Marion Vermeulen

Transmitted/Founder and Chronic Subtype C HIV-1 Use CD4 and CCR5 Receptors with Equal Efficiency and Are Not Inhibited by Blocking the Integrin α4β7

Sexual transmission of human immunodeficiency virus type 1 (HIV-1) most often results from productive infection by a single transmitted/founder (T/F) virus, indicating a stringent mucosal bottleneck. Understanding the viral traits that overcome this bottleneck could have important implications for HIV-1 vaccine design and other prevention strategies. Most T/F viruses use CCR5 to infect target cells and some encode envelope glycoproteins (Envs) that contain fewer potential N-linked glycosylation sites and shorter V1/V2 variable loops than Envs from chronic viruses. Moreover, it has been reported that the gp120 subunits of certain transmitted Envs bind to the gut-homing integrin α4β7, possibly enhancing virus entry and cell-to-cell spread. Here we sought to determine whether subtype C T/F viruses, which are responsible for the majority of new HIV-1 infections worldwide, share biological properties that increase their transmission fitness, including preferential α4β7 engagement. Using single genome amplification, we generated panels of both T/F (n = 20) and chronic (n = 20) Env constructs as well as full-length T/F (n = 6) and chronic (n = 4) infectious molecular clones (IMCs). We found that T/F and chronic control Envs were indistinguishable in the efficiency with which they used CD4 and CCR5. Both groups of Envs also exhibited the same CD4+ T cell subset tropism and showed similar sensitivity to neutralization by CD4 binding site (CD4bs) antibodies. Finally, saturating concentrations of anti-α4β7 antibodies failed to inhibit infection and replication of T/F as well as chronic control viruses, although the growth of the tissue culture-adapted strain SF162 was modestly impaired. These results indicate that the population bottleneck associated with mucosal HIV-1 acquisition is not due to the selection of T/F viruses that use α4β7, CD4 or CCR5 more efficiently.

Impact of individual-donation nucleic acid testing on risk of human immunodeficiency virus, hepatitis B virus, and hepatitis C virus transmission by blood transfusion in South Africa.

BACKGROUND: In 2005, the South African National Blood Service introduced individual‐donation (ID) nucleic acid test (NAT) screening for human immunodeficiency virus (HIV) RNA, hepatitis C virus (HCV) RNA, and hepatitis B virus (HBV) DNA. At the same time the use of ethnic origin to prioritize the transfusion of blood according to a hierarchy of residual risk was discontinued.

Comparison of Viral Env Proteins from Acute and Chronic Infections with Subtype C Human Immunodeficiency Virus Type 1 Identifies Differences in Glycosylation and CCR5 Utilization and Suggests a New Strategy for Immunogen Design

ABSTRACT Understanding human immunodeficiency virus type 1 (HIV-1) transmission is central to developing effective prevention strategies, including a vaccine. We compared phenotypic and genetic variation in HIV-1 env genes from subjects in acute/early infection and subjects with chronic infections in the context of subtype C heterosexual transmission. We found that the transmitted viruses all used CCR5 and required high levels of CD4 to infect target cells, suggesting selection for replication in T cells and not macrophages after transmission. In addition, the transmitted viruses were more likely to use a maraviroc-sensitive conformation of CCR5, perhaps identifying a feature of the target T cell. We confirmed an earlier observation that the transmitted viruses were, on average, modestly underglycosylated relative to the viruses from chronically infected subjects. This difference was most pronounced in comparing the viruses in acutely infected men to those in chronically infected women. These features of the transmitted virus point to selective pressures during the transmission event. We did not observe a consistent difference either in heterologous neutralization sensitivity or in sensitivity to soluble CD4 between the two groups, suggesting similar conformations between viruses from acute and chronic infection. However, the presence or absence of glycosylation sites had differential effects on neutralization sensitivity for different antibodies. We suggest that the occasional absence of glycosylation sites encoded in the conserved regions of env, further reduced in transmitted viruses, could expose specific surface structures on the protein as antibody targets.

Characterization of occult hepatitis B virus strains in south african blood donors

Since October 2005, all blood units collected in South Africa were screened individually for human immunodeficiency virus (HIV)‐1, hepatitis B and C virus (HBV, HCV) genomes uncovering preseroconversion window period (WP) infections for each virus and occult HBV infections (OBIs) defined as persistent HBV DNA without detectable hepatitis B surface antigen (HBsAg). Samples identified as HBsAg‐negative/DNA‐positive were confirmed by combining real‐time quantitative polymerase chain reaction, nested amplification, anti‐HBc and anti‐HBs. Amplified basic core promoter/precore, pre‐S/S, and whole genome were sequenced, analyzed, and compared to 73 HBsAg+ strains. Genotype was determined by phylogenetic analysis. From 109 samples examined, 54 were classified as OBI, 14 as WP, 20 as false‐positive, five as other classification, and 16 as undetermined due to lack of serological or follow‐up data. OBI donors were predominantly males (67%), median age 31 years, black (54%), with normal alanine aminotransferase levels. Viral load ranged between unquantifiable and 518 IU/mL (median 5 IU/mL). Genotype A1 was more frequent (23 strains) than genotype D (seven strains). Genotype A1 strains were little mutated. In the major hydrophilic region, 56.5% strains were wild type or with few amino acid substitutions. Most important, all 13 full genome sequences presented 1 to 7 mutations known to or assumed to negatively impact viral replication. In particular, 6/13 sequences had a stop codon in the HBx gene translated into deletion of 117 or 19‐25 C‐terminus amino acids not found in 15 HBeAg+ HBsAg+ strains. One WP sequence with an HBx stop codon suggested infectivity. Conclusion: Genotype A1 OBIs are different from genotype A2 and D OBIs in that there is little evidence of immune pressure as a major factor involved in OBI genesis. Limited replication appears mostly related to genetic viral defects. (HEPATOLOGY 2009.)

