The immunology ecosystem in South Africa: striking an equitable balance between fostering discovery, promoting translation and capacity building.

In the vast and diverse continent of Africa, the field of immunology holds immense significance as it navigates the complex landscape of infectious diseases and public health challenges. In this article, we speak with Professor Clive Gray, who provides powerful and valuable insights into the unique research opportunities and immunological advancements supported by Africa's unique blend of social, economic and environmental factors and also discusses the societal and cultural challenges that need to be overcome for equitable research to be achieved across the continent.

Stereotypic Expansion of T Regulatory and Th17 Cells during Infancy Is Disrupted by HIV Exposure and Gut Epithelial Damage.

Few studies have investigated immune cell ontogeny throughout the neonatal and early pediatric period, when there is often increased vulnerability to infections. In this study, we evaluated the dynamics of two critical T cell populations, T regulatory (Treg) cells and Th17 cells, over the first 36 wk of human life. First, we observed distinct CD4+ T cells phenotypes between cord blood and peripheral blood, collected within 12 h of birth, showing that cord blood is not a surrogate for newborn blood. Second, both Treg and Th17 cells expanded in a synchronous fashion over 36 wk of life. However, comparing infants exposed to HIV in utero, but remaining uninfected, with HIV-unexposed uninfected control infants, there was a lower frequency of peripheral blood Treg cells at birth, resulting in a delayed expansion, and then declining again at 36 wk. Focusing on birth events, we found that Treg cells coexpressing CCR4 and α4ß7 inversely correlated with plasma concentrations of CCL17 (the ligand for CCR4) and intestinal fatty acid binding protein, IL-7, and CCL20. This was in contrast with Th17 cells, which showed a positive association with these plasma analytes. Thus, despite the stereotypic expansion of both cell subsets over the first few months of life, there was a disruption in the balance of Th17 to Treg cells at birth likely being a result of gut damage and homing of newborn Treg cells from the blood circulation to the gut.

T-SPOT.TB Reactivity in Southern African Children With and Without in Utero Human Immunodeficiency Virus Exposure.

Infants who are human immunodeficiency virus (HIV)-exposed uninfected (iHEU) experience higher risk of infectious morbidity than infants HIV-unexposed uninfected (iHUU). We compared tuberculosis (TB) infection prevalence in 418 Bacillus Calmette-Guérin vaccinated sub-Saharan African iHEU and iHUU aged 9-18 months using T-SPOT.TB. Prevalence of TB infection was low and did not differ by HIV exposure status.

Turning Discoveries into Treatments: Immunology in Africa.

An exemplar outcome of an immunology-based intervention is vaccine development; the current COVID-19 pandemic is a case in point. Can we build an immunology research ecosystem in Africa that nurtures discovery and enables translation? We see African immunologists as key agents of change and discuss obstacles and opportunities.

Maternal T Cells in the Human Placental Villi Support an Allograft Response during Noninfectious Villitis.

During human pregnancy, proinflammatory responses in the placenta can cause severe fetal complications, including growth restriction, preterm birth, and stillbirth. Villitis of unknown etiology (VUE), an inflammatory condition characterized by the infiltration of maternal CD8+ T cells into the placenta, is hypothesized to be secondary to either a tissue rejection response to the haploidentical fetus or from an undiagnosed infection. In this study, we characterized the global TCR ß-chain profile in human T cells isolated from placentae diagnosed with VUE compared with control and infectious villitis-placentae by immunoSEQ. Immunosequencing demonstrated that VUE is driven predominantly by maternal T cell infiltration, which is significantly different from controls and infectious cases; however, these T cell clones show very little overlap between subjects. Mapping TCR clones to common viral epitopes (CMV, EBV, and influenza A) demonstrated that Ag specificity in VUE was equal to controls and significantly lower than CMV-specific clones in infectious villitis. Our data indicate VUE represents an allograft response, not an undetected infection. These observations support the development of screening methods to predict those at risk for VUE and the use of specific immunomodulatory therapies during gestation to improve outcomes in affected fetuses.

T-Cell Homeostatic Imbalance in Placentas From Women With Human Immunodeficiency Virus in the Absence of Vertical Transmission.

