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Pathogen COinfection:<br/>HIV, Tuberculosis, Malaria and Hepatitis C virus

Final Report Summary - PATHCO (Pathogen COinfection:HIV, Tuberculosis, Malaria and Hepatitis C virus)

Executive Summary:
Acquired immune deficiency (AIDS), tuberculosis (TB) and malaria are the primary infectious diseases causing death world wide. In addition to these pathogens, 170 million people are infected with hepatitis C virus (HCV), which leads to chronic liver disease. Because of shared routes of transmission, HCV co-infection is recognized as a major cause of morbidity and mortality among HIV-1 infected persons. The epidemiology and clinical features of co-infected subjects are well documented, however, there is a paucity of basic scientific studies addressing the interactions between these pathogens. There is undoubtedly a complex interplay between pathogens and the host immune response. This was highlighted when the Merck HIV-1 vaccine trial was halted due to increased HIV-1 transmission amongst vaccine recipients with previous adenovirus infection, suggesting that immune responses specific for adenovirus vector antigens were detrimental. We propose that pathogen evasion and dysregulation of host immune responses plays a key role in co-infection associated morbidity.
We will test this hypothesis by establishing in vitro and ex vivo co-infection model systems to study pathogen interactions and assess the effect(s) of co-infection on innate signalling and adaptive immune responses. We will develop new approaches to dissect pathogen interactions, ranging from the genesis of fluorescent labelled viruses to state-of-the-art tissue explant models and novel humanised mouse models. Translational studies of co-infected patients will ascertain pathogen-specific effects on innate and adaptive immune responses and the consequences for disease progression. It is imperative that such interactions are elucidated before proceeding
with new prophylactic or therapeutic strategies aimed at curtailing pathogen transmission or disease progression in co-infected individuals. We specifically address the call of understanding the basic biology of co-pathogen interactions and immunity.

Project Context and Objectives:
WP1: Distinct Mycobacterium tuberculosis (Mtb) strains have variations in cell wall components, mainly lipids, which can modulate induced immune responses and thereby modulate HIV expression. Genome sequence analysis to detect mutations in 192 Mtb strains collected from HIV infected and uninfected patients in Cape Town, and the use of computational tools to detect and analyse evidence of strain selection helped us identify potential strain with alterations in cell envelope lipid biosynthesis genes. Using liposomes to deliver lipid extracts from selected clinical strain and from precise knockouts strain, and purified lipids we were able to demonstrate that distinct Mtb glycolipids can affect immune responses and HIV infection.

WP1 Objectives:
1. To identify mutations in genes from Mtb / playing a role in cell envelope biosynthesis.
2. To characterize biochemically candidate strains isolated from the above clinical isolates.
3. To determine which Mtb strains affect HIV-1 replication in vitro and compare the innate immune responses in HIV-1 infected and uninfected cultures.
4. To validate outcomes of the above by studying the host response phenotype to engineered Mtb strains with ablated (mutant) or restored functions of key cell wall components or metabolites.

WP2: In vitro systems that model hepatic oxygen tensions highlight a role for low oxygen to limit HIV replication at the transcriptional level and are currently studying the role of HIF-regulated metabolites on HIV replication. In addition we identified that the HIV LTR promoter is regulated via HCV E1E2 glycoprotein activation of NF-kB, leading to a reduction in HIV expression. This inhibitory effect was mediated via the E2 protein and we are testing if it can act on bystander cells. We have shown that HCV is capable of infecting colorectal tissue and that this infection affects the mucosal environment inducing a reduction in the level of certain cytokines in contrast to what it is observed with HIV infection of the same tissue. When tissue is infected with both HCV and HIV, at the same time, the mucosal responses observed are different and replication of HIV is also affected.

WP2 Objectives:
1. To develop multi-cell and liver tissue explant systems to study HIV-1 and HCV co-infection.
2. To define the effect(s) of HIV-1 infection on immune cell capture and dissemination of HCV and to identify trans-infection inhibitors.
3. To study the effect(s) of HIV-1/HCV co-infection on immune cell inflammasome signaling.
4. To study the effect(s) of HIV-1/HCV co-infection on HCV specific cellular immune responses.

WP3: We study pathways of infection for two pathogens infecting the liver, the malaria parasite HCV. Plasmodium sporozoites infect the liver and where two host factors are known to contribute to infection, CD81 and SRBI, which are also receptors for HCV. In addition to SRBI and CD81, HCV uses several additional entry factors, including the tight junction proteins Claudin 1 and occluding, the LDL receptor, the Niemann-Pick C1-like (NPC1L1) choleteral receptor, and the receptor tyrosine kinases EGFR and EphA2. Using various Plasmodium parasites, including species infecting humans, and robust cell culture models, we performed a systematic analysis of the role of HCV entry factors during malaria liver infection Our data show that Plasmodium and HCV share a limited number of entry factors, namely CD81 and SRBI, but use distinct mechanisms to enter cells. These results have potential implications for the design of new vaccine strategies against malaria.

