Modelling Multiple Dosing with Drug Holiday in Antiretroviral Treatment on HIV-1 Infection

A within-host mathematical model to describe the dynamics of target cells and viral load in early HIV-1 infection was developed, which incorporates a combination of RTI and PI treatments by using a pharmacokinetics model. The local stability of uninfected steady state for the model was determined using an alternative threshold. The pharmacokinetics model was employed to estimate drug efficacy in multiple drug dosing. The effect of periodic drug efficacy of pharmacokinetic type on outcome of HIV-1 infection was explored under various treatment interruptions. The effectiveness of treatment interruption was determined according to the time period of the drug holidays. The results showed that long drug holidays lead to therapy failure. Under interruption of treatments combining RTI and PI therapy, effectiveness of the treatment requires a short duration of the drug holiday.

STUDY OF HIV-1 TRANSMISSION ACROSS HUMAN CERVICAL TISSUE TO THE LYMPHOID TISSUE USING AN ORGAN CULTURE MODEL

The female genital tract is the main route for heterosexual transmission of HIV-1. Studies with SIV in macaque model indicate that after crossing the mucosa of cervix and vagina, SIV infected cells migrate to draining lymph nodes where the virus expands before it gets disseminated into the gut and other organs of the body. We have developed a combined organ culture system composed of cervical tissue together with tonsil tissue (surrogate lymphoid tissue for lymph node) to study HIV-1 transmission across the mucosal barrier and to evaluate the fate of HIV-1 and HIV-1 infected cells after crossing cervical mucosa and migrating to regional lymph nodes. To study the transmission of virus, a defined amount of pretitered cell-free or cell-associated HIV-1BAL or HIV-1IIIB was added to the top of the cervix tissue and incubated at 37°C in a CO2 incubator. The top well was removed after 3-4 days and tonsil tissue or tonsil mononuclear cells in the bottom well were cultured for an additional 12 days. Replication of cell-free and cell-associated HIV-1BAL and HIV-1IIIB in tonsillar cells after crossing the cervix mucosa was confirmed by the presence of HIV-1 p24 in culture supernatant, the detection of HIV-1 DNA by real-time PCR, presence of p24 antigen by immunofluorescence assay and presence of HIV-1 RNA by simultaneous immunophenotyping and ultrasensitive fluorescence in situ hybridization. We have also characterized by immunofluorescence assay HIV-1 infected migratory cells exiting from the cervix tissue. CD4+ (T cells), CD11c+ (dendritic cells) and CD68+ (macrophages) cells were found to migrate from cervix tissue and were positive for HIV-1. The public health relevance of this model is that we may use the combined cervix and tonsil tissue/cell model to determine the mechanism of sexual transmission of HIV-1 in women at the cellular and molecular level and to evaluate anti-HIV microbicides

Stochastic Model for Langerhans cells and HIV Dynamics in Vivo

Working paperMany aspects of the complex interaction between HIV and the human immune system remain elusive. Our objective is to study these inter-actions, focusing on the specic roles of langerhans cells (LCs) in HIV infection. In patients infected with HIV, a large amount of virus is as-sociated with LCs in lymphoid tissue. To assess the influence of LCs on HIV viral dynamics during anti-retroviral therapy, we present and analyse a stochastic model describing the dynamics of HIV, CD4+ T-cells, and LCs interactions under therapeutic intervention in vivo. We per-form sensitivity analyses on the model to determine which parameters and/or which interaction mechanisms strongly affect infection dynamics.Many aspects of the complex interaction between HIV and the human immune system remain elusive. Our objective is to study these inter-actions, focusing on the speci c roles of langerhans cells (LCs) in HIV infection. In patients infected with HIV, a large amount of virus is as-sociated with LCs in lymphoid tissue. To assess the influence of LCs on HIV viral dynamics during anti-retroviral therapy, we present and analyse a stochastic model describing the dynamics of HIV, CD4+ T-cells, and LCs interactions under therapeutic intervention in vivo. We per-form sensitivity analyses on the model to determine which parameters and/or which interaction mechanisms strongly affect infection dynamics

