Human skin Langerhans cells are targets of dengue virus infection.

Dengue virus (DV), an arthropod-borne flavivirus, causes a febrile illness for which there is no antiviral treatment and no vaccine. Macrophages are important in dengue pathogenesis; however, the initial target cell for DV infection remains unknown. As DV is introduced into human skin by mosquitoes of the genus Aedes, we undertook experiments to determine whether human dendritic cells (DCs) were permissive for the growth of DV. Initial experiments demonstrated that blood-derived DCs were 10-fold more permissive for DV infection than were monocytes or macrophages. We confirmed this with human skin DCs (Langerhans cells and dermal/interstitial DCs). Using cadaveric human skin explants, we exposed skin DCs to DV ex vivo. Of the human leukocyte antigen DR-positive DCs that migrated from the skin, emigrants from both dermis and epidermis, 60-80% expressed DV antigens. These observations were supported by histologic findings from the skin rash of a human subject who received an attenuated tetravalent dengue vaccine. Immunohistochemistry of the skin showed CD1a-positive DCs double-labeled with an antibody against DV envelope glycoprotein. These data demonstrate that human skin DCs are permissive for DV infection, and provide a potential mechanism for the transmission of DV into human skin.

High-throughput genotyping of KIR2DL2/L3, KIR3DL1/S1, and their HLA class I ligands using real-time PCR.

Killer immunoglobulin-like receptors (KIRs) expressed on natural killer cells are critical components of innate immunity. Interactions between KIRs and their human leukocyte antigen (HLA) ligands have been shown to influence autoimmune and infectious disease course in defined populations. However, the low throughput and high cost of current methods impede confirmation of the universality of these findings. To support large epidemiology surveys, we developed a high-throughput real-time polymerase chain reaction-based assay to identify carriers of KIR3DL1, KIR3DS1, KIR2DL2, and KIR2DL3 and their HLA ligands. The platform performed with 100% sensitivity and specificity in detection of carrier and non-carrier on reference samples. The application of this platform will further clarify the nature and impact of the KIR-HLA epistatic interaction on disease course in large global population-based studies.

HLA class I allele and haplotype diversity in Ugandans supports the presence of a major east African genetic cluster.

The objective of this study was to characterize the class I human leukocyte antigen (HLA) genetic composition of the Ugandan population to better define its relationship with other African groups. Samples from 175 individuals from Kampala (Uganda) were subjected to class I HLA-A, -B, and -C sequence-based typing. The high concordance between the major alleles and haplotypes found in the current and Kenyan populations and interpopulation genetic distance analysis strongly supported the presence of an East African cluster that contained the current Ugandan population along with Kenyan Luo and Nandi populations. The congruence of major alleles in different populations would permit consideration of East Africa as an integrated setting when designing and evaluating much needed malaria, tuberculosis, and AIDS vaccines.

Are you PEPped and PrEPped for travel? Risk mitigation of HIV infection for travelers.

The HIV pandemic persists globally and travelers are at risk for infection by the Human Immunodeficiency Virus (HIV). While HIV-focused guidelines delineate risk stratification and mitigation strategies for people in their home communities, travel issues are not addressed. In this review, direct and indirect evidence on HIV risk among travelers is explored. The burgeoning practice of employing pre-exposure prophylaxis (PrEP) with anti-retroviral therapy in the non-travel setting is introduced, as well as the more established use of post-exposure prophylaxis (PEP). Challenges in applying these lessons to travelers are discussed, and a new guidelines process is scoped and recommended.

HIV neutralization assay using polymerase chain reaction-derived molecular signals.

