Michael A. Eller

DC-SIGN (CD209) Mediates Dengue Virus Infection of Human Dendritic Cells

Dengue virus is a single-stranded, enveloped RNA virus that productively infects human dendritic cells (DCs) primarily at the immature stage of their differentiation. We now find that all four serotypes of dengue use DC-SIGN (CD209), a C-type lectin, to infect dendritic cells. THP-1 cells become susceptible to dengue infection after transfection of DC-specific ICAM-3 grabbing nonintegrin (DC-SIGN), or its homologue L-SIGN, whereas the infection of dendritic cells is blocked by anti–DC-SIGN antibodies and not by antibodies to other molecules on these cells. Viruses produced by dendritic cells are infectious for DC-SIGN– and L-SIGN–bearing THP-1 cells and other permissive cell lines. Therefore, DC-SIGN may be considered as a new target for designing therapies that block dengue infection.

Effect of Human Immunodeficiency Virus Type 1 (HIV-1) Subtype on Disease Progression in Persons from Rakai, Uganda, with Incident HIV-1 Infection

BACKGROUND Human immunodeficiency virus type 1 (HIV-1) subtypes differ in biological characteristics that may affect pathogenicity. METHODS We determined the HIV-1 subtype-specific rates of disease progression among 350 HIV-1 seroconverters. Subtype, viral load, and CD4(+) cell count were determined. Cox proportional hazards regression modeling was used to estimate adjusted hazard ratios (HRs) of progression to acquired immunodeficiency syndrome (AIDS) (defined as a CD4(+) cell count of < or =250 cells/mm(3)) and to AIDS-associated death. RESULTS A total of 59.1% of study subjects had subtype D strains, 15.1% had subtype A, 21.1% had intersubtype recombinant subtypes, 4.3% had multiple subtypes, and 0.3% had subtype C. Of the 350 subjects, 129 (37%) progressed to AIDS, and 68 (19.5%) died of AIDS. The median time to AIDS onset was shorter for persons with subtype D (6.5 years), recombinant subtypes (5.6 years), or multiple subtypes (5.8 years), compared with persons with subtype A (8.0 years; P = .022). Relative to subtype A, adjusted HRs of progression to AIDS were 2.13 [95% confidence interval {CI}, 1.10-4.11] for subtype D, 2.16 [95% CI, 1.05-4.45] for recombinant subtypes, and 4.40 [95% CI, 1.71-11.3] for multiple subtypes. The risk of progression to death was significantly higher for subtype D (adjusted HR, 5.65; 95% CI, 1.37-23.4), recombinant subtypes (adjusted HR, 6.70; 95% CI, 1.56-28.8), and multiple subtypes (adjusted HR, 7.67; 95% CI, 1.27-46.3), compared with subtype A. CONCLUSIONS HIV disease progression is affected by HIV-1 subtype. This finding may impact decisions on when to initiate antiretroviral therapy and may have implications for future trials of HIV-1 vaccines aimed at slowing disease progression.

Long-term sequelae after Ebola virus disease in Bundibugyo, Uganda: a retrospective cohort study

