Bifidobacterium infantis associates with T cell immunity in human infants and is sufficient to enhance antigen-specific T cells in mice.

Bacille Calmette-Guerin (BCG) vaccine can elicit good TH1 responses in neonates. We hypothesized that the pioneer gut microbiota affects vaccine T cell responses. Infants who are HIV exposed but uninfected (iHEU) display an altered immunity to vaccination. BCG-specific immune responses were analyzed at 7 weeks of age in iHEU, and responses were categorized as high or low. Bifidobacterium longum subsp. infantis was enriched in the stools of high responders, while Bacteroides thetaiotaomicron was enriched in low responders at time of BCG vaccination. Neonatal germ-free or SPF mice orally gavaged with live B. infantis exhibited significantly higher BCG-specific T cells compared with pups gavaged with B. thetaiotaomicron. B. infantis and B. thetaiotaomicron differentially affected stool metabolome and colonic transcriptome. Human colonic epithelial cells stimulated with B. infantis induced a unique gene expression profile versus B. thetaiotaomicron. We thus identified a causal role of B. infantis in early-life antigen-specific immunity.

Factors influencing maternal microchimerism throughout infancy and its impact on infant T cell immunity.

Determinants of the acquisition and maintenance of maternal microchimerism (MMc) during infancy and the impact of MMc on infant immune responses are unknown. We examined factors that influence MMc detection and level across infancy and the effect of MMc on T cell responses to bacillus Calmette-Guérin (BCG) vaccination in a cohort of HIV-exposed, uninfected and HIV-unexposed infants in South Africa. MMc was measured in whole blood from 58 infants using a panel of quantitative PCR assays at day 1, and 7, 15, and 36 weeks of life. Infants received BCG at birth, and selected whole blood samples from infancy were stimulated in vitro with BCG and assessed for polyfunctional CD4+ T cell responses. MMc was present in most infants across infancy, with levels ranging from 0 to 1,193/100,000 genomic equivalents and was positively impacted by absence of maternal HIV, maternal and infant HLA compatibility, infant female sex, and exclusive breastfeeding. Initiation of maternal antiretroviral therapy prior to pregnancy partially restored MMc level in HIV-exposed, uninfected infants. Birth MMc was associated with an improved polyfunctional CD4+ T cell response to BCG. These data emphasize that both maternal and infant factors influence the level of MMc, which may subsequently affect infant T cell responses.

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.

Dysregulation of the Immune Environment in the Airways During HIV Infection.

HIV-1 increases susceptibility to pulmonary infection and disease, suggesting pathogenesis in the lung. However, the lung immune environment during HIV infection remains poorly characterized. This study examined T cell activation and the cytokine milieu in paired bronchoalveolar lavage (BAL) and blood from 36 HIV-uninfected and 32 HIV-infected participants. Concentrations of 27 cytokines were measured by Luminex, and T cells were phenotyped by flow cytometry. Blood and BAL had distinct cytokine profiles (p=0.001). In plasma, concentrations of inflammatory cytokines like IFN-γ (p=0.004) and TNF-α (p=0.004) were elevated during HIV infection, as expected. Conversely, BAL cytokine concentrations were similar in HIV-infected and uninfected individuals, despite high BAL viral loads (VL; median 48,000 copies/ml epithelial lining fluid). HIV-infected individuals had greater numbers of T cells in BAL compared to uninfected individuals (p=0.007); and BAL VL positively associated with CD4+ and CD8+ T cell numbers (p=0.006 and p=0.0002, respectively) and CXCL10 concentrations (p=0.02). BAL T cells were highly activated in HIV-infected individuals, with nearly 2-3 fold greater frequencies of CD4+CD38+ (1.8-fold; p=0.007), CD4+CD38+HLA-DR+ (1.9-fold; p=0.0006), CD8+CD38+ (2.8-fold; p=0.0006), CD8+HLA-DR+ (2-fold; p=0.022) and CD8+CD38+HLA-DR+ (3.6-fold; p<0.0001) cells compared to HIV-uninfected individuals. Overall, this study demonstrates a clear disruption of the pulmonary immune environment during HIV infection, with readily detectable virus and activated T lymphocytes, which may be driven to accumulate by local chemokines.