Characterization of the capacity of human polyclonal antibody to neutralize wild-type patient isolates has important implications for vaccine development. We report the development of a polymerase chain reaction-based neutralization assay that quantitatively measures each infection using HIV proviral formation. These molecular end points identified the absence or quantitative diminution of DNA provirus formation as well as a delay in the kinetics of HIV DNA provirus formation. Using both laboratory strain prototype isolates (HIV-1-MN, HIV-IIIb) and primary wild-type patients' isolates, neutralization end points were reproducibly determined. End points were reached within 72 h, thereby minimizing the impact of subsequent rounds of infection on interpretation of results. Although the neutralization titer of polyclonal sera was usually comparable using standard technology, this assay did find isolate-dependent variation in the relationship between p24 production and HIV proviral DNA formation. Finally, we noted the disparity between the ability of human sera to neutralize prototype and wild-type isolates in primary peripheral blood mononuclear cell targets. We believe this assay provides unique opportunities to characterize the initial events of virus-antibody interaction and will help to elucidate clinically relevant neutralization immunoregulatory mechanisms.

Detection and quantification of HIV type 1 RNA in nasopharyngeal washes from HIV-infected subjects.

Human immunodeficiency virus type 1 (HIV) RNA load was measured in paired samples of peripheral blood plasma and nasopharyngeal (NP) washes from 97 Thai subjects infected with subtype E or B. HIV RNA was quantifiable in 93% of peripheral blood plasma samples tested and was inversely correlated (rho =-0.524; p < 0.001) with CD4 absolute count. HIV RNA was quantifiable in 29% of NP samples tested, and the median value was less than that of plasma viral load. HIV RNA load in NP samples was correlated (rho = 0.388; p < 0.001) with viral load in peripheral blood. HIV RNA was not detected in NP washes from subjects with undetectable plasma viral load. Virus isolation attempts on two NP samples were negative. The results do not support local HIV production in the nasopharynx, but extend current knowledge of HIV shedding to include the NP compartment.

Diversity of envelope glycoprotein from human immunodeficiency virus type 1 of recent seroconverters in Thailand.

The envelope-coding sequence of human immunodeficiency virus type 1 (HIV-1) was determined for 11 Thai seroconverters between 1995 and 1996. On the basis of the env sequences, all subjects were infected with HIV subtype E. Compared with the interpatient protein diversity among HIV-1 Thai reference sequences from 1990 to 1992 (4.4%), the diversity among the 1995-1996 seroconverters was approximately double (9.5%). The tetrapeptide tip of the V3 loop was invariant for 10 of the 11 seroconverters, and identical to that observed in sequences derived from the 1990-1992 group. However, in the V3 region, sequences from 2 of the 11 subjects demonstrated more than 5 amino acid changes relative to the reference strains. This may represent the "aging" of the HIV epidemic seen in other endemic regions. These findings may have substantial implications for vaccine development and evaluation for both HIV antibody and cytotoxic T lymphocyte repertoire recognition.

Selective increases in HIV-specific neutralizing antibody and partial reconstitution of cellular immune responses during prolonged, successful drug therapy of HIV infection.

Because the immune response to HIV depends on viral gene expression, we examined the HIV-specific immune responses in persons whose viral load after highly active antiretroviral therapy (HAART) was <400 on at least 3 occasions over a 12-month interval. Eleven patients were identified. While there was little change in mean HIV-binding antibody (Ab) titers in this group, two persons mounted increases in HIV envelope-specific binding antibody. Neutralizing antibody (NAb) titers against a panel of HIV-1 primary isolates (BZ167, US1, and CM237) increased post-HAART (80% neutralization titer against US1, p = 0.06; against CM237, p = 0.04). The two persons with large increases in binding antibody also had increases in primary isolate NAb. Roughly half of HAART recipients had significant increases in neutralizing antibody to the primary isolates US1 and CM237. Compared with CD4-matched, non-HAART controls, there were significant increases in NAb against the subtype B primary isolate US1 (p < 0.0009); no increases were seen against more easily neutralized primary isolate BZ167. There were no differences after HAART in antibody-directed cellular cytotoxicity (ADCC). HAART resulted in a partial restoration of lymphoproliferative responses to recall antigens (tetanus and diphtheria). New responses developed to HIV Gag p24. No patient responded to HIV Env gp160 or gp120 either before or after HAART. The data underscore the lack of functional reconstitution of HIV-specific, CD4-mediated responses despite durable suppression of viral replication. In the setting of stable anti-HIV Ab levels, the development of increased NAb in certain individuals suggests that control of the virus by HAART may assist in immune control of HIV.

