Dynamics and control of T follicular helper cell-dependent and -independent responses to influenza virus infection and immunization
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2020
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Melbourne Authors
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Microbiology & Immunology
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PhD thesis
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Open Access
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© 2020 Wenbo Jiang
Abstract
Seasonal influenza viruses circulate globally and cause recurrent disease in humans. Worldwide, annual epidemics are estimated to cause 1 billion infections, with 3 to 5 million cases of severe illness and 290,000 to 650,000 deaths. Influenza viruses undergo rapid antigenic evolution allowing mutant viruses to escape from host immune responses acquired to parental virus strains. Current seasonal influenza vaccines are effective when vaccine strains are matched with circulating strains. However, there is little to no cross-protection against antigenic variants, emerging pandemic or zoonotic outbreak strains. There is therefore tremendous interest in the development of novel universal vaccines which induce potent, broad and durable antibody responses against most or all influenza viruses. T follicular helper cells are crucial for the generation of high affinity antibodies and the maintenance of B cell memory. But relatively little is known about Tfh in important animal models of influenza. Insights gained from the study of Tfh cell responses will facilitate the design of next generation vaccines against influenza.
In this thesis, we first developed an activation-induced marker assay for the identification of antigen specific Tfh cells in mice after influenza virus infection and hemagglutinin protein immunization. We showed that the AIM assay was robust and sensitive for the detection of murine Ag specific Tfh cells by quantifying the upregulation of surface CD154 or CD25 OX40 following either HA peptide pool or whole HA protein stimulation for 18 hours. This murine AIM assay makes it feasible to delineate Ag specific Tfh cells in mice without the need for transgenic mice or MHC II tetramers restricted to specific epitopes. Importantly, Ag specific Tfh cells can be sorted for TCR sequencing or adoptive transfer since AIM assay is a live cell assay.
Ferrets are a well established animal model for influenza research and are widely used to investigate the pathogenesis and transmission of influenza viruses and preclinically evaluate the efficacy of influenza vaccines. However, little is known about ferret Tfh cells due to the lack of ferret reactive immunological reagents. To enable the study of ferret Tfh cells, we screened commercial markers of Tfh cells, antiBCL6, CXCR5 and PD1 antibodies, and found two anti-BCL6 antibodies had cross reactivity with lymph node cells from influenza infected ferrets. We also developed two murine monoclonal antibodies against ferret CXCR5 and PD1 using a single B cell PCR based method. We were able to clearly identify Tfh cells in LNs from influenza infected ferrets using these antibodies. The development of ferret Tfh marker antibodies and the identification of ferret Tfh cells will facilitate the assessment of vaccine induced Tfh responses in the ferret model and the design of novel vaccines against influenza infection.
HA stem is an attractive target for the development of universal influenza vaccines due to its relatively conserved feature. However, HA stem is poorly immunogenic when administered alone in a soluble form. Immunogen multimerization can enhance the immunogenicity of poor immunogens even in the absence of the help of T cells, which serves as an alternative pathway to improve the immunogenicity of stem without the dependence on Tfh responses. We showed that chemically coupling a peptide derived from the head domain of PR8 HA, P35, with the weakly immunogenic HA stem protein caused aggregation of the HA stem which significantly enhanced stem specific B cell responses independent of Tfh cell help in mice. P35 conjugation represents a new pathway to boost stem specific antibody responses without introducing exotic carrier proteins which will elicit anti carrier responses.
Collectively, we investigated Tfh responses to influenza virus infection and immunization in mice and ferrets and explored the effects of immunogen multimerization on humoral immunity in the context limiting Tfh responses to HA stem. An increased understanding of Tfh dependent and independent mechanisms to enhance humoral immune responses will assist developing novel vaccines to prevent the infection of influenza and other viruses.
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T follicular helper cell, Influenza, Ferret, Stem