Determining the potential universal applicability of a diurnal response to vaccination and the associated circadian immune processes

Vaccination programmes are a core element of public health strategy worldwide and are key to preventing a wide range of life threatening infections. For example, the influenza vaccination is part of the seasonal vaccination programme carried out by GP surgeries across the UK, with adults aged 65+ years being the majority of recipients. Adults aged 65+ are also now offered a one off vaccination against pneumonia and shingles. Despite good uptake of these vaccinations among older adults (>70 % for the annual flu vaccination in the UK), the over 65s make up the highest proportion of the influenza-related hospitalisations and mortalities and Pneumonia is the 5th highest cause of death in the UK. These data suggest that the vaccines work less well in older adults and an analysis of 31 studies that measured the efficacy of the flu vaccination found in older adults this varied from 17-53%, compared with 70-90% in young adults. Most older adults are therefore not fully protected against infections by the national vaccination programme. There are many reasons for this poor response to vaccines, but a major factor is that the immune system functions less well as we age. This project will investigate a novel way to improve vaccination responses in older adults.

It has become clear in the last decade that the immune system works better at different times of day, it shows a circadian rhythm. Bearing this in mind our previous project funded by MRC carried out a study looking at whether having the influenza vaccination in the morning versus the afternoon made a difference to how well the vaccine worked (how much antibody was produced in the blood). The results showed that the vaccine worked significantly better in the morning, which gives a cost neutral and quick way to improve vaccination responses in older adults. What we don't know is whether this response is unique to the influenza vaccine and how the time of day effect is achieved. This project will examine the time of day effect on the influenza vaccine and also the pneumonia vaccine Pneumovax 23, which will allow us to compare responses with two very different types of vaccine (protein and non protein). This is important as it will indicate whether the beneficial effect of morning vaccination might be seen in a wide range of vaccines which are either protein or non-protein in nature. We will also use mice to determine exactly how the time of day affects the immune response and our ability to produce antibody to increase our understanding of this important aspect of immunity relevant to public health medicine.

The project has three objectives:

1. To determine the impact of time of day on immune memory cell formation in humans. We will vaccinate subjects (aged 60-65) either in the morning (09.00) or afternoon (16.00), in line with routine GP surgery times, with the pneumonia vaccine Pneumovax 23 and the annual influenza vaccine. Subjects will provide blood samples at baseline, 4h, 24h, 7 days, 28 days and 1 year. Using CyTof methodology we will follow the ontogeny of the cellular immune response, assessing frequency of antigen specific plasmablasts and antigen specific IgM, IgA and IgG expressing B cells. We have CyTof reagents for 12 polysaccharide components of Pneumovax and influenza A and B strains. Antibody titres for these specific vaccine components will also be assessed to determine degree of class switching, magnitude and durability of the antibody response.
2. To determine differences in antigen presenting cell migration (skin DC and Langerhans cell) to lymph nodes with time of day. Skin painting with FITC in dibutylpthalate will be used and the migration of activated DCs and Langerhans cells from skin to draining lymph nodes will be monitored at different stages of the mouse circadian/activity cycle, equivalent to morning and evening in humans.
3. To determine the effect of time of day on germinal centre function and the role of clock genes in the function of germinal centre cells. Mice will be vaccinated with T dependent and T independent antigens at the extremes of the activity cycle and effects on initial BCR signalling, B cell translocation to the germinal centre T zone, and proliferation will be tested. To interrogate B cell intrinsic effects adoptive transfer will be used and germinal centre responses, affinity maturation, antibody titres and class switching monitored by immunostaining and RTPCR. Targeted deletion of circadian cycle genes, Bmal1 and RevErba, in B cells will allow us to determine their role in the circadian vaccination response.

Who might benefit from the research and how will they benefit?
There is a broad range of potential beneficiaries of the research that can be summarised as follows:

The scientific community. As stated earlier the data from the project will provide fundamental new understanding of the effect of circadian rhythms on the generation of an adaptive immune response, specifically to two clinically relevant vaccines. As we are using 2 different vaccine formats (inactivated virus for the influenza vaccine and polysaccharide antigens in pneumovax) it will allow us to determine if both T dependent and T independent antigens (and thus vaccine components) display circadian behaviour. Researchers working on vaccine development will be able to use this simple adjustment to enhance vaccine responses.The work in mice will provide mechanistic detail of how the B cell and germinal centre response varies with time of day, providing the immunology community with new information on a major factor influencing immune regulatory pathways. The data will also raise awareness of the need to take time of day in to account in studies that analyse the immune response, whether this be in mice or humans. This will greatly reduce data variability.

Applied health researchers and public policy makers. In the short term the data from the project should lead to clinical trials to test the impact of time of day of vaccination in a range of patient groups and for different vaccines which currently give variable responses. The project will thus benefit public health by revealing a cost neutral and very easy intervention to test that addresses a major health issue. If the intervention is widely applicable then in the medium term it could be implemented very rapidly in the UK and beyond.

The health service. If we can improve vaccine efficacy in older adults and other immune compromised groups there will be substantial benefit to the NHS and social services. Older adults make up the bulk of pneumonia and influenza related hospital admissions, they stay longer in hospital and are often frailer after the infection. For many it is the cause of death. So improving vaccination responses would reduce incidence of infection and thus keep more older adults out of hospital and independent for longer.

The vaccine industry. This is perhaps less obvious as there is no IP in a time of day adjustment to protocols. However, if vaccine producers can combine this variation with other novel vaccine components (TLR5 ligands work well in older mice) to improve existing vaccines, or even to revise their interest in vaccines that have not worked well previously but could improve if time of day of vaccination was adjusted. Both could increase vaccine sales or development of new vaccines.