Socio-demographic and drug use factors associated with HIV-1 recombinants and dual infections in Northern Thai drug users: associations of risk with genetic complexity.

BACKGROUND: Dual infection with diverse HIV strains can foster the emergence of recombinants. The resulting increase in viral genetic diversity is a major challenge for vaccine development HIV treatment. In this study we aim to investigate the socio demographic factors associated with an increasing level of genetic diversity among HIV strains in a population of drug-users in Northern Thailand. METHODS: From 1999 through 2000, 2231 volunteers were enrolled in the Opiate-Users Research in Chiang Mai, Thailand. HIV subtype analysis was conducted among those HIV-1 seropositive (n=347) using a multi-region hybridization assay. Social and demographic variables were assessed using a structured questionnaire. RESULTS: Overall, 336/347 (96.8%) of the samples could be typed. 81.8% were CRF01_AE, 3.9% were subtype B, 9.2% were recombinants (mostly between CRF01_AE and B) and 5.1% were dual infections. Dual infections were more frequent among those with a lower education level (AOR: 5.2; 95% CI 1.4-20.3), those who have initiated injecting in the last 3 years (AOR: 3.9; 95% CI 1.1-14.6), and those reporting frequent needle sharing in the last 3 months (AOR: 7.0; 95% CI 1.5-34.1). Both recombinant strains and dual infection were more frequent among those reporting frequent needle sharing in the last 3 months (AOR: 5.3; 95% CI 1.6-17.1). CONCLUSION: To limit the expanding complexity of HIV-1 strains, early intervention should be aimed at reduction in needle sharing, especially among new intravenous drug users.

High prevalence of HIV infection among rural tea plantation residents in Kericho, Kenya.

Human immunodeficiency virus type 1 (HIV-1) epidemiology among residents of a rural agricultural plantation in Kericho, Kenya was studied. HIV-1 prevalence was 14.3%, and was higher among women (19.1%) than men (11.3%). Risk factors associated with HIV-1 for men were age (>or=25 years), marital history (one or more marriages), age difference from current spouse (>or=5 years), Luo ethnicity, sexually transmitted infection (STI) symptoms in the past 6 months, circumcision (protective), and sexual activity (>or=7 years). Among women, risk factors associated with HIV-1 were age (25-29 years, >or=35 years), marital history (one or more marriages), age difference from current spouse (>or=10 years), Luo ethnicity, STI symptoms in the past 6 months, and a STI history in the past 5 years. Most participants (96%) expressed a willingness to participate in a future HIV vaccine study. These findings will facilitate targeted intervention and prevention measures for HIV-1 infection in Kericho.

Pyogenic infections due to Ochrobactrum anthropi.

Ochrobactrum anthropi is a nonfermentative gram-negative bacillus that has been isolated with increasing frequency from human clinical specimens. Previously, its pathogenic niche was believed to involve the causation of catheter-associated bacteremic illnesses. We describe three cases of pyogenic infection due to O. anthropi, thereby expanding the known pathogenic potential of this organism.

Isolation of nontuberculous, non-avium mycobacteria from patients infected with human immunodeficiency virus.

Mycobacterium avium serovars account for 97% of typeable M. avium complex (MAC) organisms causing infection in patients with AIDS. We reviewed 216 consecutive cultures that yielded nontuberculous mycobacteria (NTM) from 212 patients. Only the first isolate of each species of NTM recovered from each patient was analyzed in the study. Among the 92 patients infected with the human immunodeficiency virus, 96 NTM organisms were identified; M. avium was recovered from 50 (77%) of the 65 NTM-positive cultures of blood or bone marrow, while Mycobacterium intracellular and other non-avium NTM accounted for 18% and 5% of the isolates, respectively. Little difference in the susceptibility of isolates to antibiotics was noted between HIV-positive and HIV-negative patients or between M. avium and M. intracellulare. These data demonstrate that HIV-positive patients develop disseminated disease with NTM other than M. avium more frequently than has been previously reported and that these patients do not appear to be infected with NTM that are more resistant to antimicrobial agents than are NTM isolated from HIV-negative patients.

