Take home messages on the importance of the genital mucosa in transmission and pathogenesis

Beginning with a plenary presentation on day one of the Australasian HIV&AIDS conference the complexity and importance of understanding the relationship between STIs and HIV was laid bare.

Associate professor Jo-Ann Passmore, from the University of Cape Town gave a fantastic presentation with a few key messages. She was presenting data on the vaginal mucosa of South African women prior to acquisition of HIV infection and the role of STIs:

• A healthy female genital mucosa is already an effective barrier to pathogens.

• Inflammation in the genitals exacerbates the likelihood of HIV establishing an infection as the cytokines draw in the very cells HIV needs to infect.

• The inflammation seen in the genitals is different compared to inflammation markers in the plasma.

• STIs are a driver of inflammation.

• There is heterogeneity between the various STI and inflammation (cytokine profiles) they induce.

•  Chlamydia, the most prevalent STI is very inflammatory.

• Using the inflammation markers, the researchers could identify 70% of HIV seroconverters.

Delving further into the proteins and associated inflammatory markers, Lyle Mckinnon presented data from the CAPRISA004 trial (Centre for the AIDS Programme of Research in South Africa), in Durban, South Africa which aimed to elucidate the mechanisms behind genital inflammation increasing HIV acquisition risk. They looked at the soluble mucosal proteome and associated cytokine expression in female Kenyan sex workers. Out of around 500 proteins measured, they found 3 that are involved in cell migration and 7 that decreased mucosal barrier function. These 10 proteins were the defining feature of women with elevated inflammatory cytokines and allowed them to predict which women had the highest inflammation.

Rather than being a helpful way to repair tissue and fight pathogens genital inflammation in this situation may be reducing the effective barrier of a healthy genital mucosa and increasing the risk of acquiring HIV. Genital inflammation may help to explain high rates of HIV transmission when the per –act transmission risk (1/1250 for vaginal intercourse) is low.

Next, Keith Fowke from the University of Manitoba gave a presentation on an innovative approach to reduce risk of HIV transmission – using low dose anti-inflammatory drugs.  

Keith set the scene by explaining that younger women have the highest risk of acquiring HIV based on their inflammation patterns and this may peak shortly after sexual debut or in the earlier years of sexual activity. Likewise, STIs are known to also be more inflammatory in younger people made worse by younger people having the highest rates!

In reference to female sex workers specifically; inflammation levels seem to decrease over time and markers of inflammation can increase from as little as a one month break from sex work. This suggests a model of mucosal tolerance over time with a peak risk of acquisition in between the stages of transactional sex and first identifying as a sex worker, arguably when these women are most at risk of acquiring HIV based on non-biological factors.

Taking this forward and with the acknowledgment that activated CD4+ T cells (caused by inflammation) are 1000x more susceptible to HIV infection than non activated cells it seems pertinent to consider modulating the inflammatory response as a prevention strategy.

The researchers identified 80 low risk women from Kenya and established baseline activation levels, then randomized them to take 6 weeks oral dose of either Aspirin (aka acetylsalicylic acid or ASA) or hydroxychloroquine (HCQ) which is used in the treatment of rheumatoid arthritis. They found a significant reduction in the percentage of CD4+ CCR5+ T cells in both PBMCS and cervical mononuclear cells (CMCs) for those taking aspirin and saw a similar reduction in CD4+ CCR5+ T cells in CMCs for those taking HCQ.

Besides the take home message that it works…..and aspirin in particular is already taken by many people around the world, the participants reported that taking the drugs were non-stigmatising, something which could have positive effects for adherence which in turn increases the efficacy of the prevention strategy.

Moving from one side of the perineum to the other, Andrew Harman from the Westmead Millennium Institute gave an informative and visually intriguing (pictures below) presentation on interventions at the mucosa or research working towards that end.

He outlined the research by first explaining about the large amount of heterogeneity between and within the immune cell subsets of the genital and anal tissues (dendritic cells in particular). Dendritic cells are of interest because of the role they play in transferring HIV from the epithelium to CD4 T cells.

