1. CAF01 adjuvant increases the protection conferred by a commercially available influenza split vaccine in a ferret model Introduction Desirable traits of cellular immunity to influenza include a speculated cross-protection against heterologous strains and the longer protection it confers over time. Protection relying solely on antibodies, as induced by current inactivated vaccines, is short-lived and falls below effective levels after 6 to 12 months. In contrast, T-cell immunity is considered longer-lived. The ideal vaccine to fight an influenza pandemic would result in both a humoral and a cellular immune response with only one injection of a minimal dose. Adding an adjuvant to the current vaccine to improve its immunogenicity is one of the strategies which have been considered. Recently, a novel cationic liposomal adjuvant (CAF01) was developed based on cationic dimethyldioctadecylammonium, with the immunomodulator glycolipid trehalose 6,6´-dibehenate incorporated into the liposomes. This new adjuvant induces a strong T-cell response as well as production of IgG2 antibodies leading to protection in a range of diseases including malaria, Chlamydia and tuberculosis. In this study, we investigated the effect of CAF01 on the immunogenicity and protection conferred by a commercial split vaccine against influenza in ferrets. We observed that CAF01 led to a reduction in the viral load in nasal washes, as measured by RT-PCR, compared to the commercial vaccine alone. The adjuvant also increased the levels of IgG and IgA antibodies to influenza proteins, as detected by ELISA in nasal washes and serum. The IgG antibodies in serum showed functionality in a hemagglutination inhibition assay. In addition, intracellular staining of interferon-gamma (IFN-g) demonstrated the induction of T-cell responses upon vaccination and challenge. Material and Methods 24 ferrets were immunized twice at two-weeks intervals, then challenged after a month of rest with 10 7 TCID of an homologous H1N1 virus. Vaxigrip: 8 ferrets, 2 x 80 µl commercial vaccine Vaccine + adjuvant group: 8 ferrets, 2 x 80 µl vaccine + 250 µl CAF01 Control PBS group: 8 ferrets, 2 x 250 µl PBS Viral titers in nasal washes were measured by RT-PCR, Influenza-specific antibodies were detected in serum via ELISA using homemade rabbit anti- ferret IgG, PBL positive for intracellular gamma-IFN staining after PMA stimulation were analyzed by flow cytometry. In a similar experiment, 24 ferrets received various doses of influenza vaccine with or without CAF01 and then were challenged with a homologous H1N1 virus. C.J.M. Martel a*, T.H. Jensen a, L.P. Nielsen b, E.M. Agger b, M. Blixenkrone-Møller a, P. Andersen b, B. Aasted a a Department of Veterinary Disease Biology, Faculty of Life Sciences, University of Copenhagen, Stigbøjlen 7, DK-1870 Frederiksberg C, Denmark. b Department of Infectious Disease Immunology, Statens Serum Institut, Copenhagen, Denmark. Abstract The immunogenicity and protective efficacy of current preventive vaccines against influenza are considered suboptimal, and the development of novel effective influenza vaccination strategies is urgently needed. Commercially available trivalent split vaccines are known to elicit mainly a humoral immune response, whereas the induction of cell-mediated immune responses is negligible. Recently, a cationic liposomal adjuvant (CAF01, dimethyldioctadecylammonium/trehalose 6,6’-dibehenate) was developed. In the current study, we compared the immune response in ferrets vaccinated with a commercially available influenza split vaccine with the same vaccine mixed with the CAF01 adjuvant and furthermore used two recently circulating H1N1 viruses for the challenge of the animals. CAF01 improved the immunogenicity of the vaccine, increasing the influenza-specific IgA and IgG levels as well as triggering cellular-mediated immunity, measured by flow cytometry as the production of interferon-gamma by lymphocytes. The adjuvant also enhanced the protection conferred by the vaccine, reducing the viral load measured in nasal washes by RT-PCR. The protection data obtained in the human relevant challenge model supports the potential of CAF01 in future influenza vaccines. Results and Conclusions CAF01 enhances antibody and CMI responses of the split vaccine Two weeks after the first immunization, both the groups that received the non-adjuvanted vaccine and the vaccine with CAF01 showed a significant increase of their vaccine-specific IgG antibodies titers in serum (fig 2A). These increased antibody titers persisted until challenge (week 6), with IgG antibody levels in the CAF01 group being more than 100-fold higher than in the group receiving the vaccine only. Hemagglutination inhibition assay (HAI) was performed in parallel. As shown in figure 2B, the CAF01 group also showed a rise in titre two weeks after the first immunization (fig 2B), whereas no significant increase was induced by the vaccine alone at that time point.To analyse the cell-mediated immune responses, peripheral blood leukocytes were isolated from the blood of immunized ferrets, cultivated for 24 hours with a recombinant H1 hemagglutinin from H1N1 A/New