Longitudinal follow-up of the new influenza A/H1N1 virus infection in vivo and in vitro in healthy volunteers

Influenza is a very common virus that causes considerable morbidity and
mortality in the worldwide population on a yearly basis. Moreover, it has a
perceived pandemic potential, illustrated recently by the pandemic of the new
influenza A/H1N1 virus. (1, 2) Although there is abundant knowledge on the
make-up of the influenza virus (3), many facets of the human response against
this virus remain unknown. Gaining knowledge on these responses will help us in
treating influenza infections and lead to more effective vaccines.
Additionally, it might also lead to identification of individuals that have a
high risk of morbidity and even mortality during infection.

Up till now, research of influenza responses has mainly focused on antibody
responses. This has a practical background, as antibodies can easily be
measured in blood, but also as it is thought that antibody responses are the
most important mechanism by which the immune system combats influenza viruses.
This notion is supported by successful (antibody) protection following
vaccination.
Recently, research also turned to the role of T cells in influenza infections
as they are the primary responding cells during infection, especially in the
absence of protective antibody titers. Moreover, it was stated that T-cell
responses have a better predictive value for influenza protection than antibody
titers (4). Therefore, T-cells might play a more prominent role than
anticipated so far.

However, investigation of T-cell responses, but also of B-cell responses is
hampered by limitations. Due to the huge repertoire of T- and B-cell receptors
in the human body, it is very difficult to identify (and follow) individual T-
and B-cell responses. Recently, a new technique was developed within our
department which is able to find and follow individual clonal responses without
having any prior knowledge of what these clones look like (5). By combining
this technique with recently developed in-vitro stimulation assays, we are for
the first time able to identify clones which play a functional role in
influenza infections and follow them over time.

This project is embedded in a new research line that has been set-up in the
AMC. This research line is a collaboration between the clinical immunology and
rheumatology department, the lab of genome analysis and multiple partners in
immunology and infectious diseases (e.g. Prof. Dr. R. van Lier/Prof. Dr. I. ten
Berge/ Prof.Dr. U. Beuers). We feel this study will not only give us insight
in the role of T- and B-cells during viral infection, it can also lead us to a
better understanding of the pathology of infectious diseases (such as acute
viral infections and auto-immune disease).

In this study we will for the first time combine in-vitro stimulation assays
and high throughput sequencing to identify, quantify and phenotype the clones
that play a functional role in influenza infections and follow them over time.
This will lead to the identification and characterization of functional clones.

We want to study otherwise healthy volunteers undergoing new influenza A/H1N1
infection. Since it is very likely that the H1N1 pandemic is returning to the
Netherlands in the spring of 2010, we expect to be able to recruit our
volunteers through GP practices throughout Amsterdam. They will be seen at the
AMC hospital.

This is a longitudinal study in which we want to investigate the effect of new
influenza A/H1N1 virus on the humoral immune system. We will look for
individual clonal responses in both the T-cell as well as the B-cell
compartment and follow these cell clones over time.

The study will consist of eight visits. In the first visit, the volunteers will
be included with confirmed H1N1 infection. This will be confirmed by a routine
hospital test (PCR). In all visits we will note relevant clinical parameters
and draw blood.

Taken Amount
Purpose Time points
Non-heparinised blood (PAXGene) 2 mL - Full repertoire
analysis (T/B cell) Day 0-4


Day 7


Day 14


Day 28


Day 56


Day 84


Day 112


Day 140
Heparinised blood 70-80 mL - Cellular
subset analysis (T/B cell) Day 7

- In vitro stimulation assays Day 84
Serum 3
mL - HI assay

Total amount of blood drawn per individual: 70-80ml (2x) + 2ml (8x) = 156*176ml

The following procedures will be performed:
Serum will be separated from whole blood for start of symptoms
PBMC will be isolated from heparinised blood according to standard operating
procedures.
* PBMC will be used to look for individual B- and T-cell clones in specific
subsets during acute infection and the formation of memory.
* PBMC will be used to study the effects of stimulation with the new influenza
A/H1N1 virus on B- and T-cells and the formation of functional clones.

Non-heparinised blood * collected in PAXGene Blood RNA tubes * will be isolated
according to standard operating procedures.
* RNA will be used to look for individual B- and T-cell clones in peripheral
blood during acute infection and the formation of memory.

Hemagglutination-inhibition (HI) assay will be performed on serum.
* Antibody titers will be determined to quantify the presence of specific
antigens.

In total, 160-180mL of blood is drawn.