BACKGROUND The limited data available for long-term Ebola virus disease health outcomes suggest that sequelae persist for longer than 1 year after infection. The magnitude of the present outbreak in west Africa necessitates a more complete understanding of the health effects and future medical needs of these patients. METHODS We invited adult survivors of the 2007 Bundibugyo Ebola virus outbreak in Uganda and their contacts to take part in an observational study roughly 29 months after the outbreak. We collected information about health status, functional limitations, and demographics. We collected blood samples for clinical chemistry, haematology, and filovirus antibodies using ELISA. Analyses were restricted to probable and confirmed survivors and their seronegative contacts. FINDINGS We recruited 70 survivors of the 2007 Bundibugyo Ebola virus and 223 contacts. We did analyses for 49 probable and confirmed survivors and 157 seronegative contacts. Survivors of the Bundibugyo Ebola virus were at significantly increased risk of ocular deficits (retro-orbital pain [RR 4·3, 95% CI 1·9-9·6; p<0·0001], blurred vision [1·9, 1·1-3·2; p=0·018]), hearing loss (2·3, 1·2-4·5; p=0·010), difficulty swallowing (2·1, 1·1-3·9; p=0·017), difficulty sleeping (1·9, 1·3-2·8; p=0·001), arthralgias (2·0, 1·1-3·6; p=0·020), and various constitutional symptoms controlling for age and sex. Chronic health problems (prevalence ratio [PR] 2·1, 95% CI 1·2-3·6; p=0·008) and limitations due to memory loss or confusion (PR 5·8, 1·5-22·4; p=0·010) were also reported more frequently by survivors of Bundibugyo Ebola virus. INTERPRETATION Long-term sequelae persist for more than 2 years after Ebola virus disease. Definition of health consequences related to Ebola virus disease could improve patient care for survivors and contribute to understanding of disease pathogenesis. FUNDING Chemical Biological Technologies Directorate, Defense Threat Reduction Agency.

A Phase 1/2 Study of a Multiclade HIV-1 DNA Plasmid Prime and Recombinant Adenovirus Serotype 5 Boost Vaccine in HIV-Uninfected East Africans (RV 172)

BACKGROUND Human immunodeficiency virus (HIV) vaccine development remains a global priority. We describe the safety and immunogenicity of a multiclade DNA vaccine prime with a replication-defective recombinant adenovirus serotype 5 (rAd5) boost. METHODS The vaccine is a 6-plasmid mixture encoding HIV envelope (env) subtypes A, B, and C and subtype B gag, pol, and nef, and an rAd5 expressing identical genes, with the exception of nef. Three hundred and twenty-four participants were randomized to receive placebo (n=138), a single dose of rAd5 at 10(10) (n = 24) or 10(11) particle units (n = 24), or DNA at 0, 1, and 2 months, followed by rAd5 at either 10(10) (n= 114) or 10(11) particle units (n = 24) boosting at 6 months. Participants were followed up for 24 weeks after the final vaccination. RESULTS The vaccine was safe and well tolerated. HIV-specific T cell responses were detected in 63% of vaccinees. Titers of preexisting Ad5 neutralizing antibody did not affect the frequency and magnitude of T cell responses in prime-boost recipients but did affect the response rates in participants that received rAd5 alone (P = .037). CONCLUSION The DNA/rAd5 vaccination regimen was safe and induced HIV type 1 multi-clade T cell responses, which were not significantly affected by titers of preexisting rAd5 neutralizing antibody. Trial Registration. ClinicalTrials.gov identifier: NCT00123968 .

Prospective Study of Acute HIV-1 Infection in Adults in East Africa and Thailand

BACKGROUND Acute human immunodeficiency virus type 1 (HIV-1) infection is a major contributor to transmission of HIV-1. An understanding of acute HIV-1 infection may be important in the development of treatment strategies to eradicate HIV-1 or achieve a functional cure. METHODS We performed twice-weekly qualitative plasma HIV-1 RNA nucleic acid testing in 2276 volunteers who were at high risk for HIV-1 infection. For participants in whom acute HIV-1 infection was detected, clinical observations, quantitative measurements of plasma HIV-1 RNA levels (to assess viremia) and HIV antibodies, and results of immunophenotyping of lymphocytes were obtained twice weekly. RESULTS Fifty of 112 volunteers with acute HIV-1 infection had two or more blood samples collected before HIV-1 antibodies were detected. The median peak viremia (6.7 log10 copies per milliliter) occurred 13 days after the first sample showed reactivity on nucleic acid testing. Reactivity on an enzyme immunoassay occurred at a median of 14 days. The nadir of viremia (4.3 log10 copies per milliliter) occurred at a median of 31 days and was nearly equivalent to the viral-load set point, the steady-state viremia that persists durably after resolution of acute viremia (median plasma HIV-1 RNA level, 4.4 log10 copies per milliliter). The peak viremia and downslope were correlated with the viral-load set point. Clinical manifestations of acute HIV-1 infection were most common just before and at the time of peak viremia. A median of one symptom of acute HIV-1 infection was recorded at a median of two study visits, and a median of one sign of acute HIV-1 infection was recorded at a median of three visits. CONCLUSIONS The viral-load set point occurred at a median of 31 days after the first detection of plasma viremia and correlated with peak viremia. Few symptoms and signs were observed during acute HIV-1 infection, and they were most common before peak viremia. (Funded by the Department of Defense and the National Institute of Allergy and Infectious Diseases.).

