infection. In conclusion, HIV-1 and HIV-2 prevalence was higher compared with previous studies of pregnant women in Guinea-Bissau. The prevalence of co-infection of HIV and other STIs is high. National evidence-based guidelines for the management of STIs in Guinea-Bissau are essential.

The X4 phenotype of HIV type 1 evolves from R5 in two children of mothers, carrying X4, and is not linked to transmission.

Previously, we found that emergence of the X4 viral phenotype in HIV-1-infected children was related to the presence of X4 in their mothers (C.H. Casper et al., J Infect Dis 2002; 186:914-921). Here, we investigated the origin of the X4 phenotype in the child, analyzing two mother-child pairs (Ma-Ca, Mb-Cb) where the mothers carried X4 and their children developed X4 after an initial presence of R5. We used nested polymerase chain reaction of the env V3 region to generate 203 HIV-1 clones for sequencing (Ma, n = 44; Ca, n = 73; Mb, n = 61; Cb, n = 25) from DNA of peripheral blood mononuclear cell (PBMC) lysates, altogether 167 clones, or from cDNA of plasma RNA, 36 clones. PBMC and plasma isolate sequences from each time point enabled us to assign the probable phenotype to clone sequences in a phylogenetic tree. The transmission and evolution were reconstructed using the maximum likelihood method. In mother-child pair Ma-Ca, one maternal R5 isolate clustered with the child's R5 sequences, at the earliest time when R5 was isolated in the child, confirming this as a likely source of the transmitted R5 phenotype. At age 3, an X4 population was present in the child that had evolved from the child's own R5-associated population, clearly distinct from the maternal X4 sequences. The second mother-child pair (Mb-Cb) displayed a similar pattern. Amino acid substitution patterns corroborated the conclusions from the phylogenetic tree. Thus, in both children, the X4 virus developed from their own R5 population, and was not caused by transmission of X4.

A new cell line-based neutralization assay for primary HIV type 1 isolates.

Simple and standardized assays for detection and quantification of neutralizing antibodies to primary HIV-1 isolates are needed in research on HIV-1 vaccines and pathogenesis. Here we describe a new HIV-1 neutralization assay that is based on plaque formation in U87.CD4-CCR5 and U87.CD4-CXCR4 cells, which is an attractive alternative to peripheral blood mononuclear cell-based assays. Infected cells form syncytia, that is, plaques, that can be stained with hematoxylin and enumerated by light microscopy. Neutralization is determined by the ability of a serum to reduce the number of plaque-forming units (PFU) relative to controls exposed to medium or negative serum. The intraassay variation of the plaque-forming unit determinations was tested with 15 serum-virus combinations and showed good reproducibility. The differences ranged from -19 to +27% and had a standard deviation of +/- 9.1%. On the basis of these data the cutoff for neutralization (i.e., plaque reduction) was set to 30% (3.3 standard deviations). Virus titration experiments showed that neutralization results were dependent on virus dose and therefore the neutralization assays should be performed with a virus dose of 10-100 PFU/well. The reproducibility of the new neutralization assay was tested with 4 primary viruses and 9 sera for a total of 20 virus-serum combinations. The mean difference in neutralization (i.e. plaque reduction) determinations performed on different days was as small as 11%. None of 10 Swedish sera and 1 Ugandan plasma pool from HIV-1-uninfected subjects were positive for neutralization, indicating that the assay has high specificity. In summary, the new U87.CD4 cell line-based neutralization assay for primary HIV-1 isolates is a highly reproducible, sensitive, and high-throughput assay that is well suited for large-scale HIV-1 neutralization studies.

Coreceptor change appears after immune deficiency is established in children infected with different HIV-1 subtypes.

Change of HIV-1 coreceptor use has been connected to progression of disease in children infected with HIV-1, presumably subtype B. It has not been possible to discern whether the appearance of new viral phenotypes precedes disease development or comes as a consequence of it. We studied the evolution of coreceptor use in HIV-1 isolates from 24 vertically infected children. Their clinical, virological, and immunological status was recorded and the env V3 subtype was determined by DNA sequencing. Coreceptor use was tested on human cell lines, expressing CD4 together with CCR5, CXCR4, and other chemokine receptors. The children carried five different env subtypes (nine A, five B, four C, three D, and one G) and one circulating recombinant form, CRF01_AE (n = 2). Of the 143 isolates, 86 originated from peripheral blood mononuclear cells (PBMCs) and 57 originated from plasma, received at 90 time points. In 52 of 54 paired plasma and PBMC isolates the coreceptor use was concordant. All 74 isolates obtained at 41 time points during the first year of life used CCR5. A change from use of CCR5 to use of CXCR4 occurred in four children infected with subtype A, D, or CRF01_AE after they had reached 1.5 to 5.8 years of age. There was a significant association with decreased CD4+ cell levels and severity of disease but, interestingly, the coreceptor change appeared months or even years after the beginning of the immunological deterioration. Thus CXCR4-using virus may emerge as a possible consequence of immune deficiency. The results provide new insights into AIDS development in children.

