OA17719A

OA17719A - Acellular pertussis vaccine.

Info

Publication number: OA17719A
Application number: OA1201500359
Authority: OA
Inventors: Jan Theunis Poolman
Original assignee: Crucell Holland B.V.
Priority date: 2013-03-08
Filing date: 2014-03-06
Publication date: 2017-10-11

Abstract

The invention relates to an acellular pertussis (aP) vaccine composition comprising Bordetella pertussis antigens pertussis toxoid (PT), filamentous hemagglutinin (FHA), and fimbriae types 2 and 3 (FIM), and optionally pertactin (PRN), wherein FIM is present in an amount of 12-100 ug per human dose.

Description

Background of the invention

Bordetella pertussis is the causative agent of whooping cough. Introduction of killed whole-cell B. pertussis (wP) vaccines in the 1940s has been successful in reducing the morbidity and mortality due to whooping cough in children and infants [http;//www.cdc.gov/vaccines/pubs/pinkbook/downloads/pert.pdf; Bisgard, K.M. http://www.cdc.gov/vaccines/pubs/pertussisguide/downloads^/chapterl.pdf. 2000; Edwards. K.M. & Decker, M.D. Pertussis vaccines. In Vaccines (Eds. Plotkin, S.A., Orenstein, W.A. & Offit, P.A.) Elsevier Health Sciences. 2008. 467-517; this textbook is hereinafter referred to as Vaccines. Plotkin 2008]- Nevertheless, worldwide, pertussis remains an important problem in children. Estimâtes from the WHO suggest that in 2008 about 16 million cases of pertussis occurred, and that about 195,000 children died from this disease.

Since the 1990s wP vaccines hâve been replaced by acellular pertussis (aP) vaccines in most high-income and more recently also in some middie-mcome countries. Acellular pertussis vaccines induce relatively fewer side-effects compared to wP vaccines that are associated with a high risk for fever (> 3 8°C). reactogenicit) at the injection site and, although to a lesser estent, convulsions and hypotonichyporesponsive épisodes [Zhang, Cochrane Database Syst Rev 2011],

One to two décades after introducing aP vaccines, a rise in pertussis notifications in adolescents and adults has been reported by several countries, including the US, UK, Australia, Norway and the Netherlands. Possible explanations include improved diagnostics and surveillance, adaptation of circulating B. pertussis strains to vaccines, and/or increased waning immunity associated with aP vaccines [Tanaka, Jama 2003, 290; 2968-2975; Satoh, Comp Immtinol Microbiol Infect Dis 2010, 33; e81 -88; Zepp, Lancet Infect Dis 2011 ; De Greeff, PLoS One 2010, : e!4183; Tan, Pediatr Infect Dis 7 2005, 24(5 Suppl); S10-18],

Currently, ail licensed aP vaccines consist of minimal one, but mostly multiple, up to a maximum of five (detoxified) B. pertussis virulence factors. Ail aP vaccines contain pertussis toxoid (PT). Multicomponent aP vaccines at least include

PT and the B. pertussis surface adhesin fîlamentous hemagglutinin (FHA). With increasing valency further one or more of the adhesins pertactin (PRN) and fimbriae type 2 and type 3 (FIM2 and FIM 3, together referred to as FIM or FIM2/3 herein) are présent [Edwards, In: Vaccines, Plotkin, 2008. 467-517].

WO 96/34883 describes doses of 1-10 pg of FIM per human dose, with doses of 10 and 5 pg per human dose in an aP vaccine exemplified, while only doses of 5 pg per human dose were actually tested, and the tested vaccines were considered efficacious.

It is generally believed that aP5 vaccines (acellular pertussis vaccines with the five components PT, FHA, PRN, FIM2/3; DTaP5 are aP5 vaccines further comprising tetanus toxoid and diphtheria toxoid) are the most effective aP vaccines currently available. The individual amounts of the aP components présent in commercially available registered aP5 vaccines are (in microgram per human dose): 2.5-20 for PT,

5-20 for FHA, 3 for PRN and 5 for FIM.

In several of the acellular pertussis vaccine efficacy trials conducted in Europe in the mid-1990s, efforts were made to détermine immune correlates of protection for the individual aP vaccine components. Using data from the Swedish DTaP5 (PT+FHA+PRN+FIM2/3) trial a statistically significant corrélation between clinical protection and the presence in pre-exposure sera of IgG antibodies against PRN, FIM2 and PT, but not to FHA, were demonstrated [Storsaeter, Vaccine 1998, 16: 19071916]. FIM3 appears to be a nonprotective component within DTaP5 [Poolman, Expert Reviews Vaccines 2007, 6: 47-56].

