Increased CXCR4 use of HIV-1 subtype C identified by population sequencing in patients failing antiretroviral treatment compared with treatment-naive patients in Botswana.

HIV-1 uses the coreceptors CCR5 and/or CXCR4 for cell entry. Monotropic CCR5-using variants are found early in the infection while CXCR4-using variants may appear after progression to AIDS. CXCR4 use may consist of both monotropic and dualtropic viruses. The viral phenotype is important in evaluating the response to CCR5 inhibitors, a new class of antiviral drugs. The coreceptor use of HIV-1 was investigated using population sequencing in 24 patients from Botswana, carrying HIV-1 subtype C and failing antiretroviral treatment, while 26 treatment-naive patients acted as controls. Single genome sequencing was used to discern minor HIV-1 populations in the treatment-experienced group. The Geno2Pheno method was employed to predict the coreceptor use phenotype from HIV-1 env gp120 V3 DNA sequences. The glycan-charge model adjusted for subtype C was also used for phenotype prediction. The viral phenotype of population sequences was predicted using Geno2Pheno in 24/24 treatment-experienced patients, of whom eight (33%) were predicted to harbor CXCR4-using strains as compared to 2/26 in the treatment-naive group (p=0.03). Single genome sequencing generated 4-23 clones/patient in the treatment-experienced group. Altogether, 90/295 (31%) putative CXCR4-using clones were identified. In 10/24 (42%) treated patients at least one clone was predicted to be CXCR4-using, further increasing the amount of identified treatment-experienced patients with CXCR4 use. Although subtype C is usually associated with comparatively little CXCR4 use, the frequency of CXCR4 use in treatment-experienced patients with subtype C can be higher, which may have implications for the administration of CCR5 inhibitors in this patient group.

Challenges in virological diagnosis of HIV -1 transmission from sexual abuse--HIV-1 genetic links are mandatory.

The purpose of this article is to set forth possible strategies and techniques of analysis to diagnose or identify the source of HIV transmission in victims of sexual abuse. Diagnosis of HIV-1 transmission from sexual abuse is complicated. Timely blood samples are important. The right to confidentiality of the HIV diagnosis may prevent sampling from the offender. Hideous rapes occur during war, which victimize many women. Women delivering a child, seeking an abortion, or having a miscarriage may include victims of sexual abuse. HIV-infected children, where vertical transmission has been excluded, are important for investigation. Men who have sex with men may abuse young men. HIV-infected teenagers with signs of early infection should also be considered. Hundreds of single HIV-1 sequences can be created from one or more blood samples from the case and the alleged abuser. The more HIV-1 genes and sequences that are included, the better the outcomes of the phylogenetic relation. Evidence in support of transmission may be obtained from phylogenetic tree analysis and may also free someone from suspicion.

R5 human immunodeficiency virus type 1 with efficient DC-SIGN use is not selected for early after birth in vertically infected children.

The binding of human immunodeficiency virus (HIV) to C-type lectin receptors may result in either enhanced trans-infection of T-cells or virus degradation. We have investigated the efficacy of HIV-1 utilization of DC-SIGN, a C-type lectin receptor, in the setting of intrauterine or intrapartum mother-to-child transmission (MTCT). Viruses isolated from HIV-1-infected mothers at delivery and from their vertically infected children both shortly after birth and later during the progression of the disease were analysed for their use of DC-SIGN, binding and ability to trans-infect. DC-SIGN use of a child's earlier virus isolate tended to be reduced as compared with that of the corresponding maternal isolate. Furthermore, the children's later isolate displayed enhanced DC-SIGN utilization compared with that of the corresponding earlier virus. These results were also supported in head-to-head competition assays and suggest that HIV-1 variants displaying efficient DC-SIGN use are not selected for during intrauterine or intrapartum MTCT. However, viruses with increased DC-SIGN use may evolve later in paediatric HIV-1 infections.

HIV-1 mother-to-child transmission, post-test counselling, and antiretroviral prophylaxis in Northern Viet Nam: a prospective observational study.