Protection of neutralization epitopes in the V3 loop of oligomeric human immunodeficiency virus type 1 glycoprotein 120 by N-linked oligosaccharides in the V1 region.

The V3 region of the human immunodeficiency virus type 1 envelope protein gp120 constitutes a potential neutralization target, but the oligosaccharide of one conserved N-glycosylation site in this region protects it from neutralizing antibodies. Here, we determined whether N-linked glycans of other gp120 domains were also involved in protection of V3 neutralization epitopes. Two molecular clones of HIV-1, one lacking three N-linked glycans of the V1 region (HIV-1(3N/V1)) and another lacking three N-linked glycans of the C2 region (HIV-1(3N/C2)), were created and characterized. gp120 from both mutated viral clones had higher electrophoretic mobilities than gp120 from wild-type virus, confirming loss of N-linked glycans. Wild-type virus and both mutant clones replicated equally well in established T cell lines and all three viruses were able to utilize CXCR4 but not CCR5 as a coreceptor. The induced mutations increased gp120 affinity for CXCR4 but caused no corresponding increase in viral ability to replicate in T cell lines. HIV-1(3N/V1) was neutralized at about 25 times lower concentrations of an antibody to the V3 region than were wild-type virus and HIV-1(3N/C2). Soluble, monomeric gp120 from HIV-1(3N/V1) and wild type virus had identical avidity for the V3 antibody, indicating that the V1 glycans were able to shield V3 only in oligomeric but not monomeric gp120. In conclusion, one or more N-linked glycans of gp120 V1 is engaged in protection of the V3 region from potential neutralizing antibodies, and this effect is dependent on the oligomeric organization of gp120/gp41.

Mother-to-child transmission of HIV-1: the role of HIV-1 variability and the placental barrier.

The acquired immunodeficiency syndrome (AIDS), which is caused by the human immunodeficiency virus (HIV), was first described in the United States of America in 1981 [1]. The worldwide spread of HIV has soon been recognized and AIDS has become one of the most alarming infectious diseases of our days. Its impact has been tremendous, high morbidity and mortality has caused a reversal of socioeconomic gains previously recorded in several developing countries, especially those in Sub-Saharan Africa [2]. Epidemiological data about the HIV and AIDS pandemic are updated by the Joint United Nation Programme on HIV/AIDS, UNAIDS (http://www.unaids.org). Their latest report from December 2000 states that in year 2000 approximately 5.3 million people have become newly infected with HIV, of which 2.2 were women and 600,000 children younger than 15 years of age. The estimated number of people living with HIV/AIDS globally is 36.1 million, of which 16.4 million are women and 1.4 million are children younger than 15 years of age. Approximately 25.3 million (70%) of these HIV infected people live in Sub-Saharan Africa, 5.8 million in South- and South-East Asia (15%), and 1.4 million in Latin-America (5%). During year 2000, 3 million people died of AIDS (1.3 million women and 500,000 children younger than 15 years of age). This means that an estimated total of 21.8 million persons have died of AIDS since the beginning of the epidemic, including 4.3 million children younger than 15 years of age.

Quantitative evaluation of HIV-1 coreceptor use in the GHOST3 cell assay.

The utility of the GHOST(3) cell assay has been evaluated for testing coreceptor use of primary human immunodeficiency virus type 1 (HIV-1) isolates. GHOST(3) cells were derived from the human osteosarcoma cell line, HOS, and have been engineered to stably express CD4 and one or another of the chemokine receptors CCR3, CCR5, CXCR4, Bonzo, or the orphan receptor BOB. The indicator cell line carries the HIV-2 long terminal repeat-driven green fluorescence protein (GFP) gene, which becomes activated upon infection with HIV or simian immunodeficiency virus. Viral entry is followed by Tat activation of transcription and GFP becomes expressed. Infected cells can be detected 2 or 3 days after infection by simple fluorescence microscopic observation. This simplicity is the main advantage of the GHOST(3) cell system and makes it particularly suitable for screening of a large number of isolates. In addition, the efficiency of coreceptor use can be accurately quantitated with flow cytometric analysis. Here, we evaluated the coreceptor use of 59 primary HIV-1 isolates of different subtypes.

