Assessment of oral transmission using cell-free human immunodeficiency virus-1 in mice reconstituted with human peripheral blood leucocyte

Oral-genital contact is one of the risk factors for the transmission of human immunodeficiency virus (HIV) in adults. In recent reports, oral exposure to simian immunodeficiency virus (SIV) was found to have important implications for the achievement of mucosal transmission of HIV in a rhesus monkey animal model. In the present study, we aimed first to establish a small animal model which did not develop tonsils suitable for HIV oral mucosa transmission, using non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice and NOD/SCID B2m(null) mice grafted with human peripheral blood leucocytes (hu-PBL) and stimulated with interleukin (IL)-4, and second to investigate whether oral exposure to cell-free R5 and X4 HIV-1 could lead to oral transmission of HIV through intact or traumatized mucosal tissues in humanized mice. Both low and high concentrations of cell-free R5 and X4 viruses failed to cause oral transmission with or without trauma in hu-PBL-NOD/SCID and NOD/SCID Beta2m(null) mice, which presented a number of CD4+ cells in gingival tissues and oral cavities with or without tissue injury. The present results show that IL-4-administrated NOD/SCID B2m(null) mice are useful as a small-humanized model for the study of HIV oral infection, which may help to define the window of opportunity for oral transmission by the HIV virus in animal model experiments.     

Long-term productive human immunodeficiency virus-1 infection in human infant microglia.

The course of human immunodeficiency virus 1 (HIV-1) infection in human infant microglia was studied using purified primary cultures of microglia derived from brain autopsy tissue. Previous in vitro studies have used fetal or adult brain tissue. Important differences may exist between brain tissues of different maturational ages with regard to HIV-1 replication and other neuropathogenic effects. Infant microglia were infected with four different strains of HIV-1 (JR-FL, JR-CSF, Ba-L, and IIIB). Productive infection was demonstrated by p24 antigen production, immunocytochemistry, and recovery of replication-competent virus from the supernatants of the infected cultures. Multinucleated giant cells developed in culture mimicking the neuropathological changes seen in the brains of patients with HIV encephalopathy. Productive infection was more readily established by monocyte-tropic strains (JR-FL and Ba-L) of HIV-1 than by a lymphocyte-tropic strain (IIIB). p24 antigen production in this system peaked at 47 to 51 days postinfection. Viral persistence in giant cells was demonstrated by immunocytochemistry for the gp120 and gp41 viral antigens as late as 70 days postinfection. This in vitro culture system, using infant microglia that support viral replication for more than 2 months, may provide a useful model for studying the pathogenesis of progressive HIV encephalopathy.     

Immunohistochemical studies of human immunodeficiency virus-1 in liver tissues of patients with AIDS.

A wide spectrum of hepatic lesions has been reported in AIDS, but it is not known whether the changes are related to the presence of human immunodeficiency virus type 1 (HIV-1). Therefore, we examined liver sections from 15 consecutively autopsied patients with AIDS for the presence of HIV-1 antigens p24 (core) and gp41 (envelope) by the avidin-biotin-peroxidase complex method using monoclonal antibodies. The most common histologic abnormalities noted were steatosis, portal inflammation, Kupffer cell hyperplasia, and focal hepatocellular and bile duct damage. Intra-hepatic opportunistic organisms were detected in six of 15 (40%) cases, with Mycobacterium avium-intracellulare being the commonest (four cases). Immunoreactivity for HIV-1 antigens was demonstrated in 12 of 15 cases (80%), with staining limited to Kupffer cells and other mononuclear cells characterized by a lymphoid morphology. Approximately the same number and type of cells were stained with both monoclonal antibodies and did not bear any relation to the degree of histologic abnormalities nor to the presence of opportunistic infections. The data suggest that some pathologic changes in AIDS livers are more likely the result of an indirect effect mediated by infected resident and circulating mononuclear cells than a direct cytopathic effect of HIV-1.     