Sera collected from subjects from a vaccine trial in Germany allowed estimation of the spécifie levels of antibody to PT, FHA, PRN and FIM2 that correlated with protection, which showed that only antibodies against PRN, and PT were signifîcantly associated with protection [Stehr, 1998, Pediatrics 101: 1-11; Cherry, 1998, Vaccine 16: 1901-1906]. In addition, pre-clinical studies hâve shown that the addition of PRN enhances the level of protection conferred by vaccines that contain PT and FHA in a murine intranasal infection model [Guiso, 1999, Vaccine 17:

2366-2376; DeNoel, 2005, Vaccine 23: 5333-5341] and that antibodies to PRN were crucial for opsonophagocytosis of B. pertussis [Hellwig, 2003, JID 188: 738-742], Together these data indicate that PT and PRN are the main protective antigens in current acellular pertussis vaccines.

As part of a prospective aP vaccine efficacy trial, protective IgG against PT, t

FHA, PRN and FIM2/3 was measured in consecutive sérum samples obtained from participants over an 18-month period. Over the 18-months the percent decay in IgG against PT was strongest (73% réduction in géométrie mean IgG titer) and was significantly higher than the percent réduction in antibodies against PRN, FHA and FIM. In contrast, IgG antibody to PRN had the lowest decay rate (56% réduction in géométrie mean IgG titer) [Le, 2004, JID, 190: 535-544].

Combining the two observations that 1) PT and PRN are the main protective antigens in aP vaccines and 2) that antibodies to PT hâve a significantly higher decay 10 rate than antibodies to PRN, highlights that anti-PRN antibodies are crucial in providing aP-mediated long-term protection against B. pertussis infection.

However, an emergence of B. pertussis strains not expressing PRN has been observed in the last few years around the world, for example in France, Japan, the Netherlands, the USA, Finland, Norway and Sweden [Bouchez, 2009, Vaccine 27:

6034-41; Hegerle, 2012, Clin. Microbiol. Infect. 18: E340-346; Otsuka, 2012, PloS

One 7: e31985; Advani, 2013, J. Clin. Micro 51: 422-428]. A recent study in the US showed that 11 out of 12 isolâtes of B. pertussis cultured from specimens from children hospitalized in Philadelphia during 2011 and 2012 were in faci PRN-négative [Queenan, 2013, N Engl J Med. 368: 583-4]. Whether this strain adaptation is primarily vaccine-driven is currently not known. It is possible that these PRNnegative strains can escape vaccine induced immunity, especially when anti-PT titers hâve declined, and that this has contributed to the observed increase in B. pertussis disease.

The currently licensed and marketed aP vaccines thus appear insufficiently efficacious, especially against the newly emerging PRN-negative strains. There remains a need for further aP vaccines, that hâve improved efficacy in particular against such PRN-negative B. pertussis strains.

Summary of the invention

We hâve surprisingly found that aP vaccines comprising a high dose of FIM2/3 shows a significantly improved protection against PRN-negative B. pertussis strains. The invention provides an acellular pertussis (aP) vaccine composition comprising B. pertussis antigens pertussis toxoid (PT), filamentous hemagglutinin (FHA), and fimbriae types 2 and 3 (FIM), and optionally pertactin (PRN), wherein FIM is présent in an amount of 12-100 pg per human dose.

In certain embodiments, FIM is présent in an amount of 15-80, 15-60, 20-60, or 12-50, 15-50, 20-50, 20-30, 20-25, or 12-30, or 12-25, or about 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 pg per human dose.

In preferred embodiments, the aP vaccines of the invention comprise pertactin (PRN).

In certain embodiments, PT is genetically detoxified.

In certain embodiments, the compositions of the invention further comprise antigens from one or more pathogens other than B. pertussis.

In certain embodiments thereof, the compositions comprise one or more of:

a) tetanus toxoid; b) diphtheria toxoid; c) Haemophilus influenzae type-b (Hib) oligosaccharide or polysaccharide conjugate: d) hepatitis B virus surface antigen (HBSAg); and e) inactivated polio virus (IPV). In certain embodiments, the compostions of the invention comprise an acellular pertussis vaccine composition according to the invention, tetanus toxoid and diphtheria toxoid.