WP3 Objectives:
1. To identify common Plasmodium and HCV hepatocyte receptor-dependent entry pathways.
2. To establish Plasmodia/HCV co-infection models.
3. To study the effect of Plasmodia/HCV co-infection on innate immune signalling.

WP4: Infections by HIV, HBV and HCV cause global health problems and where co-infection with multiple viruses induces more severe diseases and higher mortality. We developed mouse model systems to better study these interactions. Our results show that mice with human immune grafts can be mono-infected with IV or HBV, as well as simultaneously infected with HIV and HBV, constituting a first model system to study co-infections. Our future work will focus on investigating the cross-talk between the host and the virus to unravel the pathophysiology and eventually to test new therapeutic treatments or vaccine strategies. We have also developed a new and more sensitive quantitative assay that allows for the detection of lower copy number of proviral DNA than previously achievable which can be utilised when studying these animals models as well as human subjects.

WP4 Objectives:
1. Generation and maintenance of humanized mouse models.
2. Utilize mouse models to study HIV-1, HCV or P. falciparum mono- and co-infection(s).
3. Monitor selected co-infections within humanized mouse models.

WP5: We have shown that HCV specific T-cell response occur in HCV mono-infection compared to HIV-HCV co-infected persons. These responses are inversely correlated with HCV viral load and accelerated liver disease indicated by liver fibrosis. We have identified that HIV-1 co-infection hampers HCV CD8 T-cell differentiation. The reduced maturation of HCV-specific CD8 T cells in HIV-1 co-infection associates with reduced T-cell activation. We also demonstrate that HIV-1 reduces HCV-specific CD4 T cell numbers but has no significant effect on anti-HCV B cell responses. Polymorphisms of the host factors CD-SIGN and L-SIGN were successfully linked with risk of HCV transmission but only via the mucosal transmission route and can be linked to expression.

WP5 Objectives:
1. To identify HIV-1 and HCV-specific CD8 T cell responses in HIV-1/HCV co-infected versus mono-infected subjects and to determine the mechanisms underlying T cell failure in co-infection.
2. To compare composition and function of monocyte sub-populations in mono- and co-infections.
3. To investigate functionality and HIV-1 susceptibility of HCV-specific CD4 cells in co-infection.
4. To study frequency and functionality of HCV memory B cells in mono- and HIV-1 co-infection.
5. To determine the association of host factor genotypes with HIV-1 and/or HCV disease.

WP6 Objectives:
. To guarantee full synergy and integration among PathCo beneficiaries to provide added European value and to extend the state of the art in research on pathogen co-infection and to facilitate the following outcomes:
- To reach stated milestones and deliverables
- To achieve planned objectives
- To recognize problems, to evaluate and adopt actions to solve them.

WP7 Objectives:
1. To disseminate results amongst partners and the wider scientific community.
2. To ensure an efficient use of the resources to train young scientists to encourage their active participation in research activities and professional development

Project Results:
Many of the goals proposed within PathCo have been achieved and where we have contributed significantly to the field of co-pathogen interactions and along the lines as initially proposed. Obviously not all aims have been reached due to results obtained along the way but meaningful advances have been made regarding our overall objectives. This has been a highly collaborative venture and where specialists working within their specialised fields of interest have come together to combine their knowledge and technical approaches to address a number of aims relating to the molecular and cellular interactions between TB, HIV-1, HCV and malaria. This was also extended to the development of humanised mouse model systems for the future study of how different pathogens interact at relevant and state-of-the-art model systems. We also addressed many of the issues through studying humans infected with the pathogens and especially in combination. The main areas within PathCo studied and highlighted below are the following 1) TB interactions with HIV-1 2) HCV interactions with HIV-1 3) HCV interactions with malaria 4) development of humanised mouse model systems to study mono- or co-infections and 5) the analysis of human responses to pathogen infections associated with disease and progression, mainly HIV-1 and HCV. The highlights relating to each area are listed and summarised below:

1) TB interactions with HIV-1

Tuberculosis (TB) is the leading cause of death among HIV-1-infected individuals and where co-infection with Mycobacterium tuberculosis (Mtb) exacerbates the progression of both diseases. One of our main aims was to define the genetic and molecular basis underlying Mtb regulation of HIV-1 replication. We therefore analysed 192 Mtb isolates collected as part of previous CIDRI studies conducted in Khayelitsha, Cape Town, from both HIV-1 infected as well as HIV-1 negative individuals. Based on the hypothesis that immunological and physiological differences between HIV-1 infected and uninfected individuals might manifest as differences in the strength and focus of natural selection acting upon Mtb, we sought to detect evidence of natural selection on the genomes of clinical Mtb strains that underwent WGS. Two approaches were applied to detect one or both forms of natural selection within the Mtb coding sequences. First, the FUBAR method was used to investigate overall patterns of positive and negative selection in the Mtb genome regardless of HIV-1 infection status. Second, the MEDS method was applied to specifically determine whether signals of positive selection were different in Mtb strains isolated from HIV-1 co-infected individuals than they were in Mtb strains isolated from HIV-1 uninfected individuals. The latter revealed SNPs in Mtb lipid metabolising genes that are under HIV-1 influenced selection. We carried out a lipid analysis of the selected strain to conform effects of the SNPs on production of the cognate lipid species.