Shockingly and relevant to the Australian epidemic concentrated amongst MSM, very little is known about the role  dendritic cells play in transferring HIV to CD4 T cells in the tissues of the colon and anus. This is in comparison to the female genital tract where the role of dendritic cells is much more understood.

The main reasons for this are that healthy human tissue is difficult to obtain, (especially anogenital tissues!) dendritic cells are hard to isolate and dissociate and even if they can be isolated, they are in small numbers.

So, over many years Andrew has had to establish collaborations with plastic surgeons, colorectal surgeons, urologists and gynaecologists to gain access to the full range of anogenital tissues – see image below for process of preparing human tissue. This has led to profiling of the various immune cell subsets in anogential tissues and in particular a novel population of rectal dendritic cells that can be identified by the combination of cell surface receptors proteins. If this can be characterised further it could lead to the translational goal of producing compounds that block these cell surface receptors, therefore preventing these cells playing a role in transferring HIV to CD4 T cells. Ideally a compound of this nature could be applied as a tissue type specific microbicide in the future – watch this space.

This insightful plenary session at the 9th Australasian Viral Hepatitis Conference began with Dr Dieter Glebe from Germany presenting on the origins of hepatitis B virus, a topic that rarely crops up in the clinic but is nonetheless fascinating.

Titled “Of bats and men: species specificity of hepadnaviral infection” Dr Glebe began by explaining the existing phylogenetic understanding of the virus in mammals which can be isolated in many species of non-human primates and also new world rodents such as woodchucks and ground squirrels. However, as the strains in the new world rodents cannot infect non-human primates and vice versa, Dr Glebe looked to bats, the mammalian viral mixing pot to investigate how the virus might have moved between rodents and non-human primates. He screened 3,080 bats from 54 species around the world, finding 10 animals PCR positive and a detection rate of 6-10% of hepadnaviral DNA in individual species. Most of the viruses were isolated from the liver.

The research then focused on assessing the zoonotic potential of these bat viruses. One strain isolated from a Panamanian tent making bat seemed to be the closest match to human virus and successfully replicated in human liver cells in vitro. Fortunately, the nucleoside analog entecavir was able to inhibit replication as with the human virus. However, and slightly worryingly, antisera from hepatitis B vaccinated humans was not protective against infection with the bat virus. Dr Glebe wrapped up the presentation with a charming picture of a non-human primate happily eating away at a bat, reminding us that nature often finds a way help species “mix”.

The second and final presentation of the session was titled “HBV Reactivation: Playing with fire” presented by Professor Maggie Bassendine from Newcastle, UK. Professor Bassendine began by reminding the audience that “resolved infection” is not immune clearance but immune control (as HBV cccDNA can still be found in some hepatocytes) and hence that control can be lost under certain circumstances. This reactivation can occur after receiving immunosuppressive therapy, such as chemotherapy or drugs associated with transplantation. Unfortunately it is not possible to predict whether reactivation will occur and how severe it will be, but some agents do seem to make it more likely. For instance, when chemotherapy was given with steroids, reactivation was more common than chemotherapy given without steroids. One of the newer agents used in cancer therapy, the monoclonal antibody Rituximab seems to be particularly associated with HBV reactivation with one paper recording it occurring in 1/20 patients with resolved HBV infection.

Professor Bassendine then discussed the role of screening for resolved HBV infection before giving patients immunosuppressive therapy noting the lack of consistency between HBV clinical guidelines which recommend screening, and oncology guidelines which don’t explicitly recommend screening, but leave it at the clinician’s digression. This is concerning as 20% of oncologists have been recorded never screening patients. Professor Bassendine then concluded the presentation by a making call for universal screening to be included across all national and local guidelines and suggesting the clinicians in the audience start by investigating the current screening practices in their own local hospitals.

HIV monitoring focuses on plasma RNA viral loads and CD4 T- cell counts from the blood, but this only represents a part of the total infection in other parts of the body such as the gut and genital tract. A better understanding of mucosal immunity at these sites has implications for both prevention (microbicides) and treatment research (immune reconstitution outside of PBMCs).

