Unisec

WP4 - Work performed since the beginning of the project

Overview work package

Comparative evaluation of different adjuvants combined with whole inactivated virus influenza vaccines

The first phase of the project was devoted to establishing protocols for the side-by-side evaluation of vaccine candidates and adjuvants in the mouse model. We identified that work in the pig-model would go on in parallel with the mouse model work. The best candidates selected from the mouse model study would be discussed for a next testing step in the ferret models. The first experiment for side-by-side comparison of vaccine candidates available within UNISEC focused on comparison of adjuvanted vaccines administered intramuscularly (IM) or intranasally (IN).

All vaccines, irrespective of presence of adjuvant or route of immunization, protected completely from homologous challenge without any significant weight loss or growth of virus in the lungs, as described in the previous report. Differences among the vaccines became apparent, however, after heterologous and particularly heterosubtypic challenge. Overall, the IN administered adjuvanted vaccines provided the best protection, resulting in the lowest amount of weight loss with none of the mice having to be euthanized due to reaching the humane endpoint (20% weight loss). This was in contrast to the other experimental groups in which weight loss was more pronounced and several or all animals had to be euthanized. Interestingly, protection from clinical signs did not necessarily correlate with reduction in lung virus titers, this was particularly obvious in case of the heterologous challenge. Thus, the immune response induced by IN administered adjuvanted vaccines mainly resulted in mitigation of disease symptoms rather than in preventing lung virus growth. Hence, the vaccine protected against disease, albeit it did not necessarily prevent virus growth and infection.

Determination of immune responses revealed that serum antibodies reactive with all 3 challenge viruses were induced by all vaccines but that these antibodies could only neutralize homologous A/PR/8 virus. Mucosal IgA and influenza-specific IFNϒ-producing CD4 T cells were identified as the most likely correlates of protection in the groups IN vaccinated with adjuvanted vaccines.

The two best performing vaccines (IN administered WIV + CAF-09 or CTA1-3M2e-DD) were selected to determine the relevance of mucosal IgA. For this purpose, IgA-/- mice and control wt mice were immunized IN with plain or adjuvanted A/PR/8 WIV, as previously described, and followed by a heterosubtypic X31 H3N2 virus challenge infection. Interestingly, absence of IgA did not affect protection afforded by CTA1-3M2e-DD-adjuvanted vaccine indicating that immune responses other than IgA mediate protection was induced by this vaccine. In contrast, protection afforded by CAF-09-adjuvanted vaccine was impaired, though not abolished, in IgA-/-. This implies that IgA most likely contributes to protection induced by this vaccine formulation, but that other mechanisms  also play a role. Currently, we are investigating the role of CD4 T cells by immunizing mice with the adjuvanted vaccines and depleting CD4 T cells just prior to virus infection.

Lung resident memory CD4 T cells and host protection 

Immunizations with the CTA1-3M2e-DD fusion protein given intranasally have proven to stimulate complete protection against a live challenge infection with heterosubtypic influenza A virus strains. We reported that protection was long lasting (> 1 year) and we observed that M2e-specific memory CD4+ T cells were resident effector memory T cells in the lung. A kinetic study of the transcriptome in tetramer-specific M2e CD4 T cells will be undertaken to collect information about gene expression with a possible correlate to protection. The protective effect was dependent on IL-17A, as evidenced by the loss of roughly 50% of the protective capacity in immunized IL-17A-deficient mice, despite comparable anti-M2e IgG2a antibody levels. Moreover, memory CD4 T cells provided substantial B cell helper functions upon a live challenge infection with not only rapid development of IgG anti-M2e antibodies but also the production of IgG anti-HA-specific antibodies. This suggested that memory CD4 T cells were, indeed, effective helpers of naïve HA-specific B cells, facilitating isotype-switch from IgM to IgG-subclasses. Importantly, the M2e-specific memory T cells, upon transfer to a naïve host, had a direct protective effect on lung virus load after challenge infection.

The side-by-side experiment in the mouse model have been continued (challenge with heterologous and heterosubtypic virus) and extended to encompass also whether protection in the model is provided by IgA and/or CD4 T cells. The studies have provided encouraging results and they have identified three lead adjuvant formulations together with the WiV vaccine. CAF09, CTA1-DD and CTA1-3M2e-DD. These were all intranasal immunization protocols. The adjuvanted vaccine candidates will now enter the next phase and be tested for efficacy in the ferret model.

Comparative evaluation of different adjuvants combined with WIV vaccines.

Following on from mouse studies described earlier, vaccination experiments with WIV vaccine in combination with selected adjuvants have now been planned to proceed in the ferret model.

Evaluation of the DNA vaccine candidate in the ferret model.

The DNA vaccine has been described in more detail earlier in this report, but in summary, the influenza DNA vaccine consisted of plasmids encoding the following six genes: HA and NA A(H1N1)pdm2009, NP and M A(H1N1)pdm1918 and  HA and NA A(H3N2)05/pdm1968. The diluent was Diluvac Forte® and the vaccine was administered via the transdermal route using the IDAL® vaccinator. Preliminary results from  homologous (H1N1pdm) challenge proved promising, therefore a second heterologous (HPAI H5N1) challenge experiment was scheduled and is currently ongoing.

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