Bacille Calmette-Guérin Vaccine Strain Modulates the Ontogeny of Both Mycobacterial-Specific and Heterologous T Cell Immunity to Vaccination in Infants.

Differences in Bacille Calmette-Guérin (BCG) immunogenicity and efficacy have been reported, but various strains of BCG are administered worldwide. Since BCG immunization may also provide protection against off-target antigens, we sought to identify the impact of different BCG strains on the ontogeny of vaccine-specific and heterologous vaccine immunogenicity in the first 9 months of life, utilizing two African birth cohorts. A total of 270 infants were studied: 84 from Jos, Nigeria (vaccinated with BCG-Bulgaria) and 187 from Cape Town, South Africa (154 vaccinated with BCG-Denmark and 33 with BCG-Russia). Infant whole blood was taken at birth, 7, 15, and 36 weeks and short-term stimulated (12 h) in vitro with BCG, Tetanus and Pertussis antigens. Using multiparameter flow cytometry, CD4+ T cell memory subset polyfunctionality was measured by analyzing permutations of TNF-α, IL-2, and IFN-γ expression at each time point. Data was analyzed using FlowJo, SPICE, R, and COMPASS. We found that infants vaccinated with BCG-Denmark mounted significantly higher frequencies of BCG-stimulated CD4+ T cell responses, peaking at week 7 after immunization, and possessed durable polyfunctional CD4+ T cells that were in a more early differentiated memory stage when compared with either BCG-Bulgaria and BCG-Russia strains. The latter responses had lower polyfunctional scores and tended to accumulate in a CD4+ T cell naïve-like state (CD45RA+CD27+). Notably, BCG-Denmark immunization resulted in higher magnitudes and polyfunctional cytokine responses to heterologous vaccine antigens (Tetanus and Pertussis). Collectively, our data show that BCG strain was the strongest determinant of both BCG-stimulated and heterologous vaccine stimulated T cell magnitude and polyfunctionality. These findings have implications for vaccine policy makers, manufacturers and programs worldwide and also suggest that BCG-Denmark, the first vaccine received in many African infants, has both specific and off-target effects in the first few months of life, which may provide an immune priming benefit to other EPI vaccines.

Impact of maternal HIV exposure, feeding status, and microbiome on infant cellular immunity.

At least one-third of infants born in sub-Saharan Africa have been exposed to the effects of maternal HIV infection and antiretroviral treatment. Intrauterine HIV exposure is associated with increased rates of morbidity and mortality in children. Although the mechanisms responsible for poor infant health with HIV-1 exposure are likely to be multifactorial, we posit that the maternal environment during gestation and in the perinatal period results in altered infant immunity and is possibly the strongest contributing factor responsible for the disproportionally high infectious events among HIV-exposed infants who remain HIV uninfected. This review provides a synthesis of studies reporting the impact of intrauterine HIV exposure, feeding practices, and microbiota on immune ontogeny in the first year of life in HIV-exposed uninfected infants.

Breastfeeding mitigates the effects of maternal HIV on infant infectious morbidity in the Option B+ era.

OBJECTIVE: The effects of in-utero HIV-exposure on infectious morbidity and mortality in settings with universal maternal treatment and high breastfeeding rates are unclear. Further, the benefits of exclusive feeding options have not been assessed in the Option B+ era. We investigated these in two African settings with high breastfeeding uptake and good HIV treatment infrastructure during the first year of life. METHODS: Cox regression with time-changing variables in a birth cohort of 749 HIV-exposed uninfected and HIV-unexposed uninfected infants from Cape Town, South Africa and Jos, Nigeria. RESULTS: There was no difference in infectious morbidity incidence between HIV-exposed uninfected and HIV-unexposed uninfected infants (hazard ratio 1.01; 95% CI 0.78-1.32) after adjusting for confounding variables. Formula-fed infants had significantly higher infectious morbidity incidence when compared with exclusively breastfed infants (hazard ratio 1.64; 95% CI 1.03-2.63) and mixed-breastfed infants (hazard ratio 1.42; 95% CI 1.00-2.02) after adjusting for potential confounding variables. There was no significant difference in mortality among HIV-exposed infants and HIV-unexposed infants during the first year of life in this cohort (2.04 versus 0.94%, P = 0.38). Notably, exclusive breastfeeding for only 4 months had protective effects on morbidity up to 1 year. CONCLUSION: In settings with universal antiretroviral coverage and high breastfeeding rates, breastfeeding mitigates the effects of in-utero HIV exposure among infants during the first year of life. These findings support previous recommendations for exclusive breastfeeding among HIV-infected women and highlight the role that breastfeeding plays on the health of infants in settings where exclusive breastfeeding is not always feasible or where replacement feeding is recommended.