BACKGROUND: Implementation of universal antiretroviral therapy (ART) has significantly lowered vertical transmission rates but has also increased numbers of human immunodeficiency virus (HIV)-exposed uninfected children, who remain vulnerable to morbid effects. In the current study, we investigated whether T-cell alterations in the placenta contribute to altered immune status in HIV-exposed uninfected. METHODS: We analyzed T cells from term placenta decidua and villous tissue and paired cord blood from pregnant women living with HIV (PWH) who initiated ART late in pregnancy (n = 21) with pregnant women not living with HIV (PWNH) (n = 9). RESULTS: Placentas from PWH showed inverted CD4/CD8 ratios and higher proportions of tissue resident CD8+ T cells in villous tissue relative to control placentas. CD8+ T cells in the fetal capillaries, which were of fetal origin, were positively correlated with maternal plasma viremia before ART initiation, implying that imbalanced T cells persisted throughout pregnancy. In addition, the expanded memory differentiation of CD8+ T cells was confined to the fetal placental compartment and cord blood but was not observed in the maternal decidua. CONCLUSIONS: T-cell homeostatic imbalance in the blood circulation of PWH is reflected in the placenta. The placenta may be a causal link between HIV-induced maternal immune changes during gestation and altered immunity in newborn infants in the absence of vertical transmission.

Low Immune Activation in Early Pregnancy Is Associated With Preterm But Not Small-for-gestational-age Delivery in Women Infected With Human Immunodeficiency Virus Initiating Antiretroviral Therapy in Pregnancy: A Prematurity Immunology in HIV-infected Mothers and their Infants Study (PIMS) Case-control Study in Cape Town, South Africa.

BACKGROUND: Mechanisms underlying an association between human immunodeficiency virus (HIV) or antiretroviral therapy (ART) during pregnancy with risk of preterm delivery (PTD) and small-for-gestational-age (SGA) remain unclear. We explored the association between cellular immune activation and PTD or SGA in women with HIV initiating ART during or before pregnancy. METHODS: Women with HIV enrolled at median 15 weeks' gestation, were analyzed for immune markers, and matched on ART initiation timing (15 women initiated pre- and 15 during pregnancy). There were 30 PTD (delivery <37 weeks), 30 SGA (weight for age ≤10th percentile) cases, and 30 controls (term, weight for gestational age >25th percentile) as outcomes. Lymphocytes, monocytes, and dendritic cell populations and their activation status or functionality were enumerated by flow cytometry. RESULTS: PTD cases initiating ART in pregnancy showed decreased CD8+ T cell, monocyte, and dendritic cell activation; increased classical (CD14+CD16-) and intermediate (CD14+CD16+) monocyte frequencies; and decreased inflammatory monocytes (CD14dimCD16+) compared with SGA cases and term controls (all P < .05). Allowing for baseline viral load, the immune markers remained significantly associated with PTD but only in women initiating ART in pregnancy. Lower monocyte activation was predictive of PTD. TLR ligand-induced interferon-α and macrophage inflammatory protein-1ß levels in monocytes were significantly lower in PTD women initiating ART in pregnancy. CONCLUSION: Low immune activation, skewing toward anti-inflammatory monocytes, and lower monocyte cytokine production in response to TLR ligand stimulation were associated with PTD but not SGA among women initiating ART in, but not before, pregnancy, suggesting immune anergy to microbial stimulation as a possible underlying mechanism for PTD in women initiating ART in pregnancy.

Delaying BCG vaccination until 8 weeks of age results in robust BCG-specific T-cell responses in HIV-exposed infants.

BACKGROUND: BCG vaccination prevents disseminated tuberculosis in children, but it is contraindicated for persons with human immunodeficiency virus (HIV) infection because it can result in severe disease in this population. In tuberculosis-endemic regions, BCG vaccine is administered soon after birth, before in utero and peripartum HIV infection is excluded. We therefore assessed the immunogenicity of BCG vaccine in HIV-exposed infants who received BCG at birth or at 8 weeks of age. METHODS: HIV-exposed, uninfected infants were randomly assigned to receive BCG vaccination at birth (the early vaccination arm) or 8 weeks of age (the delayed vaccination arm). BCG-specific proliferative and intracellular cytokine responses were assessed in 28 infants per arm at 6, 8, and 14 weeks of life. RESULTS: There was no difference in BCG-specific T-cell proliferation between the study arms 6 weeks after vaccination. However, at 14 weeks of age, the frequency of interferon γ-expressing CD4(+) T cells and multifunctional BCG-specific responses in the delayed vaccinated arm were significantly higher than those in the early vaccination arm (P = .021 and P = .011, respectively). CONCLUSIONS: The immunogenicity of BCG vaccination in HIV-exposed, uninfected infants is not compromised when delayed until 8 weeks of age and results in robust BCG-specific T-cell responses at 14 weeks of age. These findings support further evaluation of this modified BCG vaccination strategy for HIV-exposed infants. CLINICAL TRIALS REGISTRATION: NCT02062580.