In certain embodiments, the compositions according to the invention further comprise an adjuvant. In certain embodiments, the adjuvant comprises an aluminium sait, such as aluminium phosphate, aluminium hydroxide, or both aluminium phosphate and aluminium hydroxide.

The invention also provides a method for inducing an immune response in, or for vaccinating, a subject against B. pertussis, comprising administering to the subject a composition according to the invention.

The invention also provides a method for protecting a subject from whooping cough that is caused by infection with a PRN-negative strain of B. pertussis, comprising administering to the subject a composition according to the invention.

The invention also provides the compositions according to the invention for use in inducing an immune response in, or for vaccinating, a subject against B. pertussis by administering the composition to the subject. The invention also provides the compositions according to the invention for the préparation of a médicament for inducing an immune response in, or for vaccinating, a subject against B. pertussis by administering the composition to the subject. In certain embodiments, the B.pertussis against which an immune response is desired comprises a PRN-negative strain.

The invention also pro vides a method for vaccinating a human subject against Bordetella pertussis, optionally a PRN-negative strain of Bordetella pertussis, comprising administering to the subject the following Bordetella pertussis antigens: pertussis toxoid (PT), filamentous hemagglutinin (FHA), and fimbriae types 2 and 3 (FIM), and optionally pertactin (PRN), wherein FIM is administered in an amount of 12-100 pg.

Brief description of the Figures

Fig. 1. Mean loglO CFU of B.pertussis per lung on day 0, 2, 5 and 8 post intranasal challenge in mice vaccinated with a DTaP5 vaccine (aP5) or aP5 + FIM. Mice were vaccinated at 4 and 7 weeks of âge and challenged intranasally at 9 weeks with different strains of B. pertussis'. WHO 18323 (Fig. IA), a clinical PRN-negative isolate PRN-STOP (Fig. IB) and a clinical PRN-negative isolate PRN-IS (Fig. IC).

* p<0.05 comparing the mean logl 0 CFU counts after vaccination with aP5 and aP5 +

FIM on each spécifie time point, using Wilcoxon Exact Test.

#p<0.01 comparing the loglO CFU response-time profile from day 0 to day 8, after vaccination with aP5 and aP5 + FIM using Analysis of Covariance.

Fig. 2. Mean LoglO CFU counts from the lung in mice challenged with PRN-negative B. pertussis strain 1195 at week 9 after vaccination with aP2 vaccine at 1/10 human dose with the addition of an increasing amount of FIM at 4 and 7 weeks of âge. For details see example 2.

Fig. 3. Mean LoglO CFU counts from the lung in mice 5 days post challenge with 4 strains of B. pertussis at week 9 after vaccination with aP5 vaccine at 1/10 human dose with or without the addition of 5 pg FIM at 4 and 7 weeks of âge. For details see example 3.

Detailed description of the mventioa _

The invention pertains to compositions comprising detoxified Bordetella pertussis virulence factors, in particular pertussis toxoid (PT), filamentous hemagglutinin (FHA), and fimbriae types 2 and 3 (FIM), and optionally pertactin (PRN), wherein FIM is présent in an amount of 12-100 pg per human dose. Such compositions can be used as an acellular Pertussis (aP) vaccine, and are demonstrated herein to be surprisingly more efficacious than a currently available best-in-class aP5 vaccine.

In particular, the increased dose of FIM as compared to the previously described and recommended dosage for this component results in improved vaccine efficacy against newly emerging PRN-negative strains of B. pertussis, as compared to a marketed aP5 vaccine and other marketed aP vaccines. This is highly surprising, as the prior art does not provide any suggestions to increase the amounts of FIM per human dose over the recommended 5 pg, and the art provides actually suggestions that increasing the amount of this component does not lead to improved efficacy.

The effect of an increased dosage of FIM in vaccines has for instance been investigated in humans and animal models. In 17-19 month old infants it was shown that there were no différences in the ffequency of adverse reactions, antibody titers or in mean fold titer rise post-immunization between two 5-component aP vaccines with either 10 pg of PT, 5 pg of FHA, 3 pg of PRN and 5 pg of FIM2/3 or including double the amount of ail these antigens (Halperin, Arch Pedi Adol Med 1994, 148: 1220-1224).

In animal models it was found that there were no différences in body weight, spleen weight, peripheral leukocyte counts and clearance of B. pertussis from the lungs, when mice were vaccinated with three kinds of pertussis vaccines that contained different amounts of FIM (Morokuma, Devel Biol Stand 1991, 73: 223232).