Effect of HIV on the Frequency and Number of Mycobacterium tuberculosis-Specific CD4+ T Cells in Blood and Airways During Latent M. tuberculosis Infection.

Human immunodeficiency virus type 1 (HIV) infection substantially increases the risk of developing tuberculosis. There is extensive depletion of Mycobacterium tuberculosis-specific CD4+ T cells in blood during early HIV infection, but little is known about responses in the lungs at this stage. Given that mucosal organs are a principal target for HIV-mediated CD4+ T-cell destruction, we investigated M. tuberculosis-specific responses in bronchoalveolar lavage (BAL) from persons with latent M. tuberculosis infection and untreated HIV coinfection with preserved CD4+ T-cell counts. M. tuberculosis-specific CD4+ T-cell cytokine (interferon γ, tumor necrosis factor α, and interleukin 2) responses were discordant in frequency and function between BAL and blood. Responses in BAL were 15-fold lower in HIV-infected persons as compared to uninfected persons (P = .048), whereas blood responses were 2-fold lower (P = .006). However, an increase in T cells in the airways in HIV-infected persons resulted in the overall number of M. tuberculosis-specific CD4+ T cells in BAL being similar. Our study highlights the important insights gained from studying M. tuberculosis immunity at the site of disease during HIV infection.

Selective reduction of IFN-γ single positive mycobacteria-specific CD4+ T cells in HIV-1 infected individuals with latent tuberculosis infection.

HIV-1 is recognized to increase the risk for tuberculosis even before CD4+ T cell deficiency is profound. To better understand how HIV-1 alters immunity to latent tuberculosis, we compared the magnitude and functional profile of mycobacteria-specific CD4+ T cells between HIV-uninfected and HIV-infected individuals, using flow cytometry. In HIV-1 infection, IFN-γ single positive mycobacteria-specific CD4+ T cells were decreased, while the frequency of polyfunctional cells (IFN-γ+IL-2+TNF-α+) remained unchanged. Moreover, the proportion of IFN-γ single positive cells correlated inversely with viral replication. Our results suggest that HIV-1 affects mycobacteria-specific cells differentially, depending on their functional capacity.

A subset of circulating blood mycobacteria-specific CD4 T cells can predict the time to Mycobacterium tuberculosis sputum culture conversion.

We investigated 18 HIV-negative patients with MDR-TB for M. tuberculosis (Mtb)- and PPD-specific CD4 T cell responses and followed them over 6 months of drug therapy. Twelve of these patients were sputum culture (SC) positive and six patients were SC negative upon enrollment. Our aim was to identify a subset of mycobacteria-specific CD4 T cells that would predict time to culture conversion. The total frequency of mycobacteria-specific CD4 T cells at baseline could not distinguish patients showing positive or negative SC. However, a greater proportion of late-differentiated (LD) Mtb- and PPD-specific memory CD4 T cells was found in SC positive patients than in those who were SC negative (p = 0.004 and p = 0.0012, respectively). Similarly, a higher co-expression of HLA-DR+ Ki67+ on Mtb- and PPD-specific CD4 T cells could also discriminate between sputum SC positive versus SC negative (p = 0.004 and p = 0.001, respectively). Receiver operating characteristic (ROC) analysis revealed that baseline levels of Ki67+ HLA-DR+ Mtb- and PPD-specific CD4 T cells were predictive of the time to sputum culture conversion, with area-under-the-curve of 0.8 (p = 0.027). Upon treatment, there was a significant decline of these Ki67+ HLA-DR+ T cell populations in the first 2 months, with a progressive increase in mycobacteria-specific polyfunctional IFNγ+ IL2+ TNFα+ CD4 T cells over 6 months. Thus, a subset of activated and proliferating mycobacterial-specific CD4 T cells (Ki67+ HLA-DR+) may provide a valuable marker in peripheral blood that predicts time to sputum culture conversion in TB patients at the start of treatment.