Differential impact of magnitude, polyfunctional capacity, and specificity of HIV-specific CD8+ T cell responses on HIV set point.

Defining the characteristics of HIV-specific CD8(+) T cell responses that lead to viral control is crucial for vaccine development. We evaluated the differential impact of magnitude, polyfunctional capacity, and specificity of the CD8(+) response at approximately 6 months postinfection on the viral set point at 12 months in a cohort of HIV-infected individuals. High frequencies of Gag and Nef responses endowed with four functions were the best predictors of a low viral set point.

Rapid disease progression in HIV-1 subtype C-infected South African women.

BACKGROUND: Whereas human immunodeficiency virus (HIV) subtype B-infected individuals generally progress to AIDS within 8-10 years, limited data exist for other clades, especially from Africa. We investigated rates of HIV disease progression of clade C-infected South African women. METHODS: Prospective seroincidence cohorts in KwaZulu-Natal were assessed for acute HIV infection monthly (n = 245) or every 3 months (n = 594) for up to 4 years. Rapid disease progression was defined as CD4 decline to <350 cells/µL by 2 years postinfection. Serial clinical and laboratory assessments were compared using survival analysis and logistic regression models. RESULTS: Sixty-two women were identified at a median of 42 days postinfection (interquartile range, 34-59), contributing 282 person-years of follow-up. Mean CD4 count dropped by 39.6% at 3 months and 46.7% at 6 months postinfection in women with preinfection measurements. CD4 decline to <350 cells/µL occurred in 31%, 44%, and 55% of women at 1, 2, and 3 years postinfection, respectively, and to <500 cells/µL in 69%, 79%, and 81% at equivalent timepoints. Predictors of rapid progression were CD4 count at 3 months postinfection (hazard ratio [HR], 2.07; 95% confidence interval [CI], 1.31-3.28; P = .002), setpoint viral load (HR, 3.82; 95% CI, 1.51-9.67; P = .005), and hepatitis B coinfection (HR, 4.54; 95% CI, 1.31-15.69; P = .017). Conversely, presence of any of HLAB*1302, B*27, B*57, B*5801, or B*8101 alleles predicted non-rapid progression (HR, 0.19; 95% CI, .05-.74; P = .016). CONCLUSIONS: Nearly half of subtype C-infected women progressed to a CD4 count <350 cells/µL within 2 years of infection. Implementing 2013 World Health Organization treatment guidelines (CD4 count <500 cells/µL) would require most individuals to start antiretroviral therapy within 1 year of HIV infection.

Increased memory differentiation is associated with decreased polyfunctionality for HIV but not for cytomegalovirus-specific CD8+ T cells.

The generation of polyfunctional CD8(+) T cells, in response to vaccination or natural infection, has been associated with improved protective immunity. However, it is unclear whether the maintenance of polyfunctionality is related to particular cellular phenotypic characteristics. To determine whether the cytokine expression profile is linked to the memory differentiation stage, we analyzed the degree of polyfunctionality of HIV-specific CD8(+) T cells within different memory subpopulations in 20 antiretroviral therapy-naive HIV-1-infected individuals at ∼34 wk postinfection. These profiles were compared with CMV-specific CD8(+) T cell responses in HIV-uninfected control subjects and in individuals chronically infected with HIV. Our results showed that the polyfunctional abilities of HIV-specific CD8(+) T cells differed according to their memory phenotype. Early-differentiated cells (CD45RO(+)CD27(+)) exhibited a higher proportion of cells positive for three or four functions (p < 0.001), and a lower proportion of monofunctional cells (p < 0.001) compared with terminally differentiated (TD; CD45RO(-)CD27(-)) HIV-specific CD8(+) T cells. The majority of TD HIV-specific CD8(+) T cells were monofunctional (median 69% [interquartile range: 57-83]), producing predominantly CD107a or MIP1ß. Moreover, proportions of HIV-specific monofunctional CD8(+) T cells positively associated with proportions of TD HIV-specific CD8(+) T cells (p = 0.019, r = 0.54). In contrast, CMV-specific CD8(+) T cell polyfunctional capacities were similar across all memory subpopulations, with terminally and early-differentiated cells endowed with comparable polyfunctionality. Overall, these data show that the polyfunctional abilities of HIV-specific CD8(+) T cells are influenced by the stage of memory differentiation, which is not the case for CMV-specific responses.