We subsequently loaded lipid extracts from the different strains onto liposomes and tested their effect on viral entry utilising non-replicating viral particles with a panel of viral envelopes encompassing both CCR5 and CXCR4 HIV-1 co-receptor usage. We observed a number of effects. First, liposomes containing lipids from strains HN878, BCG and H37Rv were able to activate immature DCs (iDC) to mature DCs (mDC) after 18h incubation. The highest level was seen with lipids from the hypervirulent H878 strain, while for liposomes from BCG and H37Rv strains, a lower degree of dendritic cell (DC) activation was observed. However, clinical pathogen strains EU127 and CDC 155 glycolipids did not have any effect on the cells. We have shown here that Mtb glycolipids can affect immune responses and HIV-1 infection at different stages. Liposome associated Mtb lipids can activate DCs, indirectly modulating HIV-1 transmission by blocking the X4 and R5 HIV-1 trans-infection via iDCs by DC-SIGN depending on the specific strain of Mycobacterium (pathogenic or not), but the effect on the trans-infection was also dependent on the degree of DC activation.

We progressed to observe that liposomes from M. tuberculosis H37v producing the virulence lipid PDIM activates the capture/transfer of the HIV via DC-SIGN, while the same from a PDIM-less strain blocks the process suggesting a role for PDIMs in trans infection. Cytokine production by monocyte derived macrophages (MDM) infected with 9 Mtb clinical strains, either in mono-infection or in co-infection with HIV-1were determined via Luminex assays to identify the nature of the immune skewing. In parallel we showed that MDM infected with 9 Mtb clinical strains, either in mono-infection or in co-infection with HIV-1 had altered cyctokine profiles. Overall, using strains as well as lipid loaded liposomes we have shown that Mtb lipids are key drivers of co-relates of infection in the context of HIV-1.

Sequencing of the genomes of M. tuberculosis samples from patients in Khayelitsha, Cape Town provided evidence that co-infection with HIV-1 influences how M. tuberculosis evolves. Genes that code for enzymes that regulate the cell wall lipid profile of the Mtb were investigated for specific mutations that might change the function of these enzymes. To investigate whether these mutations might change the cell wall lipid profile of Mtb, and if these changes might influence how M. tuberculosis impacts HIV replication during co-infection of human macrophages, M. tuberculosis strains underwent lipid profiling and were assessed in in vitro infection assays. The lipid profiles of strains were analysed via TLC revealing loss of cell wall sulpholipids from some strains. When the strains were tested in MDM infection models, a differential pattern of pro-inflammatory cytokines was induced from the various Mtb strains, and that this was accentuated when MDM were co-infected with HIV-1. In addition, the supernatant from MDM infected with different strains of Mtb were used as conditioned media in luciferase-based HIV-1 replication assays in TZM-bl cells, revealing that one lipid-variable strain particularly induced elevated HIV-1 replication compared to controls, suggesting that variation in M.tb cell wall lipids has the potential to influence HIV-1 replication in macrophages and other reservoir cells of HIV-1.

In a complementary study, the HIV-1-Mtb macrophage co-infection model was used to investigate the effect of a host-directed therapy, vitamin D, on reducing the replication of HIV-1 and Mtb and the inflammatory response during co-infection. Vitamin D deficiency is common in TB patients and we have previously shown it to be exacerbated by HIV co-infection, indicating vitamin D supplementation my reduce pathogenesis during co-infection. Two different macrophage types were used. These included an M1 phenotype, thought to elicit a more pro-inflammatory response, and the other an M2 phenotype, which is thought to elicit a less inflammatory response and be more conducive to HIV-1 infection.

In both types of macrophages, vitamin D was found to inhibit HIV-1 replication. Interestingly M2 macrophages were found to secrete higher levels of the CCL2 chemokine, which has been shown to increase HIV transcription. Vitamin D inhibited the production of CCL2 as well as HIV-1 transcription. Vitamin D treatment also reduced Mtb growth in M1 macrophages when compared to M2 macrophages, but the phenotype we not observed on M2, which underwent higher rates of cell death correlated to higher levels of HIV-co-infection.

