No abstract

Polychromatic flow cytometry allows the capture of multidimensional data, providing the technical tool to assess complex immune responses. Interrogation of the adaptive T cell response to infection or vaccination already has benefited greatly from standardized protocols for polychromatic flow cytometric analysis. The innate immune system plays an important role in health and disease, and presents potentially important therapeutic and diagnostic modalities. We describe here a high-throughput polychromatic flow cytometry-based platform that enables the rapid interrogation and large scale screening of human blood antigen presenting cell responses to Toll-like receptor (TLR) ligands and other innate immune modulators. Using this assay, we found that for certain stimuli (e.g., TLR9 and TLR3 ligands), the general protocol for intracellular cytokine cytometry had to be significantly modified to allow response detection. Furthermore, high concentrations of TLR7/8 and TLR4 stimuli caused substantial changes in lineage markers, potentially confounding analysis if one were to use a conventional "lineage-negative" cocktail. The assay we developed is reproducible and has been used to show that a given individual's TLR response pattern is relatively stable over at least several months. This protocol is in strict compliance with published guidelines for polychromatic flow cytometry, provides a common platform for scientists to compare their results directly, and may be applicable to the diagnostic evaluation of Toll-like receptor function and the rapid screening of promising therapeutic innate immune modulators.

e HIV-exposed but uninfected (HEU) infants born to HIV-infected mothers from areas in the world with a high burden of infectious disease suffer higher infectious morbidity and mortality than their HIV unexposed uninfected (HUU) peers. Vaccination provides protection from infection. The possibility exists that altered response to vaccination contributes to the higher rate of infection in HEU than in HUU infants. While short-term, cross-sectional studies support this notion, it is unclear whether or not HEU infants develop long-term protective immune responses following the WHO extended program on immunization (EPI).Vaccine-specific antibody responses were compared between HEU and HUU infants from 2 weeks until 2 years of age in a longitudinal South African cohort. Total IgG and antibodies specific for Bordetella pertussis, Haemophilus influenzae type b (Hib), tetanus toxoid, hepatitis B virus (HepB), and measles virus were measured at multiple time points throughout the first 2 years of life. Prevaccine antibodies (maternal antibodies passively acquired) specific for tetanus were lower in HEU than in HUU infants, while prevaccine antibodies to HepB were higher in HEU than in HUU infants. Both groups responded similarly to tetanus, Hib, and HepB vaccination. HEU demonstrated stronger pertussis vaccine responses, developing protective titers 1 year earlier than HUU patients, and maintained higher anti-tetanus titers at 24 months of age. Vaccine-induced antibodies to measles virus were similar in both groups at all time points. Our results suggest that the current EPI vaccination program as practiced in South Africa leads to the development of vaccine-specific antibody responses that are equivalent in HEU and HUU infants. However, our data also suggest that a large fraction of both HEU and HUU South African infants have antibody titers for several infectious threats that remain below the level of protection for much of their first 2 years of life.