Coreceptor usage of sequential isolates from cynomolgus monkeys experimentally Infected with simian immunodeficiency virus (SIVsm).

Sequential isolates from eight cynomolgus monkeys experimentally infected with simian immunodeficiency virus (SIVsm, of sooty mangabey origin) were tested for coreceptor use in the human osteosarcoma indicator cell line, GHOST(3), expressing CD4 and one or another of the chemokine receptors CCR3, CCR5, CXCR4, BOB, or the orphan receptor Bonzo. The indicator cell line carries the human immunodeficiency virus type 2 long terminal repeat-driven green fluorescence protein gene that becomes activated upon infection with HIV or SIV and fluorescence can be quantitated by flow cytometric analysis. The methodological details are described in the accompanying paper (Vödrös et al., 2001, Virology 290, in press). All SIVsm inoculum viruses and reisolates used CCR5 with a high level of efficiency. CCR5 use was stable over time. BOB and Bonzo use was less efficient than CCR5 use and, in particular, late isolates obtained at the time of immunodeficiency varied greatly in their coreceptor use and often could not establish a productive infection in BOB- or Bonzo-expressing cells. Unexpectedly, early reisolates obtained 12 days postinfection could infect the entire GHOST(3) panel including the parental cells. In one case this was due to use of CXCR4, either transfected or endogenously expressed on the GHOST(3) cells. Our results demonstrate the complex coreceptor use of SIVsm isolates. Moreover, they focus attention on the initial phase of virus replication when the availability of target cells may govern the replication pattern of the virus.

HIV phenotype correlates with the relative amounts of lymphocyte function-related molecule 1 (LFA-1) and major histocompatibility complex (MHC) class II in the virion envelope.

OBJECTIVE: The biological phenotype of HIV-1 has been associated with various aspects of its infectivity, including syncytium formation and coreceptor usage. Adhesion molecules, present on both the target cell and the virus, have also been shown to play a role in the infectious process. A possible correlation between the presence of adhesion molecules in the envelope of HIV-1 with the biological phenotype of the virus is examined. DESIGN: The envelopes of 56 isolates of HIV-1 of known biological phenotype were analyzed for the presence of lymphocyte function-related molecule 1 (LFA-1) and major histocompatibility complex (MHC) class II molecules. METHODS: The coreceptor usage of each isolate was determined in a GHOST cell or a U87.CD4 infectivity assay. The presence of LFA-1 and MHC class II in each virus envelope was then determined using a virus-binding enzyme-linked immunosorbent assay (ELISA). RESULTS: Viruses using the chemokine receptor CCR5 have relatively higher levels of MHC class II than LFA-1 in their envelopes compared with those using CXCR4. CONCLUSIONS: The finding that there is a differential incorporation of MHC class II and LFA-1 molecules by CXCR4- and CCR5-using viruses augments the list of properties contributing to the biological phenotype of HIV-1. This may explain, in part, how CXCR4-using viruses are able to bind to and infect a broader range of cell types than CCR5-using viruses, and why CXCR4-using viruses are associated with a more aggressive disease course.

Evidence of the existence of a new circulating recombinant form of HIV type 1 subtype A/J in Cameroon. The European Network on the Study of In Utero Transmission of HIV-1.

Several genetic subtypes and circulating recombinant forms (CRFs) of HIV-1 have been identified. The greatest degree of genetic diversity is displayed by variants from Central and West Africa. HIV-1 env C2-V5 and protease sequences were obtained from 15 HIV-1-infected pregnant women, who were selected from a larger cohort study in Yaoundé, Cameroon. Fourteen of 15 virus variants were shown to be recombinant, whereas a single variant appeared to be nonrecombinant subtype A. Five viruses were subtype A/J recombinants, with env genes derived from subtype A and protease genes derived from subtype J. Seven viruses clustered with reference sequences for CRF02 AG(IbNG) in both the env and protease gene fragments, and were thus subtype A/G recombinants. Two variants displayed even more complex recombination patterns. Phylogenetic analyses indicated that the five subtype A/J recombinants might be the first representatives of a previously unrecognized CRF.