Catheter-associated sepsis caused by Ochrobactrum anthropi: report of a case and review of related nonfermentative bacteria.

Ochrobactrum anthropi, formerly known as CDC group Vd, is an oxidase-producing, gram-negative, non-lactose-fermenting bacillus that oxidizes glucose and grows readily on MacConkey agar. Only occasionally isolated from human clinical specimens, this organism has rarely been found to be pathogenic. We describe the first reported case of infection due to O. anthropi in a child, that of bacteremia in a 3-year-old girl undergoing chemotherapy for retinoblastoma. In addition, we review the literature concerning cases of infection due to this and closely related bacterial species, namely Alcaligenes xylosoxidans subspecies xylosoxidans, Agrobacterium radiobacter, and "Achromobacter" group B. Finally, we attempt to clarify the confusing history and taxonomy of these organisms as well as make recommendations regarding antimicrobial therapy for infections caused by them.

Proliferative responses to human immunodeficiency virus type 1 (HIV-1) gp120 peptides in HIV-1-infected individuals immunized with HIV-1 rgp120 or rgp160 compared with nonimmunized and uninfected controls.

The proliferative responses to a series of peptides constituting the human immunodeficiency virus type 1 (HIV-1) gp120 sequence were evaluated in 19 HIV-1-infected rgp160 vaccine recipients, 17 HIV-1-infected rgp120 vaccine recipients, 15 HIV-1-infected placebo recipients, and 18 HIV-1-uninfected controls. Many regions of the gp120 molecule were found to contribute proliferative epitopes, although there were clearly regions of relative dominance and silence. Vaccine recipients tended to have broader, more robust, and more frequent peptide recognition than the placebo recipients. Despite the considerable variability in the pattern of peptide recognition among individuals, there was a striking similarity between the rgp160 and rgp120 vaccinee groups as a whole. Low-risk HIV-1-uninfected individuals may react to a few peptides within the gp120 sequence as well, despite a lack of significant response to the whole gp120 protein.

Age-adjusted CD4+ lymphocyte parameters in healthy children at risk for infection with the human immunodeficiency virus. The Military Pediatric HIV Consortium.

Values for CD4+ lymphocytes are reported to vary by age. We evaluated an ethnically diverse population of healthy children at risk for human immunodeficiency virus infection to establish normal ranges for age-adjusted CD4+ lymphocyte parameters. We identified a threshold of approximately 30% CD4+ lymphocytes which corresponded to a 5th percentile for all ages. It is important that no significant differences in absolute CD4+ lymphocyte counts on the basis of ethnic group were found.

Evaluation of human immunodeficiency virus (HIV) type 1 RNA levels in cerebrospinal fluid and viral resistance to zidovudine in children with HIV encephalopathy.

The amount of human immunodeficiency virus (HIV) type 1 RNA and the presence of a codon 215 mutation indicative of zidovudine resistance were evaluated in cerebrospinal fluid (CSF) and plasma obtained from HIV-1-infected children. The level of HIV-1 RNA in CSF was highest in children with severe encephalopathy (n = 25; median, 430 copies/mL; range, 0-2.2 x 10(5) copies/mL) followed by the moderately encephalopathic (n = 7; median, 330; range, 0-1130) and nonencephalopathic groups (n = 9; median, 0; range, 0-566) (P = .007). There was no correlation between CSF and plasma HIV-1 RNA levels. Five of 7 children with the codon 215 mutation in CSF had a progression of encephalopathy, while all 8 children with wild type codon 215 had improved or stable disease during zidovudine treatment (P = .007). These findings suggest that increased viral replication and emergence of drug-resistant HIV-1 variants within the central nervous system may play a role in progression of HIV encephalopathy.