Hepatitis B virus transmission by blood transfusion during 4 years of individual-donation nucleic acid testing in South Africa: estimated and observed window period risk

BACKGROUND: Since October 2005, a total of 2,921,561 blood donations have been screened by the South African National Blood Service for hepatitis B virus (HBV) by individual‐donation nucleic acid testing (ID‐NAT). Over 4 years, 149 hepatitis B surface antigen–negative acute‐phase HBV NAT–positive donations were identified (1:19,608). The lookback program identified one probable HBV transmission.

Refinement of a viral transmission risk model for blood donations in seroconversion window phase screened by nucleic acid testing in different pool sizes and repeat test algorithms.

BACKGROUND: In minipool nucleic acid test (MP‐NAT) screening protocols, the donations implicated in reactive test pools are released for transfusion when they are nonreactive in a repeat test on the individual samples, but in individual‐donation (ID)‐NAT screening algorithms the release of nonrepeatable reactive (NRR) donations is under discussion.

Changing epidemiology of human parvovirus 4 infection in sub-Saharan Africa

Human parvovirus 4 infections are primarily associated with parenteral exposure in western countries. By ELISA, we demonstrate frequent seropositivity for antibody to parvovirus 4 viral protein 2 among adult populations throughout sub-Saharan Africa (Burkina Faso, 37%; Cameroon, 25%; Democratic Republic of the Congo, 35%; South Africa, 20%), which implies existence of alternative transmission routes.

Sensitivity of individual‐donation and minipool nucleic acid amplification test options in detecting window period and occult hepatitis B virus infections

Several comparison studies showed that the Ultrio assay (Novartis Diagnostics) used in individual‐donation nucleic acid amplification testing (ID‐NAT) format was as sensitive as the TaqScreen assay (Roche) on minipools of six donations (MP6), but the sensitivity of HBV DNA detection has been improved in the new Ultrio Plus version of the assay. A head‐to‐head comparison study was designed to compare the clinical sensitivity of the Ultrio and Ultrio Plus assay in ID, MP4, and MP8 formats using TaqScreen MP6 as a reference assay.

Comparison of human immunodeficiency virus assays in window phase and elite controller samples: viral load distribution and implications for transmission risk

After 3 years of individual‐donation nucleic acid test (ID‐NAT) screening by the South African National Blood Service (SANBS), a repository of 73 human immunodeficiency virus antibody (anti‐HIV)‐negative window period (WP)‐yield samples and 28 anti‐HIV–positive, HIV‐RNA–negative elite controllers (ECs) became available for comparison of a p24 antigen (p24 Ag) assay (Innogenetics), two viral load assays (Siemens branch DNA [bDNA] 3.0 and Abbott real‐time polymerase chain reaction [RT‐PCR]), and three triplex NAT assays (Novartis Diagnostics Ultrio and Ultrio‐Plus and Roche TaqScreen) by replicate testing of dilutions.

Features of Recently Transmitted HIV-1 Clade C Viruses that Impact Antibody Recognition: Implications for Active and Passive Immunization

The development of biomedical interventions to reduce acquisition of HIV-1 infection remains a global priority, however their potential effectiveness is challenged by very high HIV-1 envelope diversity. Two large prophylactic trials in high incidence, clade C epidemic regions in southern Africa are imminent; passive administration of the monoclonal antibody VRC01, and active immunization with a clade C modified RV144-like vaccines. We have created a large representative panel of C clade viruses to enable assessment of antibody responses to vaccines and natural infection in Southern Africa, and we investigated the genotypic and neutralization properties of recently transmitted clade C viruses to determine how viral diversity impacted antibody recognition. We further explore the implications of these findings for the potential effectiveness of these trials. A panel of 200 HIV-1 Envelope pseudoviruses was constructed from clade C viruses collected within the first 100 days following infection. Viruses collected pre-seroconversion were significantly more resistant to serum neutralization compared to post-seroconversion viruses (p = 0.001). Over 13 years of the study as the epidemic matured, HIV-1 diversified (p = 0.0009) and became more neutralization resistant to monoclonal antibodies VRC01, PG9 and 4E10. When tested at therapeutic levels (10ug/ml), VRC01 only neutralized 80% of viruses in the panel, although it did exhibit potent neutralization activity against sensitive viruses (IC50 titres of 0.42 μg/ml). The Gp120 amino acid similarity between the clade C panel and candidate C-clade vaccine protein boosts (Ce1086 and TV1) was 77%, which is 8% more distant than between CRF01_AE viruses and the RV144 CRF01_AE immunogen. Furthermore, two vaccine signature sites, K169 in V2 and I307 in V3, associated with reduced infection risk in RV144, occurred less frequently in clade C panel viruses than in CRF01_AE viruses from Thailand. Increased resistance of pre-seroconversion viruses and evidence of antigenic drift highlights the value of using panels of very recently transmitted viruses and suggests that interventions may need to be modified over time to track the changing epidemic. Furthermore, high divergence such as that observed in the older clade C epidemic in southern Africa may impact vaccine efficacy, although the correlates of infection risk are yet to be defined in the clade C setting. Findings from this study of acute/early clade C viruses will aid vaccine development, and enable identification of new broad and potent antibodies to combat the HIV-1 C-clade epidemic in southern Africa.