Structural prerequisites for intersubtype B and D antigenicity of the third variable envelope region (V3) of human immunodeficiency virus type 1.

To elucidate the structural requirements for intersubtype antigenicity of human immunodeficiency virus type 1 (HIV-1) third variable envelope region (V3), synthetic peptides were used in enzyme immunoassays (EIAs) with serum samples from persons with proven or probable subtype B and D infections. Mathematical analyses of results from EIAs with singly substituted V3 peptides revealed important residues determining overall N-terminal V3 peptide antigenicity. This information was used to design V3 immunogens, rabbit antiserum to which were tested in EIA and for in vitro neutralization of molecular clones of HIV-1(MN) and HIV-1(MAL). Intersubtype-reactive epitopes were distributed toward the N-terminal half of the V3 loop. Lysine at position 310, arginine at position 311, and isoleucine at position 314, all derived from the MN primary sequence, were major determinants of intersubtype V3 antigenicity. Combinations of residues that enhanced antigenicity often contained lysine at position 310. Threonine at position 308 was common in the least advantageous combinations. V3 immunogens modified to achieve optimal antigenicity induced antiserum with augmented cross-neutralization of virus from MAL and MN molecular clones, suggesting one approach to subunit vaccine development.

Coreceptor usage of HIV-1 isolates representing different genetic subtypes obtained from pregnant Cameroonian women. European Network for In Utero Transmission of HIV-1.

In this study, coreceptor usage of HIV-1 other than subtype B in relation to HIV-1 transmission from mother to child was investigated. Repeated sampling of 42 HIV-1-seropositive, asymptomatic women in Cameroon during the second and third trimesters of pregnancy, at delivery, and 6 months postpartum were performed. Env subtyping was carried out from uncultured peripheral blood mononuclear cells (PBMCs) by heteroduplex mobility assay and, whenever necessary, by DNA sequencing. Virus isolates were tested for coreceptor usage on human cell lines-U87.CD4 and GHOST(3)-engineered to express stably CD4 and the chemokine receptors CCR1, CCR2b, CCR3, CCR5, or CXCR4, or the orphan receptors BOB/gpr15 or Bonzo/STRL33/TYMSTR. Transmission rate was 11.9%. Viruses were predominantly envelope subtype A and used CCR5 as coreceptor and, surprisingly, 4 of 28 (14.2%) isolates from mothers and 1 of 3 isolates from children used the orphan receptor Bonzo as well. In 2 transmitting mothers from whom sequential HIV-1 isolates were available, viral coreceptor usage evolved from CCR5 monotropic to CCR5/Bonzo dual tropic during pregnancy, and in 1 case transmission of this virus could be documented. Our data suggest that evolution of HIV-1 coreceptor usage to dual (or multi-) tropism may occur during pregnancy.

The trophoblastic epithelial barrier is not infected in full-term placentae of human immunodeficiency virus-seropositive mothers undergoing antiretroviral therapy.

To study the mechanism of the placental barrier function, we examined 10 matched samples of term placentae, cord blood, and maternal blood obtained at delivery from human immunodeficiency virus (HIV)-infected mothers with children diagnosed as HIV negative in Sweden. All placentae were histologically normal, and immunochemistry for HIV type 1 p24 and gp120 antigens was negative. Highly purified trophoblasts (93 to 99% purity) were negative for HIV DNA and RNA, indicating that the trophoblasts were uninfected. Although HIV DNA was detected in placenta-derived T lymphocytes and monocytes, microsatellite analysis showed that these cells were a mixture of maternal and fetal cells. Our study indicates that the placental barrier, i.e., the trophoblastic layer, is not HIV infected and, consequently, HIV infection of the fetus is likely to occur through other routes, such as breaks in the placental barrier.

Comparative studies on neutralisation of primary HIV-1 isolates by human sera and rabbit anti-V3 peptide sera.