Another study showed there was no différence in IgG antibody level between mice receiving either a high dose (20 pg) or a low dose (4 pg) of recombinant FIM2 or FIM3. Seven days post intranasal challenge there was no différence in bacterial loads in the lung of control mice, mice vaccinated with FIM2 or mice vaccinated with FIM3 (Xu, BMC Microbiology 2009, 9:274-281).

Our finding of increased efficacy of vaccination using a high dose of FIM in the mouse nasopharyngeal challenge model is therefore highly surprising.

Thus, there are no indications or suggestions in the prior art that increasing the amount of FIM in aP vaccines over the usual amount would resuit in improved efficacy against newly emerging PRN-negative mutant strains.

A “human dose” as used herein (sometimes referred to as a “single human dose”), means an amount of vaccine that is administered to a human in a single administration. Typically, this amount is présent in a volume of 0.1-2 ml, e.g. 0.2-1 ml, typically 0.5 ml. The indicated amounts may thus for instance be présent at a concentration of micrograms per 0.5 ml bulk vaccine. In certain embodiments a (single) human dose thus equals 0.5 ml.

The components of several aP vaccines that are or hâve been marketed are described in Tables 21-3 and 21-4 of “Vaccines. 5th édition. S. Plotkin, W. Orenstein, P. Offit, 2008, Section 2, Chapter 21 “Pertussis vaccines”, K.M. Edwards & M.D. Decker. p. 467-517, incorporated by reference herein. The aP vaccine compositions of the invention comprise PT, FHA, and FIM2/3, and preferably PRN. These components are standard components in various marketed aP vaccines, and are available from different manufacturera (see, e.g. Table 21-3 of Chapter 21 of Vaccines, Plotkin 2008), and are for instance commercially available from List Biological Laboratories, Inc (Campbell, California). The compositions of the invention comprise detoxified pertussis toxin, also known as pertussis toxoid (PT). PT can be chemically or genetically detoxified. Chemical détoxification can for instance be performed by any of a variety of conventional chemical détoxification methods, such as treatment with formaldéhyde, hydrogen peroxide, tetranitromethane, or glutaraldehyde. For instance, détoxification can be performed as described on page 17 and example 3 of WO 96/34883, incorporated by reference herein. In certain preferred embodiments, PT is genetically detoxified. This can be done by making mutations in the pertussis toxin gene to inactivate the enzymatic activity of the catalytic subunit S1 of pertussis toxin, and has for instance been described in US 7,144,576, US 7,666,436, and US 7,427,404. Particularly advantageous mutations to detoxify pertussis toxin are for instance provided in US 7,427,404, incorporated by reference herein. A particularly advantageous embodiment is pertussis toxin wherein the amino acid residue Glu 129 in thepertussis holotoxin amino acid sequence in the SI subunit is substituted by Gly (E129G) and Arg9 is substituted by Lys (R9K) (US patent 7,427,404; Buasri, BMC Microbiology 2012,12: 61). Such genetically detoxified PT (E129G, R9K) can also be conveniently isolated from a genetically engineered strain that shows enhanced production of this PT [Buasri, 2012, supra]. Advantages of using genetically detoxified mutants are that no or less use of hazardous chemicals is required for détoxification, improved préservation of the epitopes of the PT antigens and thus better immune responses thereto, and/or lower amounts of antigen can be used in the vaccine. In other embodiments, PT is chemically detoxified. Chemically or genetically detoxified PT is widely used in aP vaccines (see, e.g. Table 21-3 of Chapter 21 of Vaccines, Plotkin 2008). PT can for instance be obtained and purified as described in page 16 and example 2 of WO 96/34883, incorporated by reference herein. PT can also be obtained using methods as e.g. described in US5085862, WO96/34623, US4705868, EP0336736, WO9115505, EP0306318, EP0322533, EP0396964, EP0275689, W091/12020, EP0427462, WO9819702 and US4784589, each of which is incorporated by reference herein.

Chemically or genetically detoxified PT is available from various commercial sources. In certain embodiments, the amount of PT in a vaccine according to the invention is 2-50 pg, 5-40 pg, 10-30 pg, or 20-25 pg per human dose (typically 0.5 ml).

