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For the past 20 years our group has been based in the Peter Medawar Building for Pathogen Research at the University of Oxford, where our primary research focus has been the South African HIV epidemic. To this end we are involved in multiple studies in KwaZulu-Natal province, South Africa, the area of highest HIV prevalence in the country, along with Kimberly in the Northern Cape. Much of our previous work has looked to define the role of cytotoxic T-lymphocyte (CTL) immunity in long-term immune control of HIV infection in both adults and children, and the mechanisms by which protective HLA alleles such as HLA-B57 and HLA-B57 mediate elite control and non-progression in presenting immunodominant gag epitopes.
In the course of this work we have been able to learn about the impact of HIV disease outcome on immune differences between adults and children by particular focus on HIV-infected children and mother-to-child transmission pairs. For example, although HIV-infected children typically progress more rapidly to AIDS than adults without antiretroviral therapy (ART), a subset of ART-naïve HIV-infected children remain healthy and maintain normal-for-age CD4 counts through immune mechanisms that we identified are similar to the natural, non-pathogenic hosts of SIV.
Although this is rare, there is evidence that HIV-infected children who receive ART early in life have greater potential to achieve functional cure and our current work therefore focusses on achieving paediatric HIV cure. Since 2015 we have been engaged in the Ucwaningo Lwabantwana study, meaning ‘learning from children’ in Zulu, in KwaZulu-Natal in South Africa (see below). Colloquially known as the ‘Babycure’ study since this is the end goal. An unexpected observation during the course of the study has been that females are twice as likely to be infected from their mother in-utero than males. Indeed, the female risk rises to three-fold greater than males if the mother becomes infected herself during pregnancy.
The central focus of the group is now to define the mechanisms that underpin these immune sex differences that develop before birth, their impact on HIV cure potential, and the extent to which they are shared with adults. Further details of our areas of investigation are below.
Ucwaningo Lwabantwana: ‘Babycure’
Case reports of the ‘Mississippi Baby’, the ‘VISCONTI child, and now the ‘South African child’ provide hints that early ART in children can achieve sustained HIV suppression, or ‘cure’. The early-life immune system is tolerogenic and immune activation is tightly regulated which may limit the ability of HIV to establish a large viral reservoir in children compared to adults. Furthermore, ART taken by the mother during pregnancy further suppresses viral replication in-utero, and initiating ART shortly after birth provides these children with a uniquely low viral reservoir. The Babycure study headed by Jane Millar investigated cure potential in HIV-infected newborns initiated on ART in the first 48hrs of life in KwaZulu-Natal, South Africa. To date we are following over 200 in-utero infected infants and their mothers. This allows us to characterise in detail: the early life innate and adaptive immune responses in these children and their mothers, the virus transmitted from mother-to-child, and the impact of non-structured ART-interruptions. There is a strikingly high frequency of ART non-adherence amongst these in-utero infected children for reasons that we and others are investigating (Miller et al., PIDJ 2019; Miller et al., EClinMed 2020).
In the Babycure we have shown that female foetuses have approximately twice the risk of in-utero HIV infection compared to males, and this is due to increased female susceptibility in-utero rather than increased male mortality. Indeed, we have enrolled three cases of sex-discordant twins where only the female twin has been infected.
bnAb trials to achieve cure in paediatric HIV infection
As identified in the Babycure study, the majority of HIV-infected children will need interventions in addition to the early initiation of ART. We are currently seeking funds to trial combination broadly-neutralising antibodies (bnAb’s) as means of achieving functional cure in the early-ART-treated children enrolled onto the Babycure study, a promising approach that has been successful in SHIV-infected macaques.
Natural Killer cells: B35/53Bw4TTC2 & bnAbs
Studies in early-ART-treated infants and adults and in SIV-infected non-human primates suggest that particular NK responses may significantly reduce the viral reservoir, improving cure potential. A specific immunogenetic signature, ‘B35/53Bw4TTC2’, identified in the adult VISCONTI cohort of post-treatment controllers is hypothesised to carry a 50% likelihood of cure. We also hypothesise that in a South African population, where CMV infection Is endemic in infancy, a memory-like NK response may contribute to a more effective NK response against HIV. Our work, led by Vinicius Vieira, looks to define whether this B35/53Bw4TTC2 signature, and the associated NK responses, are present in early-ART-treated children and mothers from the Babycure cohort and whether they are associated with low viral reservoirs and high cure potential.
In our studies of HIV-uninfected children we see expansion of both CD56hi and CD16hi NK subsets in children aged 5-14yrs compared to younger children and adults. The CD56hi subset is especially depleted and dysfunctional in ART-naïve HIV-infected children (Singh et al., 2020). In view of the potential for NK cells to reduce the size of the HIV reservoir via bnAb-mediated ADCC we are studying the ontogeny of NK cell function to define the optimal timing for paediatric bnAb administration to maximise the chance of achieving cure in HIV-infected children.
Type 1 Interferons and the HIV transmission bottleneck
The disparity between the male and female immune response is clear in HIV infection: females typically achieve viral loads 0.5 log10 lower than males and are 5-fold more likely to achieve elite control. However, females are more likely to lose viral control once achieved and progress to AIDS faster than males for matched viral loads. The more aggressive female immune response seen in HIV, also seen of other infections and vaccine responses, may be explained by the higher production of type 1 interferon (IFN-I) by female immune cells. The upstream receptor responsible for recognising virus infection and this initiating IFN-I production, TLR7, is encoded on the X-chromosome and escapes X-chromosome inactivation in a proportion of cells to be doubly expressed on >40% of immune cells.