IgG binding to V3 peptides and serum neutralising responses were studied in four HIV-1 infected individuals with progressive disease over a period of 31-70 months. The 18-20 mer peptides comprised residues 299-317 (numbering of HIV1 MN) in the N-terminal half of the V3 loop of the envelope glycoprotein gp120 and were derived from the sequences of autologous, as well as heterologous isolates. All four individuals studied lacked anti-V3 IgG binding to at least one autologous V3 sequence. V3 peptides to which autologous sera lacked binding IgG were all immunogenic in rabbits and induced antisera that were broadly cross-reactive by EIA and broadly cross-neutralising to primary HIV-1 isolates. This indicates that the peptides are immunogenic per se and that the respective human hosts have selective defects in recognising the corresponding V3 sequences. Despite the absence of antibody binding to autologous V3 peptides, the human sera had neutralising antibodies to autologous (three out of four cases), as well as heterologous isolates (all cases). Moreover, in vitro exposure of the patients' isolates to autologous neutralising serum or the homologous rabbit antiserum selected for variants with amino acid substitutions close to the crown of the V3 loop or in regions outside the sequence corresponding to peptides used for immunisation. The amino acid exchanges affected V3 positions known to be antigenic and which are also prone to change successively in infected persons. It is likely that neutralising antibodies recognise both linear and conformational epitopes in the V3 loop. Apparently, there are several, but restricted, numbers of ways for this structure to change its conformation and thereby give rise to neutralisation resistant viruses.

Phenotypic characteristics of human immunodeficiency virus type 1 subtype C isolates of Ethiopian AIDS patients.

It has been estimated that, to date, about 48% of all HIV-infected people in the world carry HIV-1 subtype C virus. Therefore, it is of great importance to gain better knowledge about the genetic and biological characteristics of this virus subtype. In the present study, the biological properties of HIV-1 isolates obtained from nine Ethiopian patients with AIDS were studied. DNA sequencing of the V3 loop of gp120 classified the isolates as subtype C. In primary isolation cultures, virus infection was accompanied by syncytium formation and cell lysis. Interestingly, when examining the growth in primary monocyte-macrophage cultures, initial low-level virus replication was followed by a nonproductive state, from which virus could be rescued by cocultivation with Jurkat(tat) cells. Furthermore, none of the isolates replicated in T cell lines (CEM, MT-2, HuT-78, and H9) or in the promonocytic cell line U937 clone 2. All isolates could use CCR5 as coreceptor, whereas no isolates could use CCR2b, CCR3, CCR5, CXCR4, Bonzo/STRL33, or BOB/GPR15. The genotype of the V3 region correlated with the MT-2 negative/non-syncytium-inducing (NSI) phenotype. Comparative studies revealed that the scarcity of CXCR4 usage as well as other phenotypic characteristics of subtype C isolates distinguish this subtype. On the basis of these data, we suggest that in addition, factors other than viral phenotype may govern the pathogenic potential of subtype C isolates.

Primary human immunodeficiency virus type 2 (HIV-2) isolates, like HIV-1 isolates, frequently use CCR5 but show promiscuity in coreceptor usage.

Coreceptor usage of primary human immunodeficiency virus type 1 (HIV-1) isolates varies according to biological phenotype. The chemokine receptors CCR5 and CXCR4 are the major coreceptors that, together with CD4, govern HIV-1 entry into cells. Since CXCR4 usage determines the biological phenotype for HIV-1 isolates and is more frequent in patients with immunodeficiency, it may serve as a marker for viral virulence. This possibility prompted us to study coreceptor usage by HIV-2, known to be less pathogenic than HIV-1. We tested 11 primary HIV-2 isolates for coreceptor usage in human cell lines: U87 glioma cells, stably expressing CD4 and the chemokine receptor CCR1, CCR2b, CCR3, CCR5, or CXCR4, and GHOST(3) osteosarcoma cells, coexpressing CD4 and CCR5, CXCR4, or the orphan receptor Bonzo or BOB. The indicator cells were infected by cocultivation with virus-producing peripheral blood mononuclear cells and by cell-free virus. Our results show that 10 of 11 HIV-2 isolates were able to efficiently use CCR5. In contrast, only two isolates, both from patients with advanced disease, used CXCR4 efficiently. These two isolates also promptly induced syncytia in MT-2 cells, a pattern described for HIV-1 isolates that use CXCR4. Unlike HIV-1, many of the HIV-2 isolates were promiscuous in their coreceptor usage in that they were able to use, apart from CCR5, one or more of the CCR1, CCR2b, CCR3, and BOB coreceptors. Another difference between HIV-1 and HIV-2 was that the ability to replicate in MT-2 cells appeared to be a general property of HIV-2 isolates. Based on BOB mRNA expression in MT-2 cells and the ability of our panel of HIV-2 isolates to use BOB, we suggest that HIV-2 can use BOB when entering MT-2 cells. The results indicate no obvious link between viral virulence and the ability to use a multitude of coreceptors.