The compositions according to the invention in certain embodiments comprise pertactin (PRN), a 69 kD outer membrane protein of B. pertussis (therefore sometimes also referred to as 69K antigen). PRN can for instance be obtained and purified as described on page 18-19 and example 2 of WO 96/34883, incorporated by reference herein. It can also for instance be obtained as described in EP0162639, EP0484621, US6444211, US5276142, US5101014, EP0336736, WO96/34623, WO90/16651, and W090/56076, ail incorporated by reference herein. PRN can also be conveniently isolated from B.pertussis strains that hâve been genetically engineered to express high levels of PRN, such as described for instance in [Buasri, 2012, supra}.

In certain embodiments, the amount of PRN in a vaccine according to the invention is 0.5-100 pg, 1-50 pg, 2-20 pg, 3-30 pg, 5-20 pg, or 6-10 pg per human dose (typically 0.5 ml) [see e.g. EP 0928198].

The compositions according to the invention comprise filamentous hemagglutinin (FHA), a major 230-kDa adhesin of B. pertussis that is important for the adhérence of B. pertussis to the host ciliary épithélial cells of the respiratory tract, and an established component of marketed multivalent aP vaccines. FHA can for instance be obtained and purified as described in page 17-18 and example 2 of WO 96/34883, incorporated by reference herein. FHA can also for instance be obtained as described inWO9013313, EP0484621, WO963 4623, EP0336736, WO9115505, US4784589, and W09004641, ail incorporated by reference herein.

In certain embodiments, the amount of FHA in a vaccine according to the invention is 2-50 gg, 5-40 gg, 10-30 gg, or 20-25 gg per human dose (typically 0.5 ml).

The compositions according to the invention comprise fimbrial agglutinogens

2 and 3, also referred to as fimbriae 2 and 3 or agglutinogens 2 and 3 or Agg 2 and 3 (herein referred to as FIM2 and FIM3, or as “FIM” or “FIM2/3”, which is a combination of FIM2 and FIM3 as a mix). Typically, in a composition according to the invention, the weight ratio of FIM 2 to FIM 3 is from about 1:3 to about 3:1, e.g. from about 1:1 to about 3:1, e.g. from about 1.5:1 to about 2:1. Préparation of FIM is described in detail in page 12-13 and example 2 of WO 96/34883, incorporated by reference herein. FIM can also for instance be obtained as described in WO9634623, US4784589, US6475754, EP0555894, WO9858668, and W00207764, ail incorporated by reference herein.

The amount of FIM in a vaccine according to the invention is 12-100 gg per human dose (typically 0.5 ml). In certain embodiments, this amount is 12-50 pg, or 12-30 gg per human dose (typically 0.5 ml). In preferred embodiments of the invention, the amount of FIM is at least 15 ggper human dose (typically 0.5 ml). In certain embodiments this amount is 15-100 gg, 15-80 gg, 15-60 gg, 15-50 gg, 15-30 gg or 15-25 gg per human dose (typically 0.5 ml). In iurther preferred embodiments of the invention, the amount of FIM is at least 20 gg per human dose (typically 0.5 ml). In certain embodiments this amount is 20-100 gg, 20-80 gg, 20-60 gg, 20-50 gg, 20-30 gg, 20-25 gg, or 25-50 gg per human dose (typically 0.5 ml).

FIM can be isolated from B. pertussis, or can be recombinantly produced, or is for instance commercially available from List Biological Laboratories, Inc (Campbell, 25 California; http://www.listlabs.com, product # 186 in online catalog).

In certain preferred embodiments, a vaccine composition according to the invention comprises per human dose (or per 0.5 ml bulk vaccine): 10-25 gg PT, 10-25 gg FHA, 3-8 gg PRN, and 12-50 gg FIM. In certain preferred embodiments, a vaccine composition according to the invention comprises per human dose (or per 0.5 30 ml bulk vaccine): 20-25 pg PT, 20-25 gg FHA, 3-8 gg PRN, and 12-50 gg FIM. In certain preferred embodiments, a vaccine composition according to the invention comprises per human dose (or per 0.5 ml bulk vaccine): 20-25 gg PT, 20-25 gg FHA,

3-8 gg PRN, and 12-25 gg FIM. In a certain embodiment, the invention provides a vaccine comprising per human dose (or per 0.5 ml bulk vaccine): about 20 pg PT, about 20 pg FHA, about 3 pg PRN, and about 15-20 pg FIM.