Significance of placental damage in vertical transmission of human immunodeficiency virus

The significance of physical breaches of the trophoblastic layer of the placenta in transmission of HIV from mother to infant was evaluated in 17 HIV-infected pregnant women. Samples of peripheral blood were obtained from the women during pregnancy and at delivery, at which time a small piece of placental tissue was obtained from a random site and immediately placed into fixative. Blood samples were obtained from infants at or shortly after birth and thereafter at approximately 3-month intervals, until the age of 18 months, in order to determine their HIV infection status. HIV RNA and p24 antigen were quantified in maternal plasma and CD4 cells enumerated. Paediatric diagnosis was conducted using polymerase chain reaction, virus isolation, detection of p24 antigen, and measurement of class-specific antibodies. Placental damage was quantified and evaluated using transmission electron microscopy. Maternal viral load was low, with a mean RNA copy number of 8,237 per millilitre of plasma (range 230–37,233 copies/ml). Only two women were p24-antigenaemic, and CD4 numbers ranged from 0.09 to 2.8 × 10⁹/l. There was evidence of breaks in the trophoblastic surface to the depth of the basement membrane in all 17 placentas, and perivillous fibrinoid deposits were also observed to a varying degree in all samples. However, none of the 13 infants available for follow-up had evidence of infection with HIV. Superficial damage to the trophoblastic surface of the placenta, with exposure of the basement membrane and potential exposure of CD4-expressing cells, does not appear to be a significant factor in the transmission of HIV from mother to infant during pregnancy. © 1996 Wiley-Liss, Inc.     

Ex vivo interferon-gamma response to human immunodefiency virus-1 derived peptides in human immunodeficiency virus-1 infected patients

The pattern of human immunodeficiency virus (HIV)-1 antigen-activated production of interferon (IFN)-gamma by immunocompetent cells of HIV-1 infected patients has been studied using a simplified assay combining a small volume (25 microliter) of whole blood stimulation with various HIV-1 antigens, and cytokine measurement in the same wells of microtitre plates (enzyme-linked immunotrapping assay, ELITA). The levels of IFN-gamma were higher using this assay than in the supernatant from stimulated whole blood cultures, therefore ELITA was used in the rest of the study. Specific immune responses to HIV-1 proteins (gp120, p24) and synthetic peptides derived from these proteins and from gp41 were detected in patients, but not in healthy controls. Decreased levels of IFN-gamma were observed in CDC class B (n = 5) and C (n = 4), compared with CDC class A (n = 5), following HIV-1 antigen-specific challenge. The positive response of cells from different patients to overlapping peptides of p25 (amino acids 329-344 and 335-351) was suggestive of a new epitope of HIV-1 gag recognized by T cells in the overlap region. In conclusion, the difference in in vitro antigen-specific T-cell responses of HIV-1-infected patients was shown using the ELITA method. Our results raise the possibility of using this method in screening specific antigens in HIV-1 infection.     

Rapid screening for early detection of mother-to-child transmission of human immunodeficiency virus type 1.

The testing of dried blood spots (DBSs) for the presence of human immunodeficiency type 1 (HIV-1) proviral DNA by PCR was first described in 1991. The technology has proven to be particularly valuable for resolving the infection status in HIV-1-indeterminate infants born to HIV-1-seropositive mothers. To broaden the applicability of DBS PCR, we adapted it to a standardized, commercially available microwell plate amplification and detection kit, Amplicor HIV-1, produced by Roche Diagnostic Systems. The microwell assay is rapid and easy to perform and uses equipment that is readily available in routine diagnostic laboratories. The high level of performance of the assay was demonstrated in 1,168 duplicate tests performed on 584 DBSs from 178 uninfected and 100 HIV-1-infected individuals, including 56 children with perinatally acquired HIV-1. Of 12 infants who were followed prospectively from birth, 3 (25%) were infected in utero (PCR positive at birth) and 9 (75%) were infected intrapartum (PCR negative, culture negative at birth). Overall, HIV-1 DNA was identified in 3 of 11 (27.3%) DBSs collected from infected infants during the first 4 days of life, 8 of 9 (88.9%) DBSs collected between 10 and 15 days postpartum, and 166 of 167 (99.4%) DBSs collected after 15 days of age. All 320 DBSs from uninfected children were PCR DNA negative. These findings indicate that use of the Roche microwell DBS PCR assay provides a powerful new approach for large-scale perinatal screening programs and population-based studies of vertical transmission.     