Circulating HIV-Specific Interleukin-21+CD4+ T Cells Represent Peripheral Tfh Cells with Antigen-Dependent Helper Functions

A central effort in HIV vaccine development is to generate protective broadly neutralizing antibodies, a process dependent on T follicular helper (Tfh) cells. The feasibility of using peripheral blood counterparts of lymph node Tfh cells to assess the immune response and the influence of viral and vaccine antigens on their helper functions remain obscure. We assessed circulating HIV-specific IL-21(+)CD4(+) T cells and showed transcriptional and phenotypic similarities to lymphoid Tfh cells, and hence representing peripheral Tfh (pTfh) cells. pTfh cells were functionally active and B cell helper quality differed depending on antigen specificity. Furthermore, we found higher frequency of pTfh cells in peripheral blood mononuclear cell specimens from the ALVAC+AIDSVAX (RV144) HIV vaccine trial associated with protective antibody responses compared to the non-protective DNA+Ad5 vaccine trial. Together, we identify IL-21(+)CD4(+) T cells as pTfh cells, implicating them as key populations in the generation of vaccine-evoked antibody responses.

Higher HIV-1 incidence and genetic complexity along main roads in Rakai District, Uganda

Objective:To determine the association between the incidence of HIV-1 infection and the genetic complexity of HIV-1 strains in 2 geographic strata within Rakai District, Uganda. Methods:Study volunteers with recent HIV-1 infections during the period 1997 through 2003 were recruited from 10 communities that were geographically stratified as a main road trading center (n = 5) or a secondary road trading village (n = 5). Cryopreserved plasma was available from 384 volunteers and was the source of viral RNA for genotyping by the multiregion hybridization assay. Hazard ratios (HRs) for a single HIV subtype, a recombinant form, or dual infection for gender and geographic strata were obtained using Cox proportional hazards analysis. Results:The HIV-1 incidence rate during the period 1999 through 2002 was 1.3 per 100 person-years (PYs) in the trading centers and 1.1 per 100 PYs in the trading villages. The HR for infection with an HIV-1 recombinant strain in trading centers relative to trading villages was 2.3 (95% confidence interval [CI]: 1.0 to 6.7). Among those who changed residence between village strata, the HR for a recombinant HIV-1 infection was 8.1 (95% CI: 0.4 to 47.7). Conclusions:HIV-1 incidence and genetic complexity are associated with geographic strata and population mobility in Rakai District and are important variables to be considered in planning and recruitment for vaccine trials.

Elevated natural killer cell activity despite altered functional and phenotypic profile in Ugandans with HIV-1 clade A or clade D infection.

Background and Objective:Natural killer (NK) cells most likely contribute toward limiting HIV-1 replication, and investigation into their function throughout the course of infection is therefore important. We here aimed to determine the state of the NK cell compartment in Ugandans with untreated HIV-1 clade A or D infection in comparison with matched uninfected controls. Methods and Results:The function and phenotype of NK cells were investigated using 10-color flow cytometry. Surprisingly, NK cells displayed elevated production of interferon-γ and macrophage inflammatory protein 1β, as well as CD107a degranulation in infected subjects. This included unexpected levels of degranulation in the CD56bright subset of NK cells and high levels of macrophage inflammatory protein 1β in CD56negative NK cells. HIV-1 infection was associated with reduced expression of KIR2DL1, NKG2A, CD161, and NKp30 in CD56dim and CD56negative NK cells, whereas lowered CD161 expression was the only alteration in the CD56bright subset. Interestingly, low CD4 counts were associated with increased levels of interferon-γ and degranulation in CD56bright NK cells, as well as increased NKp44 expression in the CD56dim cells. Conclusions:NK cells in HIV-1-infected Ugandans display elevated activity, despite an altered functional and phenotypic profile. Furthermore, specific alterations in the CD56bright and CD56dim subsets occur in patients with severe CD4 loss.