One simple way of preparing a vaccine according to the invention is to add commercially available FIM to a commercially available aP5 vaccine. A vaccine of 5 the invention can also be simply prepared by adding FIM to a commercially available aP vaccine that comprises PT and FHA but not yet FIM (aP2), or a commercially available aP vaccine that comprises PT, FHA and PRN but not yet FIM (aP3). In certain embodiments, the FIM may first be adsorbed to an adjuvant if so desired, e.g. to aluminium hydroxide and/or aluminium phosphate, before adding to the other components. In other embodiments, the FIM is added to the other components without prior adsorption to adjuvant.

The term ‘about’ for numerical values as used in the présent disclosure means the value ± 10%.

The compositions of the invention may in certain embodiments also comprise 15 non-pertussis protein components, e.g. to obtain combination vaccines [Decker, M.D., Edwards, K.M. & Bogaerts, H.H. Combination vaccines. In Vaccines (Eds. Plotkin, S., Orenstein, W.A. & Offit, P.A.) Elsevier Health Sciences, 2008. 1069-1101]. In certain embodiments, the compositions according to the invention may therefore further comprise antigens derived from one or more pathogens other than B. pertussis.

In certain embodiments, the compositions according to the invention comprise one or more of the following: tetanus toxoid (TT), diphtheria toxoid (DT), Haemophilus influenzae type-b oligosaccharide or polysaccharide conjugate (Hib), hepatitis B virus surface antigen (HBsAg), inactivated polio virus (IPV).

Combination vaccines of aP with such non-pertussis components are known and widely used. Préparation of combination vaccines has for instance been described in W02010/046935, US6013264, W02007/054820, W098/000167, and EP1946769, ail incorporated by reference.

In certain embodiments, the aP5 (or aP4: PT, FHA, FIM2/3) according to the invention is in a composition that further comprises DT and TT, thus providing for a 30 DTaP5 (or DTaP4) vaccine according to the invention. DTaP5 vaccines are widely used to prevent diphtheria, tetanus and whooping cough. The vaccine according to the invention has a higher amount of FIM than the DTaP5 vaccines described before, and is more efficacious against PRN-negative B. pertussis strains than conventional DTaP5 vaccines.

One way for isolating, purifying and detoxifying DT is described in page 3334 of WO 96/34883, incorporated by reference herein. DT can also for instance be obtained as described in US4709017, US5843711, US5601827, US5917017, and WO96/34623, ail incorporated by reference.

One way for isolating, purifying and detoxifying TT is described in page 3436 of WO 96/34883, incorporated by reference herein. TT can also for instance be obtained as described in EP0209281, EP0478602, and WO96/34623, ail incorporated by reference.

Hib oligosaccharide or polysaccharide conjugate can for instance be obtained as described in W02007/054820, W02004/110480, US6333036, WO2010/046935, US4372945, US4474757, WO95/08348, WO2010/046935, US4673574, EP0161188, EP0208375, and EP0477508, ail incorporated by reference. Hib antigen can for instance be the capsular polysaccharide of Hib, or a conjugate of the polysaccharide or a derived oligosaccharide thereof to a carrier protein such as DT, TT, or CRM197, a nontoxic variant of diphtheria toxin isolated from Corynebacterium diphtheriae C7 (b 197).

HBsAg can for instance be obtained as described in EP0226846, EP0299108, US6013264, W02007/054820, WO2010/046935, and WO9324148, ail incorporated 20 by reference.

IPV can be monovalent, containing one type of poliovirus (type 1, 2 or 3), or divalent (containing two types of poliovirus, e.g. types 1 and 2, 1 and 3 or 2 and 3), or bivalent (containing three types of poliovirus, i.e. types 1, 2 and 3). Preferably, the IPV according to the invention contains inactivated poliovirus types 1, 2 and 3. IPV 25 can for instance be obtained as described in US 4525349, and WO2011/006823, incorporated by reference herein.

These non-pertussis components can be obtained from various manufacturera. Examples are described in [“Vaccines.” 5th édition. S. Plotkin, W. Orenstein, P. Offit, 2008, Section 2, Chapter 38 (Combination vaccines”, M.D. Decker, K.M. Edwards, 30 H.H. Bogaerts, p 1069-1101)].

In certain embodiments, the compositions according to the invention comprise a composition comprising the pertussis components (aP5 or aP4 vaccine) according to any one of the embodiments described above (i.e. comprising 12-100 pg FIM per human dose and PT, FHA and optionally PRN; hereinbelow referred to as “aP according to the invention”, or “aP5*” for brevity), and DT.