Distinct kinetics of Gag-specific CD4+ and CD8+ T cell responses during acute HIV-1 infection.

HIV infection is characterized by a gradual deterioration of immune function, mainly in the CD4 compartment. To better understand the dynamics of HIV-specific T cells, we analyzed the kinetics and polyfunctional profiles of Gag-specific CD4(+) and CD8(+) T cell responses in 12 subtype C-infected individuals with different disease-progression profiles, ranging from acute to chronic HIV infection. The frequencies of Gag-responsive CD4(+) and CD8(+) T cells showed distinct temporal kinetics. The peak frequency of Gag-responsive IFN-γ(+)CD4(+) T cells was observed at a median of 28 d (interquartile range: 21-81 d) post-Fiebig I/II staging, whereas Gag-specific IFN-γ(+)CD8(+) T cell responses peaked at a median of 253 d (interquartile range: 136-401 d) and showed a significant biphasic expansion. The proportion of TNF-α-expressing cells within the IFN-γ(+)CD4(+) T cell population increased (p = 0.001) over time, whereas TNF-α-expressing cells within IFN-γ(+)CD8(+) T cells declined (p = 0.005). Both Gag-responsive CD4(+) and CD8(+) T cells showed decreased Ki67 expression within the first 120 d post-Fiebig I/II staging. Prior to the disappearance of Gag-responsive Ki67(+)CD4(+) T cells, these cells positively correlated (p = 0.00038) with viremia, indicating that early Gag-responsive CD4 events are shaped by viral burden. No such associations were observed in the Gag-specific CD8(+) T cell compartment. Overall, these observations indicated that circulating Gag-responsive CD4(+) and CD8(+) T cell frequencies and functions are not synchronous, and properties change rapidly at different tempos during early HIV infection.

Reduced anti-viral IgG repertoire in HIV-exposed but uninfected infants compared to HIV-unexposed infants.

Infants who are HIV exposed but uninfected (iHEU) have higher risk of viral infections compared to infants who are HIV unexposed (iHUU). We explored the effect of intrauterine HIV exposure on the infant antibody repertoire by quantifying plasma immunoglobulin (Ig) G against 206 eukaryote-infecting viruses using phage immunoprecipitation sequencing (PhiPSeq) in iHEU and iHUU at birth and 36 weeks of life. Maternal HIV infection altered the infant IgG repertoire against eukaryote-infecting viruses at birth, resulting in significantly lower antibody breadth and diversity among iHEU compared to iHUU. Neonatal anti-viral IgG repertoire was dominated by antibodies against viruses belonging to the Herpesviridae family, although, by 36 weeks, this had shifted toward antibodies against enteroviruses, likely due to waning of maternal-derived antibodies and polio vaccine-induced antibody responses as expected. The observed reduced anti-viral IgG repertoire breadth and diversity acquired at birth in iHEU might contribute to the increased rates of viral infections among iHEU during early life.

Exposure to HIV alters the composition of maternal microchimeric T cells in infants.

Infants exposed to HIV but uninfected (iHEU) display altered cellular immunity and are at increased risk of infection through poorly understood mechanisms. We previously reported that iHEU have lower levels of maternal microchimerism (MMc), maternal cells transferred to the offspring in utero/during breastfeeding. We evaluated MMc levels in T cell subsets in iHEU and HIV unexposed infants (iHU) to determine whether a selective deficiency in MMc may contribute to altered cellular immunity. Across all infants, MMc levels were highest in CD8+ T cells; however, the level of MMc in the CD8 T cell subset was significantly lower in iHEU compared to iHU.