To further study the in vivo effects of HIV-1/Mtb co-infection of disease pathogenies, human lymph nodes isolated from patients with HIV/TB were immune-histologically investigated to characterise the level of pathogen infection and the phenotype and organization of infiltrating immune cells to confirm dogmas in the literature were truly observed in humans. These studies found that in granulomas, TB patients with HIV-1 co-infection had higher bacterial burden and reduced CD4+ T-cell counts. Depletion of CD4+ T-cells in the periphery also correlated with granulomas that contained fewer CD4+ and CD8+ T-cells, less interferon γ, and more neutrophils, interleukin 10 (IL-10) and detectable M. tuberculosis.

Finally, a systematic review was also conducted on studies investigating the impact of Mtb-HIV-1 confection on pathogen replication and cellular responses. The review found a paucity of well-conducted, significantly-powered studies to draw robust conclusions on the effect of HIV co-infection on the human granuloma. A detailed systematic characterisation remains required.

2) HCV interactions with HIV-1

HIV-1 and HCV infections are considered global health problems infecting millions of individuals worldwide with an estimated 15-30% of all HIV-1 positive individuals co-infected with HCV. Co-viral interactions have been poorly studied and we aimed to identify whether HCV envelope expression can interfere with HIV-1 viral assembly and thereby viral infection and replication.

We have demonstrated that hepatic low oxygen reduces HIV-1 replication and assessed the underlying mechanism to understand the role of oxygen tension in regulating HIV-1 reactivation. It was shown that low oxygen significantly reduces infectivity of certain cell lines to HIV-1 and subsequent viral replication. Low oxygen has no significant effect on HIV entry, reverse transcription or integration and largely targets the transcriptional step of the viral life-cycle. Basal and TAT-mediated HIV transcription are both sensitive to the dampening effects of low oxygen, demonstrating a TAT-independent mechanism.

Hypoxia Inducible Factors (HIFs) are transcription factors regulated by oxygen-dependent and independent stress signals that control a wide range of genes involved in energy metabolism, inflammation and angiogenesis. There are three HIF transcription factors (HIF-1, HIF-2 and HIF-3), each a heterodimer comprising an alpha and beta subunit. The best understood, HIF-1 and HIF-2, are regulated via an oxygen-dependent degradation domain (ODD) in their alpha sub-unit, whereas the beta sub-unit is constitutively expressed. Under ‘normal’ oxygen tensions HIFα subunits are hydroxylated by prolyl hydroxylases and targeted for proteosomal degradation. Mutation of proline residues in HIFα subunits results in a protein that is constitutively expressed and transcriptionally active. HIFs exert their transcriptional activity via binding a consensus motif ‘ACGTGC’ in host promoter or enhancer elements and screening HIV LTR sequences identifies a hypoxic responsive element (HRE) that is conserved within in representative viral strains from genotypes A – G. Importantly, all diverse HIV LTR sequences studied are sensitive to the inhibitory effects of low oxygen, suggesting that HIFs limit HIV transcription. Furthermore, we demonstrate that HIV-1 transcription from latent T cell models following TNFα or PMA activation is reduced.

One of the main themes within PathCo was the analysis of how molecules from one pathogen can interfere with the infection and replication of another. One of our main aims concerning HIV-1 and HCV interaction was to determine whether the expression of one viral envelope protein can interfere with the infection and/or replication of another. One major aim was to produce viruses expressing both envelopes and studying the viral phenotype of resultant virus. We demonstrated indeed that is the HCV E1E2 envelope gene was expressed in a cell expressing HIV-1 gp120 then chimeric expressing viruses could be generated and where host cell of infection could be generated.

Through the analysis of envelope (HIV-1 gp120 and HCV E1E) co-expressing cells the surprisingly observation was made that HCV E1E1 expression resulted in a marked reduction in HIV-1 viral production and in a dose dependent manner. It was identified that the effect was mediated via HIV-1 LTR expression and the effect was observed for all HIV-1 LTR or HCV Env subtypes tested. Generated cell-lines stably expressing the HCV E1E2 protein had a significant decrease in HIV-1 LTR activity and viral production again supporting the finding that expression can switch off HIV-1 LTR activity and dampen viral production.

Hepatitis C virus (HCV) is well known to be transmitted by blood. In recent years, an increasing number of clinical cases describing colorectal transmission of HCV have been reported. However, when the PathCo consortium was awarded, there were no laboratory results confirming this new route of transmission for HCV. Within PathCo we developed a model of colorectal tissue using gut specimens obtained from surgery that once cut in small pieces, known as tissue explants, can be challenged with virus. The tissue can then be maintained in culture to determine susceptibility to infection, and tissue responses elicited upon infection. Using this model and a luciferase-reporter HCV clone, we have shown that HCV can infect colorectal tissue, and that this infection can be inhibited by a combination of HCV specific drugs (Sofosbuvir and Ledispavir), by an HCV E1 protein-specific neutralising antibody AR4A or by a broad spectrum anti-viral compound.