BACKGROUND: Pregnant women fear being identified as HIV-1-infected and this has hampered prevention programmes and the calculation of transmission rates in Viet Nam. We introduced post-test counselling, antiretroviral prophylaxis, and formula feeding, and determined the vertical transmission rate in parts of Northern Viet Nam. METHODS: HIV infection was identified in 234 pregnant women; 182 (77.8%) accepted follow-up of their children. Counselling was given on 3-7 occasions for altogether approximately 6 h on antiretroviral prophylaxis and formula feeding to avoid transmission, and on the importance of surveillance of the child. All children were formula-fed. A polymerase chain reaction (PCR) was used for the diagnosis of HIV-1 in the children. One hundred and thirty-five of the 182 mothers allowed ≥ 3 blood samples to be taken from birth to ≥ 1 y of age, 32/182 provided a birth sample only, and 15/182 provided a sample later only. Nevirapine was given at delivery to 93/135 (69%) women, and to 128/135 (95%) children. Additionally, combination therapy was given to 15/135 (11%) who entered the study before delivery, and azidothymidine to their children for 1 week. RESULTS: Nine of 135 (6.7%) children became infected and 2/15 of the others, giving altogether 11/150 infected (7.3%). Intrauterine transmission was identified in 7/167 (4.2%) children by a positive PCR test at birth. PCR was negative at birth but positive at 1 month in 2/135 (1.5%), pointing to delivery-associated transmission. Thus, intrauterine transmission accounted for 78% (7/9). None of the uninfected children died, but 3/11 (p =0.004) of the HIV-1-infected died (in AIDS). CONCLUSION: Post-test confidential counselling, formula feeding, and antiretroviral prophylaxis resulted in low rates of delivery-associated and late HIV-1 transmissions.

Short communication: phylogenetic characterization of HIV type 1 CRF01_AE V3 envelope sequences in pregnant women in Northern Vietnam.

Characterization of HIV-1 strains is important for surveillance of the HIV-1 epidemic. In Vietnam HIV-1-infected pregnant women often fail to receive the care they are entitled to. Here, we analyzed phylogenetically HIV-1 env sequences from 37 HIV-1-infected pregnant women from Ha Noi (n=22) and Hai Phong (n=15), where they delivered in 2005-2007. All carried CRF01_AE in the gp120 V3 region. In 21 women CRF01_AE was also found in the reverse transcriptase gene. We compared their env gp120 V3 sequences phylogenetically in a maximum likelihood tree to those of 198 other CRF01_AE sequences in Vietnam and 229 from neighboring countries, predominantly Thailand, from the HIV-1 database. Altogether 464 sequences were analyzed. All but one of the maternal sequences colocalized with sequences from northern Vietnam. The maternal sequences had evolved the least when compared to sequences collected in Ha Noi in 2002, as shown by analysis of synonymous and nonsynonymous changes, than to other Vietnamese sequences collected earlier and/or elsewhere. Since the HIV-1 epidemic in women in Vietnam may still be underestimated, characterization of HIV-1 in pregnant women is important to observe how HIV-1 has evolved and follow its molecular epidemiology.

Charged amino acid patterns of coreceptor use in the major subtypes of human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 has several genetic subtypes and two coreceptor use phenotypes: R5 that uses CCR5, while X4 uses CXCR4. A high amino acid charge of the envelope glycoprotein 120 V3 region, common at positions 11 and 25, is important for CXCR4 use. We characterized charged V3 amino acids, retrieving all biologically phenotyped sequences from the HIV Sequence Database. Selecting individually unique ones randomly yielded 48 subtype A, 231 B, 180 C, 37 D and 32 CRF01_AE sequences; 482 were R5 and 46 were X4. Charged amino acids were conserved in both R5 and X4 with general and subtype-specific patterns. X4 viruses gained a higher charge from positive amino acids at positions other than in R5, and through the loss of negative amino acids. Other positions than 11/25 had a greater impact on charge (P<0.001). This describes how R5 evolves into X4 in a subtype-specific context, useful for computer-based predictions and vaccine design.

Peptide microarray-based identification of Mycobacterium tuberculosis epitope binding to HLA-DRB1*0101, DRB1*1501, and DRB1*0401.

A more effective vaccine against Mycobacterium tuberculosis is needed, and a number of M. tuberculosis vaccine candidates are currently in preclinical or clinical phase I and II studies. One of the strategies to select M. tuberculosis (protein) targets to elicit a CD8(+) or CD4(+) T-cell response is to gauge the binding of candidate peptides to major histocompatibility complex (MHC) class I or class II molecules, a prerequisite for successful peptide presentation and to expand antigen-specific T cells. We scanned 61 proteins from the M. tuberculosis proteome for potential MHC class II-presented epitopes that could serve as targets for CD4(+) T-cell responses. We constructed a peptide microarray consisting of 7,466 unique peptides derived from 61 M. tuberculosis proteins. The peptides were 15-mers overlapping by 12 amino acids. Soluble recombinant DRB1*0101 (DR1), DRB1*1501 (DR2), and DRB1*0401 (DR4) monomers were used to gauge binding to individual peptide species. Out of 7,466 peptides, 1,282, 674, and 1,854 peptides formed stable complexes with HLA-DR1, -DR2, and -DR4, respectively. Five hundred forty-four peptides bound to all three MHC class II molecules, 609 bound to only two, and 756 bound to only a single MHC class II molecule. This allowed us to rank M. tuberculosis proteins by epitope density. M. tuberculosis proteins contained "hot spots," i.e., regions with enriched MHC class II binding epitopes. Two hundred twenty-two peptides that formed MHC class II-peptide complexes had previously been described as exclusively recognized by IgG in sera from patients with active pulmonary tuberculosis, but not in sera from healthy individuals, suggesting that these peptides serve as B-cell and CD4(+) T-cell epitopes. This work helps to identify not only M. tuberculosis peptides with immunogenic potential, but also the most immunogenic proteins. This information is useful for vaccine design and the development of future tools to explore immune responses to M. tuberculosis.