Chinese human immunodeficiency virus-1 patients with different routes of transmission exhibit altered expression levels of blood dendritic cell subpopulations

Dendritic cells (DCs) play a pivotal role in the pathogenesis of human immunodeficiency virus-1 (HIV-1). Reduced numbers of blood DCs have been observed in individuals with chronic HIV-1 infection. In the present study, we analyzed the expression levels of monocytes, myeloid dendritic cell (mDC) precursors, mDCs, and plasmacytoid dendritic cells (pDCs), in HIV-1-infected patients in China who were infected via different routes of transmission, including heterosexual and homosexual sexual contact, and blood transmission through importation of blood or blood products, to further elucidate their role in HIV. Compared with HIV-negative individuals (n?=?40), relative levels of CD11c+CD14?mDCs, CD11c++CD123(low) mDCs, and CD11c?CD123+ pDCs in total peripheral blood mononuclear cells (PBMCs) were significantly lower in all HIV patients (n?=?93), and in those with blood transmission (n?=?26) and heterosexual transmission (n?=?43), while relative levels of CD11c+CD14?mDCs were significantly lower in HIV patients infected via homosexual transmission (n?=?24). The results of correlation analysis demonstrated a significant negative correlation between CD4+ T-cell counts and the relative levels of CD11c++CD123(low) mDCs in HIV-I patients infected via blood transmission. There was no significant correlation between CD4+ T-cell counts and the expression level of other DC subpopulations in PBMCs from HIV patients. The results of this study suggest that HIV-1 patients with different routes of transmission exhibit altered expression levels of blood DC subpopulations, which contributes to dysregulated immune responses and pathogenesis of HIV-1.     

Blood-brain barrier tight junction disruption in human immunodeficiency virus-1 encephalitis

The blood-brain barrier (BBB) plays a critical role in regulating cell trafficking through the central nervous system (CNS) due to several unique anatomical features, including the presence of interendothelial tight junctions that form impermeable seals between the cells. Previous studies have demonstrated BBB perturbations during human immunodeficiency virus encephalitis (HIVE); however, the basis of these permeability changes and its relationship to infiltration of human immunodeficiency virus type 1 (HIV-1)-infected monocytes, a critical event in the pathogenesis of the disease, remains unclear. In this study, we examined CNS tissue from HIV-1-seronegative patients and HIV-1-infected patients, both with and without encephalitis, for alterations in BBB integrity via immunohistochemical analysis of the tight junction membrane proteins, occludin and zonula occludens-1 (ZO-1). Significant tight junction disruption (P < 0.001), as demonstrated by fragmentation or absence of immunoreactivity for occludin and ZO-1, was observed within vessels from subcortical white matter, basal ganglia, and, to a lesser extent, cortical gray matter in patients who died with HIVE. These alterations were also associated with accumulation of activated, HIV-1-infected brain macrophages, fibrinogen leakage, and marked astrocytosis. In contrast, no significant changes (P > 0.05) were observed in cerebellar tissue from patients with HIVE compared to HIV-seronegative patients or HIV-1-infected patients without encephalitis. Our findings demonstrate that tight junction disruption is a key feature of HIVE that occurs in regions of histopathological alterations in association with perivascular accumulation of activated HIV-1-infected macrophages, serum protein extravasation, and marked astrocytosis. We propose that disruption of this key BBB structure serves as the main route of HIV-1-infected monocyte entry into the CNS.     

Functional complementation of the envelope hypervariable V3 loop of human immunodeficiency virus type 1 subtype B by the subtype E V3 loop.

The hypervariable V3 loop within gp120 of human immunodeficiency virus type 1 (HIV-1) is the major determinant of cell tropism and the entry coreceptor usage of the virus. However, the information obtained thus far has been from only subtype B from North America and Europe, and little is known about other subtypes whose V3 amino acids differ by as much as 50% from subtype B V3. In this study, we examined the functional potential of the V3 element of the HIV-1 subtype E, the most crucial variant causing the AIDS epidemic throughout southeast Asia. A panel of HIV-1LAI recombinants was constructed by the overlap extension method, by which the LAI V3 loop was precisely replaced by that of the subtype E nonsyncytium-inducing (NSI) or syncytium-inducing (SI) variant. All of the recombinant viruses infected peripheral blood mononuclear cells, whereas only those with SI V3 infected MT2 cells, a CD4(+) T cell line. Consistently, the SI V3 recombinants used CXCR4, while the NSI V3 recombinants used CCR5 for infection of HOS-CD4(+) cells. Finally, only the NSI V3 sequence conferred CC-chemokine sensitivity on the parental virus. The data support the notion that the HIV-1 V3 loop consists of a relatively independent domain in gp120 and suggest that the subtype E V3 loop indeed contains the functional element to dictate the cell tropism, coreceptor preference, and chemokine sensitivity of the virus. These findings are of immediate importance in understanding V3 structure-function relationship and for examining phenotypic evolution of HIV-1 subtype E.     