We further assessed the early mucosal responses induced by HCV infection. Modulation of the mucosal environment 24 h after viral exposure was studied by quantification of secreted cytokines/chemokines (including inflammatory, anti-inflammatory and adaptive cytokines, CC and CXC chemokines, growth factors and antimicrobial proteins) in explant culture supernatants with Luminex® bead-multiplex technology. When comparing with the cytokine profile measured for unchallenged control tissue, HCV elicited a significant decrease in the levels of MIP-1, MCP-1, IL-2, IL-6 and RANTES in culture supernatant. Hence, HCV induced down-regulation of cytokines/chemokines linked to inflammation, T cell proliferation, chemotaxis and humoral immune response.

The majority of HCV sexual transmissions have been described in human immunodeficiency virus (HIV)-infected patients. HIV-1 is known to induce chronic inflammation, however the effect of a HIV-1/HCV co-infection in the colorectal tract has not been described. Using the tissue explant model, we showed that in contrast to HCV, HIV-1 induced a mucosal inflammatory response with a significant increase of IL-2, GM-CSF, MCP-2, MIG and MCP-1 and a significant decrease of IL-1. When colorectal tissue explants were infected with HIV and HCV, we measured a significant increase in the levels of IL-2, IP-10, GM-CSF, MCP-2, IL-17 and MCP-1, and a significant reduction of IL-8 and IL-6. The intensity of the modulation of cytokines linked to HIV-induced inflammation was dampened with HIV-HCV co-infection. The infection of colorectal explants by HIV-1 has been well described by our team. Co-exposure of colorectal tissue explants to HIV-1 and HCV, induced higher levels of HIV-1 replication (p24 viral antigen) after 15 days of culture.

Sexual transmission of HCV can occur, and the colorectal tract would act as a transient “reservoir”. The early responses elicited by HIV-1 in colorectal tissue differ from those elicited after exposure to HCV and co-exposure to HIV-1 and HCV affects the early responses elicited by each virus individually. These findings are pertinent given the associations we made when studying a number of host genotypes with the risk of HCV transmission and a strong association was made with 3 promoter mutations within the DC-SIGN gene, but only for people exposed sexually to the virus and not through injection. Both results would suggest that HCV can interact with dendritic cells at the mucosal surface.

3) HCV interactions with malaria

The work aimed at investigating the common pathways used by two pathogens infecting the liver, the malaria parasite Plasmodium and the Hepatitis virus C (HCV). Plasmodium sporozoites are transmitted by mosquitoes and first infect the liver, where the parasite replicates as a pre-requisite to the development of the pathogenic blood stage infection. The mechanisms of Plasmodium entry into liver cells remain largely unknown, but two host factors are known to contribute to infection, CD81 and SRBI, which are also receptors for HCV. In addition to SRBI and CD81, the HCV uses several additional entry factors, including the tight junction proteins Claudin 1 and occludin, the LDL receptor, the Niemann-Pick C1-like (NPC1L1) cholesterol receptor, and the receptor tyrosine kinases EGFR and EphA2.

Using various Plasmodium parasites, including species infecting humans, and robust cell culture models, we performed a systematic analysis of the role of HCV entry factors during malaria liver infection. Our data revealed that Plasmodium and HCV share a limited number of entry factors, namely CD81 and SRBI, but use distinct mechanisms to enter cells. Specifically, whereas HCV uses both SRBI and CD81 in a sequence of events leading to virus entry, our results showed that SRBI and CD81 define two redundant and independent entry routes for the malaria parasite. Importantly, we identified a parasite protein that is a key determinant of host cell receptor usage, the 6-cysteine domain protein P36. Using novel invasion assays based on transgenic fluorescent parasite lines produced in rodent malaria species, we uncovered that Plasmodium sporozoites actively invade cells inside two types of vacuoles, and use two different strategies, egress from the vacuole or remodeling of the vacuole membrane, to escape degradation by the host cell lysosomes. These findings illustrate how the malaria parasite evades the host cell defense mechanisms to ensure its safe migration to the liver and the establishment of a suitable intracellular niche for replication.

Since Plasmodia and HCV utilise SR-BI and CD81 to enter hepatocytes we were interested to identify host pathways that regulate expression of these entry factors. The liver experiences a gradient of oxygen from the periportal to pericentral areas that associates with metabolic zonation. We investigated the effect of low oxygen on SR-BI, CD81, claudin-1 and occludin expression. Incubating hepatocytes under physiological oxygen tension led to a significant increase in SR-BI expression at the protein and mRNA levels. Pre-treating hepatocytes with the hypoxia signalling pathway inhibitor NSC ablated the low oxygen-induced increase in SR-BI mRNA, demonstrating a role for HIF signalling to regulate SR-BI. Importantly, we demonstrated that hypoxia promotes HCV entry and replication.

We additionally generated and characterized a panel of monoclonal antibodies that recognised hepatocyte expressed CD81. The antibodies were classified in two epitope groups targeting opposing sides of EC2. We observed a wide range of anti-HCV potencies that were independent of their epitope grouping, but associated with their relative affinity for cell-surface expressed CD81.