To test the hypothesis that a stronger IFN-I innate immune response might increase the susceptibility of females to in-utero HIV infection, we are characterising the IFN-I-sensitivity of the virus transmitted from mother-to-child in the Babycure study. This work led by Emily Adland (Adland et al., Nat Comms 2020) has shown that IFN-I-resistant viruses tend to be transmitted to female foetuses, while IFN-I-sensitive viruses are preferentially transmitted to male foetuses. The female susceptibility to in-utero HIV infection is maximal when the mother herself becomes infected during pregnancy, prompting the hypothesis that IFN-I-resistant viruses are preferentially transmitted in male-to-female adult HIV transmission, and are then outgrown by IFN-I-sensitive viruses following transmission. We are addressing this with adult transmission pairs from the ZEHRP cohort, working in collaboration with Eric Hunter at Emory University and aim to ascertain both IFN-I-sensitivity and viral replicative capacity of transmitted viruses. Viral replicative capacity is indeed lower in in-utero infected females than males, consistent with the overall notion that female foetuses are more susceptible to intrauterine mother-to-child transmission.
Preventing in-utero transmission: Hofbauer cells
The placenta is a foetal organ constituting the interface between the foetus and maternal tissue. It is locked in a constant battle between immune tolerance of the allogenic foetus and immunological functionality to protect it from in-utero infection. Placental macrophages, or Hofbauer cells (HBC), are present from 18 days gestation and are the only detectable placental immune cell for the majority of gestation. They sit on the foetal side of the single-cellular syncytiotrophoblast layer separating maternal and foetal blood and putatively play an integral role in setting the immune environment of the placenta. HBC express CD4, CCR5, CXCR4, and other HIV-implicated molecules like DC-SIGN and Siglec-1, suggesting that they might facilitate in-utero HIV infection. Through collaborations with Manu Vatish at Oxford we are characterising HBC from both HIV-unexposed and HIV-exposed sex-discordant twins. The project led by Oliver Sampson also includes collaborations with William James at Oxford to use human-induced pluripotent stem cells as a model of tissue-resident macrophages to investigate the role of sex-hormones in modulating HBC functionality and to utilise CRISPR/Cas9 to assess the involvement of sex-related genes. Better understanding of the route of in-utero HIV transmission will inform strategies to protect babies from mother-to-child transmission during pregnancy, especially in settings like South Africa where sustained ART adherence is problematic.
Probing immune sex differences through transcriptomics and epigenetics
With sex differences widely documented to play a significant role in the immune response, we are looking to further investigate the poorly understood impact of sex differences on the developing immune system through a close collaboration with Alex Shalek at MIT, being the first group to bring their portable low cost single-cell RNA-seq Seq-well platform to the UK. This work, led by Nicholas Lim, looks to analyse single-cell transcriptomic profiles of cord blood mononuclear cells from HIV-unexposed and HIV-exposed sex-discordant twins to elucidate the contribution of biological sex to the neonatal immune response in the presence of HIV at an unprecedented resolution.
To explore the impact of epigenetics on differential immune gene expression we are collaborating with Veron Ramsuran at the University of KwaZulu-Natal to compare the methylation patterns of immune genes in mononuclear cord blood cells from sex-discordant twins, Previous studies have shown that males have five-fold more methylated autosomal CpG sites than females, the majority of which are acquired in-utero and maintained long-term. We are currently exploring the role of androgens in mediating these striking epigenetic differences. The main sex hormone difference between male and female foetuses is greater androgen synthesis in males, and previous studies suggest that androgens are likely to contribute to the substantial sex differences observed in autosomal DNA methylation in early life.
Viral sequence evolution
Our longitudinal sampling of maternal and child virus in the babycure study has provided full-length and gag-pro sequences to accompany the in-vitro IFN-I-sensitivity and viral replicative capacity data. Through detailed bioinformatic analysis run by Nicholas Grayson we are seeking to define the specific sequence changes that alter IFN-I-resistance and that affect viral replicative capacity. Given that viruses preferentially transmitted to females in utero are IFN-I resistant and have lower replicative capacity than those transmitted to males, we are investigating whether there are viral amino acid polymorphisms that are associated with male or female sex. The viral sequences present in mothers and transmitted in utero also enable us to investigate other questions relating to the mother-to-child transmission bottleneck, such as how many founder viruses are transmitted, and the relationship between the type of viruses transmitted in utero and timing of maternal infection. Monitoring the in-host evolution of child virus in relation to maternal HLA and child HLA also provides insights into the ontogeny of HIV-specific T-cell activity in early life, and when CTL-mediated immune pressure in the child can drive the selection of escape mutants in the child (Leitman et al, J Exp Med, 2017). In close collaboration with the Wellcome Trust Centre for Human Genomics in Oxford we use the latest high throughput bait capture techniques to specifically enrich HIV samples and generate next generation sequence data to allow us to examine viral diversity.
Elite Control and Post-treatment control of HIV in children and adults
To define mechanisms by which functional cure of HIV can be achieved, Vinicius Vieira in the group is building up a cohort of elite controllers (EC), whom maintain <50 HIV RNA c/ml without treatment, as a potential model for post-treatment control. Like adult EC, paediatric EC has a strong female bias, but unlike adult EC who achieve aviraemia within weeks of infection, paediatric EC take 6-7yrs to achieve aviraemia. Other clues to the immune responses required to achieve functional cure in children come from studies of post-treatment controllers in adults. To this end we are studying the mothers in the Babycure study who seroconvert during pregnancy and initiate ART 4-12weeks after infection, similar to the well-characterised VISCONTI cohort of post-treatment controllers.