Longitudinal gut microbiota composition of South African and Nigerian infants in relation to tetanus vaccine responses.

Infants who are exposed to HIV but uninfected (iHEU) have higher risk of infectious morbidity than infants who are HIV-unexposed and uninfected (iHUU), possibly due to altered immunity. As infant gut microbiota may influence immune development, we evaluated the effects of HIV exposure on infant gut microbiota and its association with tetanus toxoid vaccine responses. We evaluated the gut microbiota of 82 South African (61 iHEU and 21 iHUU) and 196 Nigerian (141 iHEU and 55 iHUU) infants at <1 and 15 weeks of life by 16S rRNA gene sequencing. Anti-tetanus antibodies were measured by enzyme-linked immunosorbent assay at matched time points. Gut microbiota in the 278 included infants and its succession were more strongly influenced by geographical location and age than by HIV exposure. Microbiota of Nigerian infants, who were exclusively breastfed, drastically changed over 15 weeks, becoming dominated by Bifidobacterium longum subspecies infantis. This change was not observed among South African infants, even when limiting the analysis to exclusively breastfed infants. The Least Absolute Shrinkage and Selection Operator regression suggested that HIV exposure and gut microbiota were independently associated with tetanus titers at week 15, and that high passively transferred antibody levels, as seen in the Nigerian cohort, may mitigate these effects. In conclusion, in two African cohorts, HIV exposure minimally altered the infant gut microbiota compared to age and setting, but both specific gut microbes and HIV exposure independently predicted humoral tetanus vaccine responses.IMPORTANCEGut microbiota plays an essential role in immune system development. Since infants HIV-exposed and uninfected (iHEU) are more vulnerable to infectious diseases than unexposed infants, we explored the impact of HIV exposure on gut microbiota and its association with vaccine responses. This study was conducted in two African countries with rapidly increasing numbers of iHEU. Infant HIV exposure did not substantially affect gut microbial succession, but geographic location had a strong effect. However, both the relative abundance of specific gut microbes and HIV exposure were independently associated with tetanus titers, which were also influenced by baseline tetanus titers (maternal transfer). Our findings provide insight into the effect of HIV exposure, passive maternal antibody, and gut microbiota on infant humoral vaccine responses.

Premature skewing of T cell receptor clonality and delayed memory expansion in HIV-exposed infants.

While preventing vertical HIV transmission has been very successful, HIV-exposed uninfected infants (iHEU) experience an elevated risk to infections compared to HIV-unexposed and uninfected infants (iHUU). Here we present a longitudinal multimodal analysis of infant immune ontogeny that highlights the impact of HIV/ARV exposure. Using mass cytometry, we show alterations in T cell memory differentiation between iHEU and iHUU being significant from week 15 of life. The altered memory T cell differentiation in iHEU was preceded by lower TCR Vß clonotypic diversity and linked to TCR clonal depletion within the naïve T cell compartment. Compared to iHUU, iHEU had elevated CD56loCD16loPerforin+CD38+CD45RA+FcεRIγ+ NK cells at 1 month postpartum and whose abundance pre-vaccination were predictive of vaccine-induced pertussis and rotavirus antibody responses post 3 months of life. Collectively, HIV/ARV exposure disrupted the trajectory of innate and adaptive immunity from birth which may underlie relative vulnerability to infections in iHEU.

Inflammatory markers in pregnancy are associated with postpartum weight in South African women living with HIV on antiretroviral therapy.

BACKGROUND: Postpartum weight (PPW) contributes to long-term obesity, a growing concern in persons with HIV (PWH). We investigated whether inflammatory markers in pregnancy may be involved in postpartum (PP) obesity in PWH. SETTING: A total of 57 pregnant PWH enrolled at ≤14 weeks gestation (T1) in Gugulethu antenatal care clinic in Cape Town and followed through 48 weeks PP were included. METHODS: Plasma soluble (s) CD14, sCD163, leptin, tumour necrosis factor receptor 1 (TNFR-1), resistin, adiponectin, and interleukin-6 (IL-6) were assayed in duplicate using the Luminex platform. We considered each inflammatory marker at T1 (n=57) and T3 (29-36 weeks gestation, n=31) as a separate exposure of interest. Linear mixed effects models were fit to examine whether each exposure was associated with average PPW and PPW trajectories; linear regression was used for associations with PPW change between T1 and 48 weeks. RESULTS: Median age was 32 years (IQR, 29-35), 98% were multigravida, and 49% had a BMI≥30 kg/m2. Higher T1 sCD14 levels were associated with higher average weight through 48 weeks PP (ß = 0.002, p=0.04), and T3 sCD14 with higher PPW gain (ß = 0.007, p=0.04). Leptin (ß = 0.414, p<0.01), TNFR-1 (ß = 11.048, p<0.01) and resistin (ß = 0.714, p=0.01) at T3 were associated with higher average PPW, and IL-6 (ß = 2.266, p=0.02) with PPW gain. CONCLUSION: These findings suggest that low-grade inflammation in pregnancy may play a role in postpartum obesity, pointing to potential mechanisms with implications for long-term cardiometabolic health in PWH.