4) Development of humanised mouse model systems to study mono- or co-infections

Infections by viruses such as Human immunodeficiency virus (HIV), Hepatitis B (HBV) and Hepatitis C (HCV) cause global health problems with over 380 million people infected worldwide. Co-infection with multiple viruses induces more severe diseases with rapid progressions and increased mortality rates. To understand the cause for these illnesses, it is essential to model them in living systems, however these viruses only infect humans and chimpanzees. To avoid using non-human primates, we have developed a model system in which human cells are stably grafted to mice, rendering them susceptible to infections with HIV, HCV, or HBV. Our results showed that these humanized mice can be mono-infected with HIV or HBV, as well as simultaneously infected with HIV and HBV, constituting a first model system to study co-infections in vivo. Mono-infections with each virus caused similar phenotypes to those observed in patients: HIV resulted in depletion of T lymphocytes, HBV resulted in chronic hepatitis. The HIV/HBV co-infected mice maintained HIV, while clearing HBV, resulting in a similar phenotype to the HIV mono-infected model.

5) Human responses to pathogen infections (HIV-1/HCV)

CD8+ T-cell responses in HIV-1/HCV co-infection:
Key elements of the human immune system to successfully eradicate viral infections are cytotoxic T-cells (CD8+ T-cells), which either trigger an antiviral response in infected cells or force them to die. In case of patients infected with Hepatitis C Virus (HCV) or Human Immunodeficiency Virus (HIV) T-cells often fail to clear these viruses for different reasons. Thus these patients develop a chronic infection which leads over time to fatal comorbidities. To understand why T-cells fail to clear the virus and how these T-cells can be manipulated to eventually eradicate these viruses T-cells of chronically infected patients need to be analyzed. One key feature of T-cells in e.g. chronic HCV infected patients is their state of functional impairment including diminished antiviral efficacy, in general called T-cell exhaustion. The mechanisms underlying T-cell exhaustion are as well still not fully understood. Patients chronically infected with HCV are paradigms to study T-cell exhaustion of CD8+ T-cells whereas HIV infection leads to reduced numbers of T-helper cells (CD4+ T cells). Patients infected with both viruses thus enable the analysis of the role of CD4+ T-cell help in the context of CD8+ T cell exhaustion.

Within PathCo the expression of differentiation markers (CD45RA, CCR7, CD27, CD28), inhibitory receptors (PD-1, CD160, KLRG1, 2B4) and IL7Ra-chain (CD127) were determined to characterize virus-specific T-cells and analyze their functional status. The determined expression patterns implicate that a coinfection with HIV might impact HCV-specific CD8+ T-cell differentiation which is probably due to inhibited antiviral T-cell activation in HIV/HCV coinfection. Antigen availability is an additional factor that influences T-cell differentiation. However, the effect of a cessation of chronic antigen stimulation on CD8+ T-cell differentiation and function was hitherto unknown. Currently available antiviral therapies lead to a great reduction in viral load as well as antigen availability. Thus T-cells from patients infected with HCV and HIV and treated with respective antivirals were studied. Indeed there is a strict dependency between antigen availability and differentiation of virus-specific CD8+ T-cells which influences their functional capacity.

CD4+ T-cell responses in HIV/HCV co-infection:
We studied a unique population based outbreak of HIV-1/HCV co-infection that occurred in a rural community in central China following implementation of a paid plasma donation scheme within a narrow period between 1993 and 1995. All subjects in our cohort (SM cohort) were infected from a narrow genetic source of HIV-1 and HCV strains circulating over a short period of time due to contaminated pooled blood from donors. The HIV-1-infected subjects were classified as HIV-1 slow progressors not requiring HAART or having received HAART for a short duration; meanwhile, HCV-infected subjects were not treated with interferon or direct acting antiviral agents. Thus, this cohort provides a unique setting to study the natural history of concurrent HIV-HCV co-infection, to assess the impact on viral specific immune responses, disease progression, and the evolution of both viruses.

First, we investigated the impact of HIV-1 on HCV immune Responses and its association with liver Disease Progression in this cohort. We found that HIV-1 accelerated liver disease progression and decreased HCV specific T-cell immunity. The magnitude of HCV specific T-cell responses inversely correlated with lower HCV RNA load and reduced liver injury as assessed by non-invasive markers of liver fibrosis. HIV co-infection reduced the frequency of HCV specific CD4+ T cells with no detectable effect on CD8+ T cells or neutralizing antibody levels. We didn’t observe a significant impact of HCV infection on HIV-1 disease progression. Our study highlights the impact of HIV-1/HCV co-infection on HCV specific CD4+ T cell responses and suggests a crucial role for these cells in controlling chronic HCV replication and liver disease progression.

