Bioluminescent Reporting of In Vivo IFN-γ Immune Responses during Infection and Autoimmunity

IFN-γ is a key cytokine of innate and adaptive immunity. It is important to understand temporal changes in IFN-γ production and how these changes relate to the role of IFN-γ in diverse models of infectious and autoimmune disease, making the ability to monitor and track IFN-γ production in vivo of a substantial benefit. IFN-γ ELISPOTs have been a central methodology to measure T cell immunity for many years. In this study, we add the capacity to analyze IFN-γ responses with high sensitivity and specificity, longitudinally, in vitro and in vivo. This allows the refinement of experimental protocols because immunity can be tracked in real-time through a longitudinal approach. We have generated a novel murine IFN-γ reporter transgenic model that allows IFN-γ production to be visualized and quantified in vitro and in vivo as bioluminescence using an imaging system. At baseline, in the absence of an inflammatory stimulus, IFN-γ signal from lymphoid tissue is detectable in vivo. Reporter transgenics are used in this study to track the IFN-γ response to Pseudomonas aeruginosa infection in the lung over time in vivo. The longitudinal development of the adaptive T cell immunity following immunization with Ag is identified from day 7 in vivo. Finally, we show that we are able to use this reporter transgenic to follow the onset of autoimmune T cell activation after regulatory T cell depletion in an established model of systemic autoimmunity. This IFN-γ reporter transgenic, termed “Gammaglow,” offers a valuable new modality for tracking IFN-γ immunity, noninvasively and longitudinally over time

Strong CD4 T cell responses to Zika virus antigens in a cohort of Dengue virus immune mothers of congenital Zika virus syndrome infants

Background: There is an urgent need to understand the complex relationship between cross-reactive anti-viral immunity, disease susceptibility, and severity in the face of differential exposure to related, circulating Flaviviruses. Co-exposure to Dengue virus and Zika virus in Brazil is a case in point. A devastating aspect of the 2015-2016 South American Zika outbreak was the dramatic increase in numbers of infants born with microcephaly to mothers exposed to Zika virus during pregnancy. It has been proposed that this is more likely to ensue from Zika infection in women lacking cross-protective Dengue immunity. In this case series we measure the prevalence of Dengue immunity in a cohort of mothers exposed to Zika virus during pregnancy in the 2015-2016 Zika outbreak that gave birth to an infant affected by microcephaly and explore their adaptive immunity to Zika virus. Results: Fifty women from Sergipe, Brazil who gave birth to infants with microcephaly following Zika virus exposure during the 2015-16 outbreak were tested for serological evidence of Dengue exposure and IFNγ ELISpot spot forming cell (SFC) response to Zika virus. The majority (46/50) demonstrated Dengue immunity. IFNγ ELISpot responses to Zika virus antigens showed the following hierarchy: Env>NS1>NS3>C protein. Twenty T cell epitopes from Zika virus Env were identified. Responses to Zika virus antigens Env and NS1 were polyfunctional with cells making IFNγ, TNFα, IL-4, IL-13, and IL-10. In contrast, responses to NS5 only produced the immune regulatory TGFβ1 cytokine. There were SFC responses against Zika virus Env (1-20) and variant peptide sequences from West Nile virus, Dengue virus 1-4 and Yellow Fever virus. Conclusion: Almost all the women in our study showed serological evidence of Dengue immunity, suggesting that microcephaly can occur in DENV immune mothers. T cell immunity to Zika virus showed a multifunctional response to the antigens Env and NS1 and immune regulatory responses to NS5 and C protein. Our data support an argument that different viral products may skew the antiviral response to a more pro or anti-inflammatory outcome, with an associated impact on immunopathogenesis

Impact of HLA polymorphism on the immune response to bacillus anthracis protective antigen in vaccination versus natural infection

The causative agent of anthrax, Bacillus anthracis, evades the host immune response and establishes infection through the production of binary exotoxins composed of Protective Antigen (PA) and one of two subunits, lethal factor (LF) or edema factor (EF). The majority of vaccination strategies have focused upon the antibody response to the PA subunit. We have used a panel of humanised HLA class II transgenic mouse strains to define HLA-DR-restricted and HLA-DQ-restricted CD4+ T cell responses to the immunodominant epitopes of PA. This was correlated with the binding affinities of epitopes to HLA class II molecules, as well as the responses of two human cohorts: individuals vaccinated with the Anthrax Vaccine Precipitated (AVP) vaccine (which contains PA and trace amounts of LF), and patients recovering from cutaneous anthrax infections. The infected and vaccinated cohorts expressing different HLA types were found to make CD4+ T cell responses to multiple and diverse epitopes of PA. The effects of HLA polymorphism were explored using transgenic mouse lines, which demonstrated differential susceptibility, indicating that HLA-DR1 and HLA-DQ8 alleles conferred protective immunity relative to HLA-DR15, HLA-DR4 and HLA-DQ6. The HLA transgenics enabled a reductionist approach, allowing us to better define CD4+ T cell epitopes. Appreciating the effects of HLA polymorphism on the variability of responses to natural infection and vaccination is vital in planning protective strategies against anthrax