Major histocompatibility complex class II molecule-human immunodeficiency virus peptide analysis using a microarray chip.

Identification of major histocompatibility complex (MHC) class II binding peptides is a crucial step in rational vaccine design and immune monitoring. We designed a novel MHC class II molecule-peptide microarray binding assay and evaluated 346 peptides from already identified human immunodeficiency virus (HIV) epitopes and an additional set (n = 206) of 20-mer peptides, overlapping by 15 amino acid residues, from HIV type 1B (HIV-1B) gp160 and Nef as a paradigm. Peptides were attached via the N-terminal part to a linker that covalently binds to the epoxy glass slide. The 552 peptides were printed in triplicate on a single peptide microarray chip and tested for stable formation of MHC class II molecule-peptide complexes using recombinant soluble DRB1*0101(DR1), DRB1*1501(DR2), and DRB1*0401(DR4) molecules. Cluster analysis revealed unique patterns of peptide binding to all three, two, or a single MHC class II molecule. MHC class II binding peptides reside within previously described immunogenic regions of HIV gp160 and Nef, yet we could also identify new MHC class II binding peptides from gp160 and Nef. Peptide microarray chips allow the comprehensive and simultaneous screening of a high number of candidate peptide epitopes for MHC class II binding, guided by subsequent quality data extraction and binding pattern cluster analysis.

Children born to HIV-1-infected women in Sweden in 1982-2003: trends in epidemiology and vertical transmission.

To describe the HIV-1 epidemic among childbearing women and their children in Sweden, a population-based analysis of data on all known mother-child pairs in Sweden with perinatal exposure to HIV-1 1982-2003 was conducted. The mother-to-child transmission (MTCT) rate in children prospectively followed from birth decreased from 24.7% in 1985-1993 to 5.7% in 1994-1998 and 0.6% in 1999-2003. The use of antiretroviral treatment of the mother during pregnancy and/or prophylactic antiretroviral intervention increased from 2.3% to 91.6% during the same period, and the elective cesarean delivery rate increased from 8.0% to 80.3%. No MTCT of HIV-1 occurred in Sweden after 1999.Fifty-one vertically HIV-1-infected children aged 2.7 to 17.6 years were living in Sweden by 31 December 2003, 71% being treated with antiretroviral agents. No HIV-1-related child death has been reported in Sweden after 1996. The conclusion is that MTCT of HIV-1 can be almost eliminated when appropriate resources are available. A national pregnancy screening program for HIV-1 running since 1987 with a high acceptance rate and the implementation of measures to prevent MTCT since 1994 have resulted in a significant decrease in the number of infected children. Inasmuch as knowledge of the infection status of the mother is crucial for reduction in MTCT of HIV-1, continued antenatal screening is important even in a low-prevalence country such as Sweden.

CCR5 use by human immunodeficiency virus type 1 is associated closely with the gp120 V3 loop N-linked glycosylation site.

Human immunodeficiency virus type 1 (HIV-1) enters cells through the chemokine receptors CCR5 (R5 virus) and/or CXCR4 (X4 virus). Loss of N-linked glycans and increased net charge of the third variable loop (V3) of the gp120 envelope glycoprotein have been observed to be important steps towards CXCR4 use. All reported sequences using CCR5 or CXCR4 exclusively, or using both, were gathered from the Los Alamos HIV Database and analysed with regard to the V3 N-linked glycosylation motifs (sequons) and charge. The V3 loop glycan had a sensitivity of 0.98 and a 0.92 positive predictive value in the context of CCR5 use. The difference from X4 was remarkable (P<10(-12)). Especially, the sequon motif NNT within the V3 loop was conserved in 99.2 % of the major clades. The results suggest a close association between the V3 loop glycan and CCR5 use and may provide new insight into HIV-1 tropism and help to improve phenotype-prediction models.