Secondly, we evaluated the impact of the virus source, T cell receptor usage, and antigen sensitivity on HIV-1 virus evolution and immune control – uncover the immune control of HIV-1 mediated via the HLA-B51 in this cohort. We established an in vitro virus evolution assay by co-culturing HLA-B51 cytotoxic T-cell clones specific for the Pol 283-290 TI8 epitope and HIV-1 infected MT2 cells. We found that viral evolution and viral control are heavily dependent on epitope variant sequences of the source virus. Better control of virus with epitope Pol 290-V by T cells is associated with slower mutations in the epitope; This slower occurrence of epitope mutations, is presumed due to less strong immune pressure on the infecting virus bearing epitope Pol 290-V mounted by CTLs. This study highlights the importance of the fine balance between CTL potency and the infecting virus, which may contribute to the long-term HIV viral control.

Study of B cell responses in mono- and co-infected individuals:
To identify the effect of HIV-1 infection on HCV specific B cell responses. Sera from the SM cohorts consisting of HCV mono-infection and HCV/HIV-1 co-infected were used to study B cell responses induced. After sequencing and cloning all patients HCV E1E2 genes, we established a set of representative E1E2 yeast-surface display libraries for analysis of serum profiling from the SM cohorts. Through doing so we identified at least three major immune-reactive domains in the region of Domain 1 (E1 315-341), Domain 2 (E2 407-445), and Domain 3 (E2 602-650). We did not find any clear evidence that HIV co-infection could affect serum antibody profiling against HCV glycoproteins. However, we did identify an observable difference in terms of antibody profiling among HCV genotypes. Genotype 1b infected patients’ targeted epitopes mainly on Domain 1 whereas the genotype 2a patients’ sera were reactive mainly with Domain 2. More interestingly, sera from HCV 2a/1b co-infected patients interacted with Domain 3. From in-depth mapping by flow cytometry, utilising domain 3-expressing yeast clones, we found that this region was predominantly bound by all HCV genotype sera, in which sera from HCV genotype 2a/1b co-infected patients showed the highest binding activity. However, genotype 1b patients’ sera showed weak binding to domain 1-expressing yeasts, and genotype 2a patients’ sera showed only moderate binding to domain 2-expressing yeasts. Overall, domain 3 showed the highest antigenicity in HCV chronic infection, domain 2 is only recognized by HCV 2a-infected serum, and domain 1 is mainly recognized by HCV 2a/1b co-infected serum.

Study of host genotypes associating with HCV transmission:
We identified a number of SNP genotypes that associate with susceptibility to HCV infection, in both HIV-1 positive and negative individuals. This included SNPS within the promoter region of the C-type lectin DC-SIGN gene and which correlated with mucosal but not intravenous transmission of HCV. In addition, a SNP within the LDL receptor gene associated with risk of infection through both transmission routes. These results led us to conclude that performing a powered GWAS screen of HCV at risk but uninfected compared to infected individuals would be required to confirm both these findings as well as identify further genetic associations with risk of HCV transmission.

Potential Impact:
There a number of major implications stemming from the work undertaken in PathCo and the results generated. These relate to the 5 main areas studied 1) TB interactions with HIV-1 2) HCV interactions with HIV-1 3) HCV interactions with malaria 4) development of humanised mouse model systems to study mono- or co-infections and 5) the analysis of human responses to pathogen infections associated with disease and progression, mainly HIV-1 and HCV. The highlights relating to each area are listed and summarised below:

1) TB interactions with HIV-1

Sequencing data and evolutionary analysis provides an insight into a mechanism by which HIV influences the evolution of Mtb. Biochemical and preliminary immunological assays indicate that co-infection Mtb likely accelerates HIV-1 replication, and importantly this effect may differ depending on the Mtb strain an individual is infected with.

We have demonstrated that Mtb glycolipids expressed in liposomes differentially influence HIV-1 trans-infection via DC-SIGN: depending of the Mtb strain, and the activation of DCs. Further characterisation of Mtb glycolipid-liposomes will be performed to identify which lipids have more impact in HIV-1 infection. These results reveal that Mtb glycolipids can modulate HIV-1 infection through either modulating DC immune activation as well as HIV-1 capture and transfer to susceptible cells. These results may indicate how some pathogenic and non-pathogenic strains of Mtb can differentially affect pro-inflammatory responses and thereby modulate HIV-1 infection and/or replication.

Work on the impact of vitamin D on the immune response to TB and HIV suggests that HIV-1 and Mtb replication can be reduced by supplementation during a deficient state. Importantly experiments using different macrophage types, indicates that the inflammatory status of an individual, which can be modified by vitamin D supplementation and underlying infection, has a direct impact on the initial control of Mtb and HIV-1 and the potential outcome of infection.