Laura Romas from the University of Manitoba, Canada presented data on the rectal mucosa from HIV-exposed seronegative (HESN) partners in discordant MSM couples and susceptibility to HIV-1 infection, the first time this has been investigated. She found 25 differentially expressed proteins (of 289 identified) that were overabundant in HESN MSM (p< 0.05), and had functions related to acute phase response, antimicrobial defence and Ig-mediated immunity. These proteins did not show a significant correlation (p>0.05) with clinical variables (frequency oral/anal sex, HIV-neutralizing IgA, and viral load of HIV+ partner). One in particular, Antiprotease 1 (AP1) reduced HIV infection by 50% in PBMC culture at physiologically relevant concentrations. Planned further research will aim to elucidate if increased AP1 expression is as a result of HIV-exposure or genetic variability. This could have very interesting implications for MSM prevention strategies and microbicide development.

The GI tract is know as an early site of viral replication and associated immunopathogenesis. Whilst ART stimulates CD4 T – cell recovery in blood, its effects on the GI tract are much less pronounced. However, as earlier initiation of ART seems to preserve more peripheral CD4 T – cells, Claire Deleage from the USA investigated the effect of early ART initiation on the GI tract (lamina propria CD4 T-cells) with a small cohort of Thai patients treated during Fiebig stage 1/2 or 3+ versus chronic controls. Unfortunately, there was no significant recovery of CD4 T-cells in the lamina propria after treatment initiation. After 6 months of HAART all acutely infected treated patients had significant depleted lamina propria CD4 T-cells that persisted through 24 months of treatment. Markers of GI damage, inflammation and immune activation returned to baseline levels after 6 months of ART following an increase during acute HIV-1 infection (day 0). Despite the positive effect of early ART initiation on GI damage markers, the stark lack of CD4 T-cell recovery is disappointing. Fully understanding the complexities of HIV infection in GI tract remains a great challenge, but it is essential if strategies to promote CD4 T-cell recovery in gut are ever to materialise.

“To know your Enemy, you must become your Enemy.” The quote by Sun Tzu perfectly captures the theme of the session titled Defining and Targeting Residual Virus on cART.

And so Dr Keele presented an innovative molecular tag of SIV to help better understand the location, size and variability of persistent SIV infection in non-human primates. The “barcode” approach presented allows multiple viral variants to establish infection and then be identified over the course of disease progression. This will hopefully lead to the researchers tracking down the actively replicating viruses that are responsible for maintaining the reservoir and studying how this morphs once ART is introduced and subsequently ceased. Although non-human primate models seem far removed from the clinic, these models are essential for a better understanding of how and where HIV is hiding in the body.

Much closer to home comes the follow-up of a unique patient known as C135 who was part of the Sydney Blood Bank Cohort (SBBC) and infected with HIV 33 years ago. Infected with an attenuated ∆-nef strain of virus, C135 presented as an elite controller with 3 genetic polymorphisms associated with viral control, including protective HLA class I and HLA class II alleles, and CCR5∆32 heterozygosity – a truly distinct combination when accounting for the attenuated virus. Samples taken 15 years after blood transfusion indicated established HIV-1 infection, but in samples from 1997 onwards infectious HIV-1 has never been recovered from PBMC cultures and no HIV-1 DNA found in CD4+ T cells from either PBMC or gut biopsies. The patient has remained off ART since infection and presents a strong case for either viral clearance or at least sustained virological remission. Unfortunately, the current difficulty of sampling all possible reservoir sites in the body such as the CNS makes confirming viral clearance particularly challenging.

The session concluded with a late breaker from Dr Søgaard from Denmark, who presented the results of a clinical trial to assess the effects of romidepsin (an HDAC inhibitor) on the latent reservoir in 6 HIV-infected patients. The phase I/II clinical trial,  gave six aviremic HIV-infected adults intravenous romidepsin (5 mg/m²) once weekly for 3 weeks while maintaining cART. 36 adverse events were reported although the majority were mild and resolved spontaneously. Viral load increased from undetectable at baseline to readily quantifiable levels at multiple post-infusion timepoints in 5 of 6 patients (range 46-103 copies/mL after 2^nd infusion, p=0.007) – the first time this has been seen with an HDAC inhibitor. Furthermore, the emergence of quantifiable plasma HIV-1-RNA corresponded directly with the cyclic romidepsin infusions. However, the HIV-1 RNA levels returned to baseline a few days post infusion and no significant change was detected in total viral DNA across the 6 patients suggesting a transient effect of the drug and no permanent decrease in the size of the viral reservoir. Further trials with larger numbers of patients and/or in conjunction with other therapeutic interventions are eagerly awaited.

