Combined effects of HIV and obesity on the gastrointestinal microbiome of young men who have sex with men.

OBJECTIVES: The prevalence of obesity is rising among people living with HIV, which may synergistically increase inflammation and the risk of associated diseases. Disruption of gut bacterial communities may be one of the key drivers of this inflammation; however, the combined effects of HIV and obesity on the microbiome have not been explored. METHODS: This study included 381 men who have sex with men. Thirty-nine were HIV-positive and obese (H+O+), 143 were HIV-positive and nonobese, 64 were HIV-negative and obese, and 135 were HIV-negative and nonobese. Microbiome composition was assessed by targeted sequencing of the V4 region of the 16S ribosomal RNA (rRNA) gene using rectal swab samples. Inverse probability of treatment-weighted marginal structural models were used to investigate differences in microbial composition between groups while controlling for numerous clinical and behavioural confounders. RESULTS: Significant variability in microbial composition was explained by the combination of HIV and obesity, over and above each condition alone (R2 for the marginal contribution of the H+/O+ group = 0.008; P = 0.001). H+O+ participants had the highest ratios of Prevotella to Bacteroides, a pro-inflammatory enterotype that has been described in HIV infection and obesity independently. H+O+ participants had lower levels of Bacteroides and Veillonella than all other groups, suggesting a synergistic effect of HIV and obesity on these genera. CONCLUSIONS: Our findings support the hypothesis that HIV and obesity act together to disrupt gut microbial communities, which may help explain higher levels of generalized inflammation among people living with both HIV and obesity.

HLA-G 14 bp deletion/insertion polymorphism and mother-to-child transmission of HIV.

The human leukocyte antigen HLA-G, highly expressed at the maternal-fetal interface, has a pivotal role in mediating immune tolerance. In this study we investigated the influence of HLA-G 14 bp insertion polymorphism in human immunodeficiency virus (HIV)-1 mother-to-child HIV-1 transmission. The 14 bp insertion polymorphism was analyzed among 99 HIV-1 positive mothers and 329 infants born to HIV-positive mothers in Zambia, among whom vertical transmission status and timing had been determined. HLA-G 14 bp insertion polymorphism was detected using a custom TaqMan single nucleotide polymorphisms (SNPs) genotyping assay. Logistic regression was conducted to examine the associations between HLA-G alleles and the risk of HIV transmission. The 14 bp insertion allele was more frequent in HIV exposed-uninfected (EU) infants than in infected infants, and was associated with reduced risk of both in utero (IU) and intrapartum (IP) HIV transmission, after adjusting for maternal cluster of differentiation 4 (CD4) cell count and plasma viral load. Maternal HLA-G 14 bp insertion genotype and HLA-G concordance between mother and child were not associated with the risk of perinatal HIV transmission. The presence of the 14 bp insertion associates with protection toward IU and IP HIV infection in children from Zambia, suggesting that HLA-G could be involved in the vertical transmission of HIV.

Quantitation of human immunodeficiency virus type 1 in breast milk.

The distribution and stability of human immunodeficiency virus type 1 (HIV-1) in breast milk (BM) components remain largely unknown. Inhibitory effects, if any, of BM on HIV RNA and DNA PCR amplification are poorly understood. We have addressed these issues by using virus-spiked BM samples from HIV-negative women. BM samples from HIV-negative women were spiked with HIV-1 virions or cells containing a single integrated copy of HIV DNA (8E5/LAV). After incubation under different experimental conditions, viral RNA was detected by the Roche Amplicor UltraSensitive assay in whole-milk, skim milk, and lipid fractions. We found excellent correlation between HIV-1 input copy and recovery in whole milk (r = 0.965, P < 0.0001), skim milk (r = 0.972, P < 0.0001), and the lipid fraction (r = 0.905, P < 0.001). PCR inhibition was observed in less than 10% of the spiked samples. Similar levels of inhibition were noted in BM samples collected from HIV-infected women. HIV proviral DNA was detected in BM samples using real-time PCR (linear correlation between the threshold cycle versus log DNA copy number, >0.982). The effects of incubation duration and temperature and repeated freeze-thaw cycles on HIV RNA recovery were analyzed. HIV RNA levels were remarkably stable in whole milk after three freeze-thaw cycles and for up to 30 h at room temperature. Our findings improve the understanding of the dynamics of HIV detection in BM and the conditions for BM sample collection, storage, and processing.