Intersubtype differences in the effect of a rare p24 gag mutation on HIV-1 replicative fitness.

Certain immune-driven mutations in HIV-1, such as those arising in p24(Gag), decrease viral replicative capacity. However, the intersubtype differences in the replicative consequences of such mutations have not been explored. In HIV-1 subtype B, the p24(Gag) M250I mutation is a rare variant (0.6%) that is enriched among elite controllers (7.2%) (P = 0.0005) and appears to be a rare escape variant selected by HLA-B58 supertype alleles (P < 0.01). In contrast, in subtype C, it is a relatively common minor polymorphic variant (10 to 15%) whose appearance is not associated with a particular HLA allele. Using site-directed mutant viruses, we demonstrate that M250I reduces in vitro viral replicative capacity in both subtype B and subtype C sequences. However, whereas in subtype C downstream compensatory mutations at p24(Gag) codons 252 and 260 reduce the adverse effects of M250I, fitness costs in subtype B appear difficult to restore. Indeed, patient-derived subtype B sequences harboring M250I exhibited in vitro replicative defects, while those from subtype C did not. The structural implications of M250I were predicted by protein modeling to be greater in subtype B versus C, providing a potential explanation for its lower frequency and enhanced replicative defects in subtype B. In addition to accounting for genetic differences between HIV-1 subtypes, the design of cytotoxic-T-lymphocyte-based vaccines may need to account for differential effects of host-driven viral evolution on viral fitness.

Relationship between functional profile of HIV-1 specific CD8 T cells and epitope variability with the selection of escape mutants in acute HIV-1 infection.

In the present study, we analyzed the functional profile of CD8+ T-cell responses directed against autologous transmitted/founder HIV-1 isolates during acute and early infection, and examined whether multifunctionality is required for selection of virus escape mutations. Seven anti-retroviral therapy-naïve subjects were studied in detail between 1 and 87 weeks following onset of symptoms of acute HIV-1 infection. Synthetic peptides representing the autologous transmitted/founder HIV-1 sequences were used in multiparameter flow cytometry assays to determine the functionality of HIV-1-specific CD8+ T memory cells. In all seven patients, the earliest T cell responses were predominantly oligofunctional, although the relative contribution of multifunctional cell responses increased significantly with time from infection. Interestingly, only the magnitude of the total and not of the poly-functional T-cell responses was significantly associated with the selection of escape mutants. However, the high contribution of MIP-1ß-producing CD8+ T-cells to the total response suggests that mechanisms not limited to cytotoxicity could be exerting immune pressure during acute infection. Lastly, we show that epitope entropy, reflecting the capacity of the epitope to tolerate mutational change and defined as the diversity of epitope sequences at the population level, was also correlated with rate of emergence of escape mutants.

Purification of primary human placental leukocytes to study maternal-fetal interactions.

Decidual leukocytes play key roles in maternal-fetal tolerance and immunity. Here, we present detailed methods to purify, culture, and functionally analyze human placental dNK, dTreg, dTem, and dMɸ from decidua parietalis, the maternal part of the placental membranes; decidua basalis, the maternal part of the placenta; and placental villi. These sites have high clinical relevance in the development of villitis and chorioamnionitis. This allows in-depth phenotypic and functional investigation of placental immune populations and their interactions with extravillous trophoblasts. For complete details on the use and execution of this protocol, please refer to Ikumi et al.,1 Tilburgs et al.,2 Salvany-Celades et al.,3 Crespo et al.,4 van der Zwan et al.5.