Superior Immunogenicity of Inactivated Whole Virus H5N1 Influenza Vaccine is Primarily Controlled by Toll-like Receptor Signalling

In the case of an influenza pandemic, the current global influenza vaccine production capacity will be unable to meet the demand for billions of vaccine doses. The ongoing threat of an H5N1 pandemic therefore urges the development of highly immunogenic, dose-sparing vaccine formulations. In unprimed individuals, inactivated whole virus (WIV) vaccines are more immunogenic and induce protective antibody responses at a lower antigen dose than other formulations like split virus (SV) or subunit (SU) vaccines. The reason for this discrepancy in immunogenicity is a long-standing enigma. Here, we show that stimulation of Toll-like receptors (TLRs) of the innate immune system, in particular stimulation of TLR7, by H5N1 WIV vaccine is the prime determinant of the greater magnitude and Th1 polarization of the WIV-induced immune response, as compared to SV- or SU-induced responses. This TLR dependency largely explains the relative loss of immunogenicity in SV and SU vaccines. The natural pathogen-associated molecular pattern (PAMP) recognized by TLR7 is viral genomic ssRNA. Processing of whole virus particles into SV or SU vaccines destroys the integrity of the viral particle and leaves the viral RNA prone to degradation or involves its active removal. Our results show for a classic vaccine that the acquired immune response evoked by vaccination can be enhanced and steered by the innate immune system, which is triggered by interaction of an intrinsic vaccine component with a pattern recognition receptor (PRR). The insights presented here may be used to further improve the immune-stimulatory and dose-sparing properties of classic influenza vaccine formulations such as WIV, and will facilitate the development of new, even more powerful vaccines to face the next influenza pandemic

Influenza Vaccines:What Do We Want and How Can We Get It?

Influenza vaccines have been in use for more than 60 years and have proven to be efficacious in protecting from influenza infections during epidemics and the recent H1N1 pandemic. The development of influenza vaccines has so far been largely based on empirical grounds, which leaves room for vaccine improvement by implementation of recent insights in innate and adaptive immunity. Also, evaluation and approval of new vaccines rely on rather broad correlates of protection such as the hemagglutination inhibition titre, thereby neglecting qualitative aspects of the immune response. Here we discuss how current inactivated influenza vaccine formulations differ in the type of immune response they elcit, their protective capacity, and what causes these differences. Finally, we will discuss how this knowledge can guide the development of new adjuvants that optimize the protective efficacy of influenza vaccines.</p