Virologic and immunologic determinants of heterosexual transmission of human immunodeficiency virus type 1 in Africa.

More than 80% of the world's HIV-infected adults live in sub-Saharan Africa, where heterosexual transmission is the predominant mode of spread. The virologic and immunologic correlates of female-to-male (FTM) and male-to-female (MTF) transmission are not well understood. A total of 1022 heterosexual couples with discordant HIV-1 serology results (one partner HIV infected, the other HIV uninfected) were enrolled in a prospective study in Lusaka, Zambia and monitored at 3-month intervals. A nested case-control design was used to compare 109 transmitters and 208 nontransmitting controls with respect to plasma HIV-1 RNA (viral load, VL), virus isolation, and CD4(+) cell levels. Median plasma VL was significantly higher in transmitters than nontransmitters (123,507 vs. 51,310 copies/ml, p < 0.001). In stratified multivariate Cox regression analyses, the risk ratio (RR) for FTM transmission was 7.6 (95% CI: 2.3, 25.5) for VL > or = 100,000 copies/ml and 4.1 (95% CI: 1.2, 14.1) for VL between 10,000 and 100,000 copies/ml compared with the reference group of <10,000 copies/ml. Corresponding RRs for MTF transmission were 2.1 and 1.2, respectively, with 95% CI both bounding 1. Only 3 of 41 (7%) female transmitters had VL < 10,000 copies/ml compared with 32 of 93 (34%) of female nontransmitters (p < 0.001). The transmission rate within couples was 7.7/100 person-years and did not differ from FTM (61/862 person-years) and MTF (81/978 person-years) transmission. We conclude that the association between increasing plasma viral load was strong for female to male transmission, but was only weakly predictive of male to female transmission in Zambian heterosexual couples. FTM and MTF transmission rates were similar. These data suggest gender-specific differences in the biology of heterosexual transmission.

Regions of human immunodeficiency virus type 1 nef required for function in vivo.

In vivo studies in monkeys and humans have indicated that immunodeficiency viruses with Nef deleted are nonpathogenic in immunocompetent hosts, and this has motivated a search for live attenuated vaccine candidates. However, the mechanisms of action of Nef remain elusive. To define the regions of human immunodeficiency virus type 1 (HIV-1) Nef which mediate in vivo pathogenicity, a series of mutated isogenic viruses were inoculated into human thymic implants in SCID-hu mice. Mutation of several regions, including the myristoylation site at the second glycine and a region encompassing amino acids 41 through 49 of Nef, profoundly affected pathogenicity. Surprisingly, mutations of prolines in either of the two distant PXXP SH3 binding domains did not affect pathogenicity, indicating that these regions are not required for Nef activity in developing T-lineage cells. These data suggest that some functions of Nef described in vitro may not be relevant for in vivo pathogenicity.

Human immunodeficiency virus inhibits multilineage hematopoiesis in vivo.