Caledonia/20/99, and then stained for intra-cellular IFN-g. The percentage of IFN-g -positive lymphocytes of the total number of lymphocytes was measured by flow cytometry. At week 6, the group that received CAF01 showed a significant increase in the percentage of IFN-g positive lymphocytes reaching levels of approximately 1.5% positive cells (fig 2C). CAF01-adjuvanted split vaccine confers protection against H1N1 influenza challenge Four weeks after the second immunisation, ferrets received a H1N1 challenge through the nasal cavities. Nasal washes were performed, and the number of viral genome copies in each wash was measured by quantitative RT-PCR (fig 4). As shown in fig 3A, the group that received the vaccine adjuvanted with CAF01 showed a median number of viral copies 10 to 100-fold lower than the group that received the vaccine only and the mock-vaccinated. Figure 3B shows that in another experiment, the viral load was halved at peak replication day in the animals that received vaccine + CAF01 compared to those which received the vaccine alone. Figure 3C shows the percentage of animals excreting virus during the first week of the challenge in each group (cumulative results of three similar experiments). Only half of the animals which received the CAF01 and the vaccine together had detectable viral copies in their nasal washes at day 3 and 4 whereas all animals in the two other groups were found to excrete the virus at day 4. The animals having received the vaccine + CAF01 combination also completely cleared the virus by day 5 post challenge, 24 hours earlier than the group that received the conventional vaccine only. CAF01 allows for a reduction in vaccine dose Groups of four ferrets received two intramuscular injections of Sanofi- Pasteur’s 2008-2009 Vaxigrip vaccine at a two week interval, containing either 15 µg, 1.5 µg or 0.15 µg of HA from A/Brisbane/59/2007 (H1N1). Two weeks after the first immunization, all three groups that received the vaccine with the adjuvant displayed similar, noticeable levels of antibodies (fig 4A) significantly higher in the groups that received CAF01.The hemagglutination inhibition assay confirmed this observation (fig 4B). There was a trend indicating that the ferrets that received the adjuvant seemed to develop low levels of nasal IgA (fig 4C). At day 3, there was a tendency towards diminished viral titers in groups that received the vaccine adjuvanted with CAF01 compared to the groups that received the same dose of vaccine alone (fig 4D). This tendency turned out to be significant at day 4 for all three doses tested. More strikingly, at the 0.15 µg dose adjuvanted with CAF01, the reduction in viral load was the same observed with the unadjuvanted 15 µg dose. With the 15 µg dose, the adjuvant led to a 100-fold reduction of the viral load compared to the same dose of vaccine without CAF01. Figure 2. Immune response to vaccine antigens. Ferrets were immunized twice at two week-intervals with CAF01 adjuvanted and non-adjuvanted influenza vaccine (n=8). A. Serum IgG antibodies measured by IgG specific ELISA at various timepoints. The titer was defined as the highest positive dilution. B. Hemagglutination inhibition assay using influenza A (H1N1) New Caledonia 1999. C. Percentage of IFNg positive lymphocytes at week after 24 hours of stimulation in cell culture with a recombinant H1 hemagglutinin Peripheral blood leucocytes were isolated and stimulated overnight with a recombinant H1 hemagglutinin from A/New Caledonia/20/99. Left plots: Gating on lymphocyte population. Right plots: Gating on IFN-g-positive lymphocytes. Figure 1. Set-up of the experiments. Overview of the total experiment, from the first immunization of the animal to its euthanasia, including blood sampling, challenge inoculation and immunizations. Figure 3. Viral excretion. 6 to 10 months-old ferrets were immunized twice at two week-intervals with CAF01 adjuvanted and non-adjuvanted influenza vaccine, then challenged with 10 7 TCID 50 of A/New Caledonia/20/99 (H1N1) four weeks after the second immunization (n=8). A. Number of viral copies found in nasal washes of infected animals during the first five days of challenge, measured by quantitative RT- PCR. B. Number of copies found in nasal washes of individual infected animals at peak replication day (day 3), measured by quantitative RT-PCR. C. Percentage of animals excreting the virus in their nasal washes during the challenge period (cumulative results of 3 similar experiments, for a total of 24 animals in each group). Figure 4. A/Brisbane/59/2007 study. 6 to 10 months-old ferrets were immunized twice at two week- intervals with CAF01 adjuvanted and non-adjuvanted influenza vaccine, then challenged with 10 7 TCID 50 of A/Brisbane/59/2007 (H1N1) four weeks after the second immunization (n=4). A. Vaccine- specific serum IgG titers measured in serum by ELISA. B. Hemagglutination inhibition assay serum titers using influenza A/Brisbane/59/2007. C. Vaccine-specific IgA titers measured in nasal washes by ELISA. D. Number of viral copies found in nasal washes of individual infected animals at peak replication days (day 3 and 4), measured by quantitative RT-PCR. C.