Development of influenza vaccines in the face of pandemic threat

Het influenza virus is de verwekker van de griep en veroorzaakt in de wintermaanden epidemieen op het noordelijk halfrond. Sporadisch veroorzaakt het influenza virus een pandemie, waarbij het virus zich binnen korte tijd wereldwijd verspreidt met extra ziekte-en sterftegevaIIen tot gevoIg. Tijdens de 'Spaanse griep' pandemie van 1918 zijn naar schatting tussen 50 en 100 miljoen mensen omgekomen. Het ontstaan van een pandemie hangt samen met de introductie van een nieuw subtype influenza virus vanuit het dierenrijk in de menselijke populatie. Er bestaat dan nog geen immuniteit tegen dit nieuwe virus, waardoor het zich snel kan verspreiden. Het is onvoorspelbaar wanneer zo'n introductie plaats gaat vinden, en welk subtype influenza virus dit zal betreffen, maar dat het in de toekomst weer een keer gaat gebeuren is zeer waarschijnlijk. De uitbraken van het dodelijke H5N1 vogelgriep virus onder pluimvee in Zuidoost-Azie, waarbij ook mensen gelnfecteerd raakten, vormden een directe pandemische dreiging. De eerste uitbraak vond plaats in 1997. Sinds 2003 is het virus echter niet meer weggeweest en heeft zich in die tijd verspreid over meerdere continenten. De wereld gezondheidsorganisatie (WHO) hanteert nog steeds een verhoogde alarmfase, een stap onder het niveau waarbij overdraagbaarheid van mens naar mens gesignaleerd wordt. Tot nu overleed 60~, van de mensen met een bewezen H5N1 infectie. Als er een air-borne H5Nl virus ontstaat dat van mens naar mens overdraagbaar is -dit kan dotr mutaties van het virus of door vermenging (reassortment) met een menselijk influenza virus-kan dit tot een levensgevaarlijke pandemie lei den. Vaccinatie biedt de beste bescherming tegen infectie met het influenza virus. Echter, het ontwikkelen van een vaccin voor een eerstvolgende pandemie wordt bemoeilijkt door tal van zaken. Hieronder vallen de onzekerheid over de exacte identiteit (subtype) van het nieuwe pandemische virus, de onbekendheid van het tijdstip waarop de pandemie ontstaat, en de snelle wereldwijde verspreiding van het virus. Daamaast is de totale vaccin productiecapaciteit gelimiteerd en zijn de productiefaciliteiten gesitueerd in met name gei'ndustrialiseerde landen. Tijdens een pandemie kan daarom niet iedereen ter wereld tijdig over vaccins beschikken. Het vergroten van de beschikbaarheid van vaccins is dan ook een van de grote uitdagingen voor vaccinonderzoekers, In principe zouden door een verkleining van de benodigde dosis per vaccin, meer vaccindoses in dezelfde tijd geproduceerd kunnen worden, The continuous threat ot a new influenza pandemic urges the development of optimally effective vaccines for better pandemic preparedness. Many challenges accompany vaccine development for pandemics. These relate to factors such as the uncertainty of the timing and causative virus subtype of a next pandemic, the speed of global spreading of the virus, and limitations in the vaccine production capacity. One important challenge is the accomplishment of a dose-reduction to diminish the anticipated vaccine shortage. This may be achieved by use of a vaccine formulation that induces stronger and better immune responses than those generally induced by seasonal flu vaccines. Developing a completely new and better formulation is a risky and time-consuming process, while the threat of an H5Nl pandemic required immediate pandemic preparedness. We therefore set out to identify -among the non-adjuvanted vaccine formulations available for clinical use-the best formulation in terms of the quantity and quality of the induced antibody response and to clarify its working mechanism. We tried to further increase vaccine immunogenicity by adding an adjuvant, and to improve vaccine stability by freeze-drying, for storage in anticipation of a pandemic. In a first set of experiments, we compared classic influenza vaccine formulations, in use for vaccination against seasonal influenza, in a head-to-head immunization trial in mice. Inactivated whole virus vaccine (WIV) was superior in inducing virus-specific antibodies, as compared to split-virus (SV), subunit (SU) or virosomal (VS) vaccine. The antibody response to WIV correJctted with better neutralization of the virus in vitro (Chapter 2 and 3). Also, WIV induced a Thl type immune response, characterized by high levels of IgG2aI c subtype antibodies in mice, in contrast to SV, SU or VS vaccine. The latter vaccines all induced a Th2-type response, in which IgGl antibodies dominate. The presence of IgG2a I c correlates with better protection against a viral challenge than the presence of IgGl antibodies, according to literature and also to our own experience (see Chapter 5). Inbred mice used in our experiments are genetically almost identical. We showed that wrv consistently induced a Thl-type response in two different inbred mouse strains with genetic predisposition for an antibodydominated immuno-phenotype (Balb I c) and a cell-dominated immunophenotype (C57BI/ 6), respectively. This holds promise that wrv might induce a similar response type in an outbred population, where more genetic variation exists (Chapter 2). Taken together, in naIve mice WIV proved superior in terms of magnitude and phenotype of the induced immune response.

Influenza Vaccines: What Do We Want and How Can We Get It?

Influenza vaccines have been in use for more than 60 years and have proven to be efficacious in protecting from influenza infections during epidemics and the recent H1N1 pandemic. The development of influenza vaccines has so far been largely based on empirical grounds, which leaves room for vaccine improvement by implementation of recent insights in innate and adaptive immunity. Also, evaluation and approval of new vaccines rely on rather broad correlates of protection such as the hemagglutination inhibition titre, thereby neglecting qualitative aspects of the immune response. Here we discuss how current inactivated influenza vaccine formulations differ in the type of immune response they elcit, their protective capacity, and what causes these differences. Finally, we will discuss how this knowledge can guide the development of new adjuvants that optimize the protective efficacy of influenza vaccines