Human immunodeficiency virus type 1 (HIV-1)-infected individuals often exhibit multiple hematopoietic abnormalities reaching far beyond loss of CD4(+) lymphocytes. We used the SCID-hu (Thy/Liv) mouse (severe combined immunodeficient mouse transplanted with human fetal thymus and liver tissues), which provides an in vivo system whereby human pluripotent hematopoietic progenitor cells can be maintained and undergo T-lymphoid differentiation and wherein HIV-1 infection causes severe depletion of CD4-bearing human thymocytes. Herein we show that HIV-1 infection rapidly and severely decreases the ex vivo recovery of human progenitor cells capable of differentiation into both erythroid and myeloid lineages. However, the total CD34+ cell population is not depleted. Combination antiretroviral therapy administered well after loss of multilineage progenitor activity reverses this inhibitory effect, establishing a causal role of viral replication. Taken together, our results suggest that pluripotent stem cells are not killed by HIV-1; rather, a later stage important in both myeloid and erythroid differentiation is affected. In addition, a primary virus isolated from a patient exhibiting multiple hematopoietic abnormalities preferentially depleted myeloid and erythroid colony-forming activity rather than CD4-bearing thymocytes in this system. Thus, HIV-1 infection perturbs multiple hematopoietic lineages in vivo, which may explain the many hematopoietic defects found in infected patients.

Loss of T cell receptor Vbeta repertoires in HIV type 1-infected SCID-hu mice.

Late-stage HIV-1 disease in humans has been associated with perturbations of the T cell receptor (TCR) Vbeta repertoire. It is not known if the observed loss of certain Vbeta families is attributable directly to HIV-1 infection or whether this is a consequence of multiple opportunistic infections. Putative HIV-1-associated superantigens have been postulated to be the cause of the perturbed TCR Vbeta repertoire and the subsequent CD4+ T cell depletion in HIV-1-infected humans. In this study, we examined the human TCR Vbeta repertoire in SCID-hu mice, housed in a pathogen-free environment and infected with a molecularly cloned virus strain, to ascertain directly the effect of HIV-1 on the human TCR Vbeta repertoire in the absence of other infectious agents. We demonstrate that mock-infected human thymus/liver (Thy/Liv) implants in SCID-hu mice have complete TCR Vbeta repertoires, reflective of a normal human thymus. However, HIV-1-infected implants in SCID-hu mice had depleted TCR Vbeta repertoires, corresponding with thymocyte depletion. These results indicate that HIV-1-specific mechanisms are the cause of the TCR Vbeta repertoire depletion in infected implants. However, these thymocyte depletions were not restricted to specific TCR Vbeta subsets. These results are not consistent with the hypothesis that HIV-1 acts as a superantigen in vivo. The disruption of the TCR Vbeta repertoire in the human Thy/Liv implants of the SCID-hu mice suggests that HIV-1 infection may be influencing T cell development in the thymus, contributing to both the overall CD4+ T cell depletion in AIDS and limited TCR repertoire diversity.

The SCID-hu mouse: an in-vivo model for HIV-1 pathogenesis and stem cell gene therapy for AIDS.

Animal models are critical to the investigation of human immunodeficiency virus type 1 (HIV-1) pathogenesis. However, normal animal models are either uninfectable with HIV-1, or if infected, do not display HIV-1 induced pathology. Here, we describe how the severe combined immunodeficient mouse (SCID), implanted with human fetal thymus and liver, has been used to model HIV-1 pathogenesis and anti-retroviral gene therapy. Unable to reject the human tissue, these chimeric mice provide the investigator with a human hematolymphoid organ which, following infection by HIV-1, may more closely mimic the situation seen in humans than standard in-vitro culture systems.

Replication and pathogenicity of human immunodeficiency virus type 1 accessory gene mutants in SCID-hu mice.

The functional roles of the human immunodeficiency virus type 1 (HIV-1) accessory genes (nef, vpr, vpu, and vif) are as yet unclear. Using the SCID-hu model system, we have examined the infectivity, replicative capacity, and pathogenicity of strains of the molecular clone HIV-1NL4-3 that contain deletion mutations in these individual accessory genes. We determined that deletion of these genes had differential effects on both infectivity and pathogenicity. Deletion of vpr had little or no effect on viral infectivity, replication, and pathogenicity; however, deletion of vpu or vif had a significant effect on infectivity and moderate effects on pathogenicity. nef-minus strains were the most attenuated in this system, demonstrating significantly lower levels of infectivity and pathogenicity. However, deletion of these individual genes attenuated but did not abrogate the pathogenic properties of HIV-1. Mutant viruses still retained the ability to induce thymocyte depletion to various degrees if implants were infected with higher doses of virus or observed for longer periods of time. The relative contributions of these genes to in vivo pathogenic potential should be taken into consideration when one is contemplating a live attenuated vaccine for HIV-1.