The session gave an overview of viral reservoirs, factors affecting latency and the complexity of latency between and within tissue types.

Dr Persaud from the United States began the session with the unfortunate news and analysis of the re-emergence of HIV infection in the “Mississippi child”. The researchers detected HIV-1 plasma RNA 27 months after earlier assays showed no virus present. Phylogenetic analysis of the virus confirmed it to be a 98% similar to the virus isolated from the mother, strongly suggesting reemergence from “viral remission” rather than the child becoming newly infected via another source.

Dr Van Lint from Belgium then gave a brisk yet detailed overview of viral latency mechanisms. She referenced studies which showed HIV-1 preferentially integrates into transcriptionally active regions of the genome and areas associated with clonal expansion when infecting T-cells.

Dr Van Lint also gave an overview of existing “anti-latency” agents such as vorinostat (SAHA), and newer agents such as Romidepsin, which may be more potent – very much needed if the approach is to be effective in virally suppressed patients on ART.

The presentation was finished with a description of the many cell-associated and viral factors involved in latency and thus the multitude of potential targets for anti-latency therapeutics. However, as research emerges on the complexities of HIV viral latency, it also highlights the heterogeneity of the mechanisms involved in latency within different tissue and cell types and even between individual cells. Any future therapies will need to accommodate this if they are to have any lasting benefit.

Dr Verdin from the United States reiterated the complex nature of viral latency and then described an innovative system using Green Florescent Protein (GFP) and other reporter genes to visualise a profile of genes associated with viral latency (both promoting and suppressing).

This produced a milieu of genes and related protein complexes involved, some known previously, others completely new to the viral latency field. An unexpected outcome of the increased understanding of the breadth of cell-associated factors involved in latency was the closing suggestion of considering therapeutic agents to increase or promote viral latency rather than suppress it (unlike the current experimental agents being trialled which aim to reduce viral latency). By targeting the integrated proviral DNA and “locking it in place”, away from transcription and subsequent virus production, the outcome could be similar to ART, but perhaps more potent?

Dr Chomont from the United States followed on by describing the heterogeneity within latently infected CD4+ T cells and the proportion of subsets infected within different patient groups such as post-treatment controllers and long-term non-progressors.

Both have higher proportions of latently infected cells with a more differentiated phenotype (which have a shorter turn over period within the body). He then described the role of early treatment on latently infected CD4+ T cells with mixed results.

For example Buzon et al. 2014 showed that early ART initiation did not affect the proportion of the more naive (and longer lasting) cells being infected.

However, research discussed by Dr Jintanat Ananworanich earlier in the day suggested that very early initiation of ART (2/3 weeks after infection) was associated with a reduction in the size of the reservoir in all CD4+ T-cell subsets, including the longer lasting central memory cells.

Dr Chomont also remarked that this benefit could remain even if treatment is delayed until 4-8 weeks after infection - still very early initiation within a real world setting however.

The session was concluded by Dr Melissa Churchill, from the Burnet Institute, Melbourne who presented on the role of tissue reservoirs and in particular the CNS. She described the role of the CNS as a probable viral reservoir citing multiple indirect studies, but also acknowledging the lack of conclusive and direct evidence.

Latency in the CNS is likely and, just as was reported in earlier presentations, so is the heterogeneity in latency between the different cell types susceptible to HIV infection in the CNS; astrocytes, macrophages, and microglial cells.

Dr Churchill concluded by discussing the potential role of current curative strategies (such as using histone deacetylase [HDAC] inhibitors) on the CNS with some caution – cell death within the CNS even if targeted specifically to latently infected cells would likely have negative consequences for the tissue and therefore the patient.