The novel capripoxvirus vector lumpy skin disease virus efficiently boosts modified vaccinia Ankara human immunodeficiency virus responses in rhesus macaques.

Poxvirus vectors represent promising human immunodeficiency virus (HIV) vaccine candidates and were a component of the only successful HIV vaccine efficacy trial to date. We tested the immunogenicity of a novel recombinant capripoxvirus vector, lumpy skin disease virus (LSDV), in combination with modified vaccinia Ankara (MVA), both expressing genes from HIV-1. Here, we demonstrated that the combination regimen was immunogenic in rhesus macaques, inducing high-magnitude, broad and balanced CD4(+) and CD8(+) T-cell responses, and transient activation of the immune response. These studies support further development of LSDV as a vaccine vector.

Robust immunity to an auxotrophic Mycobacterium bovis BCG-VLP prime-boost HIV vaccine candidate in a nonhuman primate model.

We previously reported that a recombinant pantothenate auxotroph of Mycobacterium bovis BCG expressing human immunodeficiency virus type 1 (HIV-1) subtype C Gag (rBCGpan-Gag) efficiently primes the mouse immune system for a boost with a recombinant modified vaccinia virus Ankara (rMVA) vaccine. In this study, we further evaluated the immunogenicity of rBCGpan-Gag in a nonhuman primate model. Two groups of chacma baboons were primed or mock primed twice with either rBCGpan-Gag or a control BCG. Both groups were boosted with HIV-1 Pr55(gag) virus-like particles (Gag VLPs). The magnitude and breadth of HIV-specific cellular responses were measured using a gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISPOT) assay, and the cytokine profiles and memory phenotypes of T cells were evaluated by polychromatic flow cytometry. Gag-specific responses were detected in all animals after the second inoculation with rBCGpan-Gag. Boosting with Gag VLPs significantly increased the magnitude and breadth of the responses in the baboons that were primed with rBCGpan-Gag. These responses targeted an average of 12 Gag peptides per animal, compared to an average of 3 peptides per animal for the mock-primed controls. Robust responses of Gag-specific polyfunctional T cells capable of simultaneously producing IFN-γ, tumor necrosis alpha (TNF-α), and interleukin-2 (IL-2) were detected in the rBCGpan-Gag-primed animals. Gag-specific memory T cells were skewed toward a central memory phenotype in both CD4(+) and CD8(+) T cell populations. These data show that the rBCGpan-Gag prime and Gag VLP boost vaccine regimen is highly immunogenic, inducing a broad and polyfunctional central memory T cell response. This report further indicates the feasibility of developing a BCG-based HIV vaccine that is safe for childhood HIV immunization.

Evaluation of CD103 (αEß7) integrin expression by CD8 T cells in blood as a surrogate marker to predict cervical T cell responses in the female genital tract during HIV infection.

Mucosal homing receptors expressed by blood T cells may be useful surrogates for measuring mucosal T cell immune responses at the site of HIV transmission. Here, we investigated whether HIV-specific responses by T cells expressing the mucosal integrin receptor CD103 in blood reliably predicted corresponding HIV-specific responses at the cervix. The frequency of CD8+ T cells expressing CD103 in blood correlated significantly with the number of CD103+CD8+ T cells from the cervix suggesting that CD103 was involved in trafficking of T cells from blood to the cervical mucosa. TGF-ß concentrations in plasma were significantly associated with the frequency of CD103 expression by blood but not cervical CD8 T cells. The majority of Gag-responsive CD8 T cells were CD103+ in both blood and at the cervix. Despite this, the magnitude of Gag-specific IFN-γ responses by CD103+CD8+ T cells in blood did not predict similar Gag-specific responses at the cervix.