In certain embodiments, the compositions according to the invention comprise aP according to the invention and TT.

In certain embodiments, the compositions according to the invention comprise aP according to the invention and IPV (referred to herein as “aP5*-IPV”).

In certain embodiments, the compositions according to the invention comprise aP according to the invention, DT and TT (referred to herein as “DTaP5*”).

In certain embodiments, the compositions according to the invention comprise aP according to the invention, DT, TT and Hib (referred to herein as “DTaP5*-Hib).

In certain embodiments, the compositions according to the invention comprise aP according to the invention, DT, TT and IPV (referred to herein as “DTaP5*-IPV”).

In certain embodiments, the compositions according to the invention comprise aP according to the invention, DT, TT and HBSAg (referred to herein as “DTaP5*HepB”).

In certain embodiments, the compositions according to the invention comprise aP according to the invention, DT, TT, Hib and HBSAg (referred to herein as “DTaP5*-Hib-HepB”).

In certain embodiments, the compositions according to the invention comprise aP according to the invention, DT, TT, Hib and IPV (referred to herein as “DTaP5*Hib-IPV”).

In certain embodiments, the compositions according to the invention comprise aP according to the invention, DT, TT, HBSAg and IPV (referred to herein as “DTaP5*-HepB-IPV”).

In certain embodiments, the compositions according to the invention comprise aP according to the invention, DT, TT, Hib, HBSAg and IPV (referred to herein as “DTaP5*-Hib-HepB-IPV”).

Further non-pertussis components could optionally be added, e.g. components that are sometimes combined with aP in combination vaccines, such as antigens from meningococci and/or pneumococci.

For the combination vaccines, the amounts of the non-pertussis components may be varied. Generally, the amounts of these components as typically présent in individual or combination vaccines can be used according to the instant invention. See for instance “Vaccines.” 5th édition. S. Plotkin, W. Orenstein, P. Offit, 2008, Section 2, for the various components and combination vaccines; in particular, Chapter 38 describes combination vaccines including aP vaccines with the components mentioned above [Decker, pp 1069-110l]; Chapter 10 describes DT [Vitek, pp 139156]; Chapter 31 describes TT [Wassilak, pp 805-840]; Chapter 25 describes IPV vaccines [Plotkin, pp 605-630]; Chapter 11 describes Hib vaccines [Chandran, pp 157-176]; and Chapter 13 describes Hepatitis B vaccines (based on HBsAg) [Mast, pp 205-242], ail incorporated by reference. Non-limiting examples of suitable amounts (it is also common to express amounts of DT and TT in IU or in Lf (flocculation units), see e.g. [Decker, In: Vaccines, pp 1069-1101, supra}, but here we provide micrograms) of the antigens per dose would for instance be: 1-100 pg, e.g. 2-40 pg, e.g. 6-25 pg, e.g. 15-25 pgDT; 1-50 pg, e.g. 2-20 pg, e.g. 5-10 pgTT; 1-100 pg, e.g. 3 to 40 pg of HBsAg protein per milliliter; 0.1-100 pg, e.g. 0.2 to 50 pg, e.g. 1 to 25 pg, e.g. 2-10 pg of the Hib capsular polysaccharide or oligosaccharide thereof in the form of a conjugate to a carrier protein; wild type-derived IPV-containing products (wt-IPV) are generally formulated to contain 40-8-32 D-Ag units per dose for poliovirus types 1, 2 and 3, respectively. However, these amounts may also be varied, e.g. lower amounts such as 10-20 D-Ag units for IPV type 1 can also be used, and the amounts for IPV types 2 and 3 can also be varied (see e.g. EP 2066344). Amounts may also vary according to the intended use, e.g. booster vaccines may in certain embodiments contain less units of certain components than priming vaccines.

The protein components in the compositions according to the invention are intended to induce an immune response upon administration to an eligible subject. It will be clear to the skilled person that wherever is referred herein to proteins or mutants thereof, e.g. toxoids, parts of the proteins may also be used and can hâve équivalent or in some cases preferred properties for inducing immune responses. Further, the proteins may contain (additional) mutations, such as délétions, insertions, substitutions, etc. Thus, immunogenic fragments and variants of the indicated protein components are included within the meaning of the proteins indicated herein.