In vivo pathogenic properties of two clonal human immunodeficiency virus type 1 isolates.

We have investigated the in vivo pathogenic properties of two molecularly cloned strains of human immunodeficiency virus type 1 (HIV-1), HIV-1NL4-3 and HIV-1JR-CSF, in human fetal thymus/liver implants in severe combined immunodeficient mice. Studies comparing their in vivo replication kinetics and abilities to induce CD4+ thymocyte depletion were performed. HIV-1NL4-3 replicated in vivo with faster kinetics and induced greater levels of CD4+ thymocyte depletion than did HIV-1JR-CSF. These results demonstrate that different viral isolates have different pathogenic properties in this system. In the SCID-hu model, this pathogenesis most likely occurs in the absence of an immune response. Therefore, we investigated whether the absence of immune selection resulted in extensive genetic variation and the generation of viral quasispecies. To this end, DNA corresponding to the fourth variable domain region of the viral envelope gp120 protein recovered from biopsy samples at 6 weeks postinfection was sequenced. Little genetic variation was noted in either HIV-1JR-CSF- or HIV-1NL4-3-infected implants. The mutation levels demonstrated in both viral strains were more reflective of the acute rather than the chronic phase of HIV-1 infection in humans. These results suggest that the SCID-hu mouse model can be used to study the in vivo pathogenicity of different HIV-1 isolates in the absence of host immune selective pressures.

Requirement of human immunodeficiency virus type 1 nef for in vivo replication and pathogenicity.

The role of human immunodeficiency virus type 1 (HIV-1) accessory genes in pathogenesis has remained unclear because of the lack of a suitable in vivo model. The most controversial of these genes is nef. We investigated the requirement for Nef for in vivo replication and pathogenicity of two isolates of HIV-1 (HIV-1JR-CSF and HIV-1NL4-3) in human fetal thymus and liver implants in severe combined immunodeficient mice. HIV-1JR-CSF and HIV-1NL4-3 differ in their in vitro phenotypes in that HIV-1JR-CSF does not induce syncytia and is relatively noncytopathic, while HIV-1NL4-3 is highly cytopathic and readily induces syncytia. The nef mutants of both isolates grew with kinetics similar to those of parental virus strains in stimulated peripheral blood lymphocytes but demonstrated attenuated growth properties in vivo. HIV-1NL4-3 induced severe depletion of human thymocytes within 6 weeks of infection, whereas its nef mutant did not. Thus, HIV-1 Nef is required for efficient in vivo viral replication and pathogenicity.

The SCID-hu mouse as a model for HIV-1 infection.

During normal fetal ontogeny, one of the first organs to harbour CD4-positive cells is the thymus. This organ could therefore be one of the earliest targets infected by human immunodeficiency virus type 1 (HIV-1) in utero. HIV-1-infected cells and pathological abnormalities of the thymus have been seen in HIV-1-infected adults and children, and in some fetuses aborted from infected women. Studies of HIV-1 pathogenesis have been hampered by lack of a suitable animal model system. Here we use the SCID-hu mouse as a model to investigate the effect of virus infection on human tissue. The mouse is homozygous for the severe combined immunodeficiency (SCID) defect. The model is constructed by implanting human fetal liver and thymus under the mouse kidney capsule. A conjoint human organ develops, which allows normal maturation of human thymocytes. After direct inoculation of HIV-1 into these implants, we observed severe depletion of human CD4-bearing cells within a few weeks of infection. This correlated with increasing virus load in the implants. Thus the SCID-hu mouse may be a useful in vivo system for the study of HIV-1-induced pathology.