Characterization of human foreskin Langerhans cells

Background: It is known that medical male circumcision (MMC) decreases HIV acquisition by up to 60%. One hypothesis is that MMC removes a foreskin (FS) that harbors different immune cells that are HIV target cells such as CD4+ macrophages, T, Langerhans (LCs), and dendritic cells (DCs). However, there have been different reports on whether the inner FS or outer FS has more HIV target cells. While LCs have been implicated in HIV transmission, their role remains controversial. Studies have shown that LCs can transmit the virus to T cells, which increases infection. On the contrary, others have reported that LCs prevent infection by degrading the virus through a langerin-dependent pathway. One of the factors that plays a major role in HIV transmission is their state of maturity and activation, which can be influenced by co-infection and other immunological processes. The aim of this study was to isolate, quantify and characterize Langerhans cells in the inner FS and outer FS from men undergoing MMC and to evaluate the phenotype of matured and activated LCS. Differences in the proteome of the inner FS and outer FS tissues were further investigated. Methodology: FS were obtained from men undergoing voluntary MMC from clinics and hospitals in the Western Cape (Age 18 years or older). Epidermal FS cells were extracted using crawl (migratory) assay and liberase enzyme digestion. Langerhans cells were isolated by density gradient centrifugation, sorted, quantified and immune-profiled by flow cytometry. CD1a and CD207 were used to identify Langerhans cells while HLA-DR, CD80, CD86 and CD40 were used as markers of maturity and activation. The gene expression profile of sorted LCs was also examined by single-cell sequencing with seq-well. Lastly, the differences in the proteome of the inner FS and the outer FS migrated epidermal cells were assessed using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Results: Langerhans cells were an average of 85% pure post-sorting. The numbers of Langerhans cells between the inner FS vs. outer FS were not statistically different (mean: 0.56% vs. 0.68% (SD=0.37) from migratory cells and 0.28% vs. 0.45% (SD=0.18) from enzyme digest, p-value >0.05, n=9). Sequencing showed that the sorted cells pooled from 5 participants (inner and outer FS) had different gene expression profiles. Furthermore, two groups of cells were identified from the sorted LCs based on their gene expression profile. The identified cells were monocyte-like and melanocyte-like cells. The monocyte-like cells were identified as LCs based on their gene expression profile while the melanocyte-like cells were identified as the contaminating cells as the cell purity was not 100%. Upon activation with tumor necrosis factor alpha (TNF-α), activated LCs isolated by the migration assay had similar proportions of cells expressing surface maturity and activation markers (HLA-DR, CD40 and CD80/86) when compared to the unstimulated controls (inactivated) (mean: 73.53% vs. 75.66%, n=9, p-value >0.05, SD=4.4) However LCs that were isolated by the migration assay expressed markers of activation at a higher level compared to LCs isolated by Liberase enzyme digestion (mean: 79.4% vs. 40%, p-value < 0.05 n=9, SD=23). Proteomics showed that the inner FS had an over-abundance of proteins involved in the interleukin 7 response and mRNA catabolic processes, while the outer FS had more spindle zones and cornified envelope proteins that were over-abundant when comparing inner and outer FS from 5 participants. Discussion and Conclusion: The study successfully extracted, sorted and immunoprofiled Langerhans cells using different methods and from different FS compartments (inner FS versus outer FS). When LCs were spontaneously migrated and isolated using the “crawl method”, they showed a more mature and activated phenotype compared to non-migrating “skin resident” immune cells. No differences were found between cells that were stimulated with inflammatory cytokines relative to unstimulated migratory cells in proportion of cells expressing activation markers. However, it was observed that cells isolated by liberase enzyme digestion showed significantly lower proportions of activation markers relative to migratory cells. Using LC-MS/MS-based proteomics; the inner FS exhibited high expression of proteins involved in the interleukin 7 response while the outer FS exhibited high expression of structural proteins, which suggests that the inner FS might be more involved in immunity as interleukins can stimulate immune response while the outer FS has a more structural role than the inner FS