Feeding practices of HIV-1-infected mothers: the role of counsellors.

UNLABELLED: In this issue of Acta Paediatrica, Chopra et al. report that voluntary counselling is central to preparing mothers for making a proper informed choice about adequate feeding practices to prevent their infants from acquiring HIV infection. The recommendations given and the way in which counselling is performed are the most important determinants of a mother's decision about how to feed her infant. In this article, we summarize the main arguments for and against breastfeeding by HIV-infected mothers. CONCLUSIONS: Further studies are needed to determine the alternatives to breastfeeding in countries where there is no access to safe formula feeding or to antiretroviral drugs. HIV-positive mothers should be made aware of the available feeding alternatives through adequate counselling from properly trained persons.

Link between the X4 phenotype in human immunodeficiency virus type 1-infected mothers and their children, despite the early presence of R5 in the child.

Coreceptor use was determined for human immunodeficiency virus type 1 (HIV-1) isolates of various subtypes from 11 women during pregnancy and their infected children. Isolates from peripheral blood mononuclear cells (n=79) and from plasma (n=59) were available. The clinical and immunological stages of HIV-1 infection were recorded. Coreceptor use was tested on human cell lines expressing CD4 and different chemokine receptors. The R5 virus predominated, and only 9 isolates from 2 mothers used CXC chemokine receptor 4. All children carried the R5 virus at the time of diagnosis of HIV-1 infection. In 2 children of mothers carrying the X4 virus, the virus switched from R5 to X4 or to R5X4 by age 18 months (child no. 9) and age 48 months (child no. 10), whereas no children followed up to a similar age whose mothers were carrying the R5 virus experienced such a switch (P=.048). This points to a link between the presence of X4 virus in the mother and the emergence of X4 virus in her child.

Determination of cytomegalovirus DNA load for monitoring of cytomegalovirus disease and antiviral treatment in solid organ transplant patients, comparing limiting-dilution PCR and hybrid capture assay with cytomegalovirus isolation.

OBJECTIVE: To improve the identification of patients at risk of developing cytomegalovirus (CMV) disease. MATERIALS AND METHODS: In a prospective study of 50 kidney or liver transplant patients who developed fever, 133 EDTA blood samples were analyzed, using two tests to measure CMV DNA: a 10-fold limiting dilution of an extract of 2 million leukocytes for CMV PCR, and a CMV hybrid capture assay. Both tests were compared with virus isolation, using an equivalent amount of leukocytes as a base for all three tests. RESULTS: The limiting-dilution CMV PCR and the hybrid capture assay presented relatively similar changes of sensitivity and specificity at different CMV DNA concentrations. The kinetics of the positive and negative predictive values were also comparable. A higher CMV DNA load corresponded to an increased risk of developing CMV disease. Furthermore, an increase in the endpoint dilution of a positive CMV PCR also corresponded to more severe disease. After antiviral treatment, the CMV PCR decreased by at least 100-fold (2 log10) in 10 cases and by 10-fold (1 log10) in five cases. Thus, there was a decrease in 15 of 18 (83%) patients. Similarly, with the hybrid capture assay, the amount of CMV DNA decreased about 100-fold in five patients and decreased by about 0.5 genome equivalents in five cases, i.e. in 10 of 12 (83%) patients. CONCLUSION: Both methods proved clinically useful for detecting patients at risk of developing CMV disease and for monitoring antiviral treatment in solid organ transplant patients.

Regular pattern of respiratory syncytial virus and rotavirus infections and relation to weather in Stockholm, 1984--1993.

OBJECTIVE: To seek the possible epidemiologic relationship between the two dominant pediatric infectious agents, respiratory syncytial virus (RSV) and rotavirus, and to analyze the relationship of RSV to influenza virus infections and climate. METHODS: In the laboratory register, we retrospectively identified pediatric cases less than 5 years of age from the period 1984--93 (including the winter of 1994). RSV was diagnosed by immunofluorescence in nasopharyngeal samples and rotavirus infections by electron microscopy of feces. RESULTS: We observed a regular and significant pattern of early RSV epidemics (December to February), alternating every other year with later ones (March to April). There were twice as many hospital admissions during early compared to late epidemics. There was a similar but reverse pattern of early and late rotavirus seasons. Influenza A virus outbreaks occurred during the same period as early RSV epidemics. Several weather factors, such as temperature, precipitation, wind force and humidity were analyzed in relation to RSV epidemics without disclosing an important relationship. Cloudiness was, however, found to be associated with RSV peaks. CONCLUSIONS: The possibility of predicting RSV epidemics may be useful for medical planning.