Cooperativity of HIV-specific cytolytic CD4+ T cells and CD8+ T cells in control of HIV viremia

ABSTRACT CD4+ T cells play a pivotal role in the control of chronic viral infections. Recently, nontraditional CD4+ T cell functions beyond helper effects have been described, and a role for cytolytic CD4+ T cells in the control of HIV infection has been suggested. We define here the transcriptional, phenotypic, and functional profiles of HIV-specific cytolytic CD4+ T cells. Fluidigm BioMark and multiparameter flow cytometric analysis of HIV-specific cytolytic CD4+ T cells revealed a distinct transcriptional signature compared to Th1 CD4+ cells but shared similar features with HIV-specific cytolytic CD8+ T cells. Furthermore, HIV-specific cytolytic CD4+ T cells showed comparable killing activity relative to HIV-specific CD8+ T cells and worked cooperatively in the elimination of virally infected cells. Interestingly, we found that cytolytic CD4+ T cells emerge early during acute HIV infection and tightly follow acute viral load trajectory. This emergence was associated to the early viral set point, suggesting an involvement in early control, in spite of CD4 T cell susceptibility to HIV infection. Our data suggest cytolytic CD4+ T cells as an independent subset distinct from Th1 cells that show combined activity with CD8+ T cells in the long-term control of HIV infection. IMPORTANCE The ability of the immune system to control chronic HIV infection is of critical interest to both vaccine design and therapeutic approaches. Much research has focused on the effect of the ability of CD8+ T cells to control the virus, while CD4+ T cells have been overlooked as effectors in HIV control due to the fact that they are preferentially infected. We show here that a subset of HIV-specific CD4+ T cells cooperate in the cytolytic control of HIV replication. Moreover, these cells represent a distinct subset of CD4+ T cells showing significant transcriptional and phenotypic differences compared to HIV-specific Th1 cells but with similarities to CD8+ T cells. These findings are important for our understanding of HIV immunopathology.

Innate and Adaptive Immune Responses Both Contribute to Pathological CD4 T Cell Activation in HIV-1 Infected Ugandans

HIV-1 disease progression is associated with persistent immune activation. However, the nature of this association is incompletely understood. Here, we investigated immune activation in the CD4 T cell compartment of chronically HIV-1 infected individuals from Rakai, Uganda. Levels of CD4 T cell activation, assessed as co-expression of PD-1, CD38 and HLA-DR, correlated directly to viral load and inversely to CD4 count. Deeper characterization of these cells indicated an effector memory phenotype with relatively frequent expression of Ki67 despite their PD-1 expression, and levels of these cells were inversely associated with FoxP3+ regulatory T cells. We therefore use the term deregulated effector memory (DEM) cells to describe them. CD4 T cells with a DEM phenotype could be generated by antigen stimulation of recall responses in vitro. Responses against HIV-1 and CMV antigens were enriched among the DEM CD4 T cells in patients, and the diverse Vβ repertoire of DEM CD4 T cells suggested they include diverse antigen-specificities. Furthermore, the levels of DEM CD4 T cells correlated directly to soluble CD14 (sCD14) and IL-6, markers of innate immune activation, in plasma. The size of the activated DEM CD4 T cell subset was predictive of the rate of disease progression, whereas IL-6 was only weakly predictive and sCD14 was not predictive. Taken together, these results are consistent with a model where systemic innate immune activation and chronic antigen stimulation of adaptive T cell responses both play important roles in driving pathological CD4 T cell immune activation in HIV-1 disease.