HIV target cells and innate immune factors in the human female genital tract

World-wide, human immunodeficiency virus (HIV) transmission most often takes place in the genital tract during heterosexual intercourse, but the site where HIV most commonly establishes primary infection in the female genital tract remains unknown. Several factors including viral load in the HIV-infected individual, presence of genital infections, genotype and possibly sex hormone levels influence the risk of HIV acquisition. In addition, cationic polypeptides with anti-HIV activity in vitro including secretory leukocyte protease inhibitor (SLPI), Elafin/Trappin-2, human neutrophil peptide (HNP) 1-3 and LL-37 may play dual roles in vivo with both an antiviral effect and target cell-recruiting properties. For this thesis, we investigated the distribution of potential HIV target cells in three anatomical regions (ectocervix, endocervix and endometrium) of the female genital tract by immunostaining, computerized image analysis and confocal microscopy. Furthermore, innate immune peptides were quantified in genital secretions using ELISA and in ectocervical tissue using real-time RT-PCR and immunohistochemistry. To characterize antigen presenting cell subsets and their expression of HIV-binding C-type lectin receptors (CLRs) ectocervical tissue biopsies were collected from HIV-negative women at low-risk of HIV infection and HIV-negative female sex workers considered to be at high-risk of acquiring such infection. Distinct cell populations were identified including CD1a + Langerin + Langerhans cells (LCs) in the epithelium and CD11c + DC-SIGN + myeloid dendritic cells (mDCs), CD68 + DC-SIGN + Mannose receptor + (MR + ) mDCs and/or macrophages as well as CD123 + plasmacytoid dendritic cells (pDCs) in the submucosa of the ectocervix. The high-risk women had significantly higher expression of CLRs than the low-risk subjects. To map the distribution of potential cellular targets and receptors binding to HIV in the endocervix and endometrium, tissue biopsies were collected from HIV-uninfected low-risk women undergoing hysterectomy. LCs were localized mainly in the columnar epithelium, whereas CD4 + CCR5 + T cells were present both within and adjacent to the endometrial and endocervical epithelium. CD11c + DC-SIGN + MR + and CD68 + DC-SIGN + MR + mDCs and macrophages were confined to the submucosa of both tissue types but were localized in close proximity to the epithelial surface. To assess the expression of SLPI, Trappin-2, HNP 1-3 and LL-37, genital fluid samples and ectocervical biopsies were collected from HIV-uninfected low-risk woman and HIV-uninfected sex workers (HIV high-risk). LL-37 and Trappin-2 levels were significantly lower among low-risk women currently using combined oral contraception. Compartmentalization of the investigated factors classed HNP 1-3 as the most abundant factor in genital fluids of low-risk woman, whereas SLPI had the highest expression in ectocervical tissue. No association between tissue expression and soluble levels of the investigated factors was seen at the individual level in either HIV low-risk or high-risk women. In summary, the spatial distribution of potential HIV target cells and innate immune factors may play an important role in HIV transmission events. Thus, a better understanding of this environment may contribute to designing HIV-inhibiting compounds

Modeling CD4+ T cells and CTL response in HIV-1 infection with antiretroviral therapy

The majority of an immune system infected by HIV (Human Immunodeficiency Virus) is CD4+ T cells. The HIV-1 transmission through cell to cell of CD4+ T cells supports the productive infection. On the other hand, infected CD4+ T cells stimulate cytotoxic T-lymphocytes cells to control HIV-1 infection. We develop and analyze a mathematical model incorporating the infection process through cell to cell contact of CD4+ T cells, CTL compartment and the combination of RTI and PI treatments. By means of the alternative reproduction ratio, it is analyzed the stability criteria and the existence of endemic equilibrium. Numerical simulations are presented to study the implication of the combination of RTI and PI therapy. The results indicate that RTI drug shows more significant effect in reducing HIV-1 infection compared to PI drug

In Vivo Assessment of Natural Killer Cell Responses during Chronic Feline Immunodeficiency Virus Infection

Accumulating evidence suggests that natural killer (NK) cells may have an important role in HIV-1 disease pathogenesis; however, in vivo studies are lacking. Feline immunodeficiency virus (FIV) infection of cats provides a valuable model to study NK cell function in vivo. The immune response against Listeria monocytogenes (Lm) is well characterized, allowing its use as an innate immune probe. We have previously shown that locally delivered IL-15 can improve Lm clearance in FIV-infected animals, and this correlated with an increase in NK cell number. In the present study, chronically FIV-infected and SPF-control cats were challenged with Lm by unilateral subcutaneous injection next to the footpad and then treated with 5-bromo-2′-deoxyuridine (BrdU). The Lm draining and contralateral control lymph nodes were evaluated for NK, NKT, CD4+ and CD8+ T cell number, proliferation, apoptosis, and NK cell function. Listeria monocytogenes burden was also assessed in both control and Lm draining lymph nodes. NK, NKT, CD4+ T and CD8+ T cells in the Lm-challenged lymph node of FIV-infected cats did not increase in number. In addition, after Lm challenge, NK cells from FIV-infected cats did not increase their proliferation rate, apoptosis was elevated, and perforin expression was not upregulated when compared to SPF-control cats. The failure of the NK cell response against Lm challenge in the draining lymph node of FIV-infected cats correlates with the delayed control and clearance of this opportunistic bacterial pathogen