Delayed-type hypersensitivity skin testing in human immunodeficiency virus-infected pediatric patients.

OBJECTIVE: To evaluate whether pediatric patients infected with human immunodeficiency virus (HIV) can mount appropriate delayed-type hypersensitivity (DTH) skin responses to recall antigens and whether these responses can be correlated with clinical or immunologic parameters. DESIGN: Prospective evaluation of DTH responses in HIV-infected children. Uninfected children born to HIV-infected mothers served as control subjects. Antigens used for yearly DTH testing included Candida albicans (1:100, 1:10); mumps virus; Trichophyton; purified protein derivative of tuberculin; and tetanus toxoid (1:100, 1:10). At the time of each DTH test, patients were staged according to two Centers for Disease Control and Prevention pediatric HIV classification systems, and T-cell subsets were obtained. RESULTS: Twenty-seven HIV-infected patients with a median age at entry of 74.1 (range, 12 to 156) months were followed. Forty-four DTH skin tests in 21 symptom-free HIV-infected patients (PI) and 18 tests in 10 HIV-infected patients with symptoms (P2), as well as 43 DTH skin tests in 18 patients who had either mild or moderate clinical symptoms or immunosuppression and 19 tests in 13 patients with severe symptoms or immunosuppression, were evaluated. Sixteen DTH skin tests were performed in 14 uninfected patients. HIV-infected patients tended to have fewer DTH responses to antigens and of smaller size than did uninfected patients. When controlled for age, few differences in DTH responsiveness were seen between HIV-infected and uninfected patients. Anergy was associated with symptomatic disease, evidence of advanced clinical or immunologic disease, and low CD4+ percentages (p <0.05). CONCLUSIONS: HIV-infected children are able to mount antigen-specific cell-mediated immune responses that are qualitatively similar to those of age-matched control subjects. Loss of DTH responsiveness correlates with both clinical and immunologic evidence of HIV disease progression.

Cross-subtype neutralizing antibodies induced in baboons by a subtype E gp120 immunogen based on an R5 primary human immunodeficiency virus type 1 envelope.

Global human immunodeficiency virus type 1 (HIV-1) diversity may require engineering vaccines to express antigens representing strains prevalent in the target population of vaccine testing. The majority (90%) of incident infections in Thailand are genetic subtype E, with a small percentage of subtype B infections in the intravenous drug user populations. We have evaluated and compared the binding and HIV-1 neutralizing properties of serum antibodies induced in baboons by CHO cell-expressed monomeric gp120 derived from a CCR5-using (R5) subtype E primary HIV-1CM235 or a CXCR4-using (X4) subtype B T-cell line-adapted (TCLA) HIV-1SF2 isolate. In contrast to the subtype-specific HIV-1 neutralizing antibodies induced with recombinant HIV-1SF2 gp120 (rgp120SF2), rgp120CM235 immunization induced antibodies capable of neutralizing both subtype E and subtype B TCLA HIV-1 isolates. However, neither immunogen induced antibodies capable of neutralizing primary HIV-1 isolates. Antibody induced by rgp120CM235 preferentially bound natively folded gp120 and retained strong cross-reactivity against multiple gp120 strains within subtype E as well as subtype B. In contrast, antibody responses to rgp120SF2 were directed predominantly to linear epitopes poorly exposed on native gp120 and had more limited cross-recognition of divergent gp120. Fine epitope mapping revealed differences in antibody specificities. While both rgp120CM235 and rgp120SF2 induced antibodies to regions within C1, V1/V2, V3, and C5, unique responses were induced by rgp120CM235 to multiple epitopes within C2 and by rgp120SF2 to multiple epitopes within C3, V4, and C4. These data demonstrate that strain and/or phenotypic differences of HIV-1 subunit gp120 immunogens can substantially alter antibody binding specificities and subsequent HIV-1 neutralizing capacity.