Env length and N-linked glycosylation following transmission of human immunodeficiency virus Type 1 subtype B viruses

Whether there is selection for specific viral Env variants upon HIV-1 transmission is controversial. We examined the V1V2 and V1V4 regions of Env in 10 new and 8 previously described transmission pairs infected with HIV-1 subtype B, including a total of 9 pairs in which the infecting partner had developed substantial viral diversity prior to transmission. We found that during transmission of HIV-1 subtype B, as well as for other subtypes reported in the past, viral populations in recipients undergo substantial genetic bottlenecks, as well as weak evidence for a propensity to replicate viruses with shorter variable loops and fewer potential N-linked glycosylation sites.     

Identification of amino acid substitutions associated with neutralization phenotype in the human immunodeficiency virus type-1 subtype C gp120

Neutralizing antibodies (Nabs) are thought to play an important role in prevention and control of HIV-1 infection and should be targeted by an AIDS vaccine. It is critical to understand how HIV-1 induces Nabs by analyzing viral sequences in both tested viruses and sera. Neutralization susceptibility to antibodies in autologous and heterologous plasma was determined for multiple Envs (3-6) from each of 15 subtype-C-infected individuals. Heterologous neutralization was divided into two distinct groups: plasma with strong, cross-reactive neutralization (n = 9) and plasma with weak neutralization (n = 6). Plasma with cross-reactive heterologous Nabs also more potently neutralized contemporaneous autologous viruses. Analysis of Env sequences in plasma from both groups revealed a three-amino-acid substitution pattern in the V4 region that was associated with greater neutralization potency and breadth. Identification of such potential neutralization signatures may have important implications for the development of HIV-1 vaccines capable of inducing Nabs to subtype C HIV-1.     

APOBEC3H haplotypes and HIV-1 pro-viral vif DNA sequence diversity in early untreated human immunodeficiency virus-1 infection

We examined single nucleotide polymorphisms (SNP) in the APOBEC3 locus on chromosome 22, paired with population sequences of pro-viral human immunodeficiency virus-1 (HIV-1) vif from peripheral blood mononuclear cells, from 96 recently HIV-1-infected treatment-naive adults. We found evidence for the existence of an APOBEC3H linkage disequilibrium (LD) block associated with variation in GA → AA, or APOBEC3F/H signature, sequence changes in pro-viral HIV-1 vif sequence (top 10 significant SNPs with a significant p = 4.8 × 10(-3)). We identified a common five position risk haplotype distal to APOBEC3H (A3Hrh). These markers were in high LD (D' = 1; r(2) = 0.98) to a previously described A3H "RED" haplotype containing a variant (E121) with enhanced susceptibility to HIV-1 Vif. This association was confirmed by a haplotype analysis. Homozygote carriers of the A3Hrh had lower GA->AA (A3F/H) sequence editing upon pro-viral HIV-1 vif sequence (p = 0.01), and lower HIV-1 RNA levels over time during early, untreated HIV-1 infection, (p = 0.015 mixed effects model). This effect may be due to enhanced susceptibility of A3H forms to HIV-1 Vif mediated viral suppression of sequence editing activity, slowing viral diversification and escape from immune responses.     

Susceptibility of human testis to human immunodeficiency virus-1 infection in situ and in vitro

Semen represents the main vector for human immunodeficiency virus (HIV) dissemination worldwide and has been shown to harbor replication-competent virus despite otherwise effective highly active anti-retroviral therapy, which achieves undetectable viral load in plasma. Despite this, the origin of seminal HIV particles remains unclear, as does the question of whether the male genital tract organs contribute virus to semen. Here we investigated the presence of HIV receptors within the human testis using immunohistochemistry and quantitative real-time polymerase chain reaction. We also analyzed the infectivity of a dual tropic HIV-1 strain in an organotypic culture, as well as the impact of viral exposure on testosterone production. Our study establishes that CXCR4+, CCR5+, CD4+, and DC-SIGN+ cells are present within the interstitial tissue of human testis and that these molecules persist throughout our organotypic culture. Our data also reveal that the human testis is permissive to HIV-1 and supports productive infection, leaving testosterone production apparently unaffected. Infected cells appeared to be testicular macrophages located within the interstitial tissue. That the testis itself represents a potential source of virus in semen could play a role in preventing viral eradication from semen because this organ constitutes a pharmacological sanctuary for many current antiretrovirals.