Finally, immunohistochemistry was applied to unique clinical samples in the form of lymph nodes from HIV/TB co-infected individuals. This work was essential to confirm reports in the literature that are often gained from animal models, hold for humans. Moreover, a systematic review of the literature suggests that well conducted studies on this topic are limited, and that a more systematic characterisation is required. Overall, this work has generated new and important knowledge around immunological and microbiological processes surrounding HIV/TB co-infection.

2) HCV interactions with HIV-1

Viral envelope trans-complementation was observed when HIV-1 gp120 and HCV E1E2 Env glycoproteins were expressed in the same cell, indicating that viral particles can be produced which have the capacity to expand the HIV-1 reservoir and potential alter the viral phenotype. Furthermore, the HCV E1E2 glycoprotein molecule can down-modulate expression from the HIV-1 LTR, resulting in reduced virus gene expression and virus production. Both thee mechanism have major implications for co-infection and how infectivity with one virus can modulate infection with the other. Understanding these mechanisms better and the physiological significance could lead to the identification of new molecules or pathways than can be targeted in future strategies aimed at controlling either infection and with significance for better understanding pathogenesis and disease progression in dual-infected individuals. The same can be said when considering the effects of hypoxia on mono- and co-pathogen infections.

One of the main contributions has been the demonstration for the first time that sexual transmission of HCV occurs through infection of the colorectal mucosa and affects the mucosal environment. This was demonstrated with our mucosal tissue explant model. The modulation of mucosal responses in the presence of another virus such as HIV-1 highlights the importance of mucosal baseline inflammation. Furthermore, co-transmission has an impact on viral replication fitness in the mucosal compartment. PathCo has shown the importance of developing new mucosal tools to assess the early events following viral mucosal exposure prior to establishment of infection in the periphery of the site of transmission, and the impact of co-infections on viral replication fitness.

3) HCV interactions with malaria

Work performed within PathCo provide a framework to test whether common interventions could target both pathogens, and shed new light on the mechanisms of malaria liver infection, with potential implications for the design of new vaccine strategies. Our work validates anti-CD81 antibodies and anti-SRB1 antibodies as inhibitors of P. falciparum and P. vivax sporozoites, respectively. The finding that hypoxia regulates SRBI expression and HCV infection has potential implications for better understanding P. vivax malaria infection. The identification of a Plasmodium protein functionally linked with host receptor usage opens exciting perspectives to explore receptor-ligand interactions, with major implications for the design of new vaccines targeting the initial stage of liver invasion. Finally, new CD81 mAbs generated in the context of PathCo will open novel perspectives to investigate CD81 function, including during HCV and Plasmodium entry, and have the potential to provide insights into tetraspanin biology in general.

4) Development of humanised mouse model systems to study mono- or co-infections

Our results showed that humanized mice can be infected with HIV and HBV (separately or in combination), to model different phases of the disease. Our future work will focus on investigating the cross-talk between the host and the virus to unravel the pathophysiology of the disease and eventually to test new therapeutic treatments or vaccine strategies.

5) Human responses to pathogen infections (HIV-1/HCV)

During the course of this project potent novel direct acting antivirals (DAAs) for an effective cure of HCV emerged. More than 90% of DAA-treated patients sustainably clear the virus. Data gained in this project shows that less differentiated HCV-specific CD8+ T-cells are maintained even for a long duration after HCV elimination and have the capacity to re-expand upon re-infection. In the case of HCV/HIV co-infected patients HCV infection can be cured as well but there is still no cure for HIV infection. Currently, the available antiretroviral therapy (ART) suppresses viral replication and disease progression but without sustainably eliminating the virus. Lessons learned in this project confirm the importance of virus-specific CD8+ T-cells for viral clearance and highlight virus-specific CD8+ T-cells as promising targets for the rational design of immunotherapeutic strategies in chronic HIV-1 as well as other human viral infections.

The key obstacles for current HIV and HCV vaccine development are the enormous diversity of HIV/HCV strains in the population and the fact that the virus is continually adapting to escape immune responses. We revealed that despite their diminished frequency and function by HIV-1 infection, in-vitro expanded HCV-specific CD4+ T cells controlled virus replication as efficiently as HCV specific CD8+ T cells in both mono and co-infected individuals. These results have important implications for immunotherapy as they suggest that CD4+ T cells can rapidly regain their antiviral activity after in-vitro expansion without further specific interventions, such as blockade of specific inhibitory receptors, e.g.PD-1 supporting adoptive T cell therapy. Additionally, using an in vitro virus evolution assay we demonstrated that identical CTL clones selected different patterns of epitope mutations in infecting virus bearing different epitope variants, which implies the importance of considering the nature of the circulating virus for therapeutic and preventative strategies in both HIV and HCV infection.

The identification of a number of novel genetic SNPs associating with the risk of HCV transmission, three in the promoter region of the DC-SIGN gene and one in the LDL receptor gene indicate a better understanding of the HCV transmission process but more importantly indicate new and novel targets when considering intervention strategies aimed at preventing HCV transmission.

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