Compositions according to the invention can be used as acellular pertussis vaccines, or as components of combination vaccines, which generate immune responses to one or more of the components in the compositions upon administration to eligible subjects. The immune réponse may comprise a cellular and/or a humoral response. Such immune responses preferably confer protection against infection with pathogen or against disease or at least reduces the severity of the symptoms caused by the pathogen from which the respective components are derived (i.e. in any case preferably against B.pertussis, and preferably also against PRN-negative mutants thereof). The compositions according to the invention can thus suitably be referred to as vaccines. A dose of a vaccine is the amount that is administered in a single administration to a subject. A subject may suitably be an animal or a human, and in certain preferred embodiments the subject is a human. Many vaccines are suitably, and actually preferably, administered more than one time to the same individual with sufficient time interval to obtain a boosting effect in the individual, e.g. at least four weeks, to several years up to about two décades between administrations. Multiple immunizations are usually administered to naive infants. The compositions according to the invention may also be administered more than one time, e.g. in a non-limiting embodiment two or three or more times with at least 4 weeks interval, for instance a one or two month time interval between each administration. One non-limiting example is administration according to the EPI schedule, at 6 weeks, 10 weeks and 14 weeks of âge. Another regimen would be at 2 months, 4 months, 6 months of âge. In certain embodiments a booster vaccination is given 10-20 years later, e.g. during adolescence. Futher decennial booster vaccinations may be given. In certain embodiments, aP according to the invention is administered two or three times in the first year of life, a further boost is administered the second year of life, and a further booster is administered at four to five years of âge, after which an adolescent boost is administered at approximately twelve years of âge. Also the Td (a TT-DT containing vaccine given to adolescents) booster recommendation may be followed, i.e. every ten years and replace Td with Tdap, wherein the acellular pertussis component is aP according to the invention. However, it will be clear to the skilled person that the vaccination scheme of the aP vaccines according to the invention may be suitably varied, as is clear from the wide variety of immunization schedules (regimens) of marketed aP vaccines by different national authorities (e.g., Table 21-5 in “Vaccines.” 5th édition. S. Plotkin, W. Orenstein, P Offit, 2008, Section 2, Chapter 21 “Pertussis vaccines”, K.M. Edwards & M.D. Decker. p. 467-517).

The compositions according to the invention may also be suitably used as booster vaccines for populations that hâve been previously vaccinated by other vaccines, be those wP or aP vaccines of different composition or combination vaccines comprising wP or aP of different composition than the vaccines of the invention. Such boosters may for instance be used for vaccination of adults or elderly, that hâve not been vaccinated against B.pertussis for more than a decade. It could be 5 useful to repeat such booster vaccinations, e.g. about once every five, ten or fifteen years. It has also been recommended to administer tetanus toxoid, reduced diphtheria toxoid and acellular pertussis vaccine (Tdap) to prégnant women with every pregnancy irrespective of previous Tdap history. In certain embodiments, the aP according to the invention is administered to an infant, a child, an adolescent, an adult, an elderly, or a prégnant woman, e.g. as aP or as Tdap.

The compositions according to the invention are pharmaceutical compositions. Such compositions comprise a composition according to the invention and typically a pharmaceutically acceptable carrier or excipient. In the présent context, the term 15 pharmaceutically acceptable means that the carrier or excipient, at the dosages and concentrations employed, will not cause unwanted or harmful effects in the subjects to which they are administered. Such pharmaceutically acceptable carriers and excipients are well known in the art [Remington. The Science and Practise of Pharmacy, Mack Publishing Company 1990; Frokjaer, S. & Hovgaard, L.

Pharmaceutical Formulation Development of Peptides and Proteins, 2000; Handbook of Pharmaceutical Excipients, Pharmaceutical Press 2000]. The compositions preferably are formulated and administered as a stérile solution. Stérile solutions are prepared by stérile filtration or by other methods known per se in the art. The solutions can then be lyophilized or filled into pharmaceutical dosage containers. The pH of the solution generally is in the range of pH 3.0 to 9.5, e.g pH 5.0 to 7.5. The components of the composition typically are in a solution having a suitable pharmaceutically acceptable buffer, and the solution may also contain a sait. In certain embodiments, detergent is présent. In certain embodiments, the vaccine may be formulated into an injectable préparation. These formulations contain effective amounts of the protein components, are either stérile liquid solutions, liquid suspensions or lyophilized versions and. optionally contain stabilizers or excipients. Several examples of suitable formulations for the storage and for pharmaceutical administration of aP vaccines or combination vaccines are known (e.g., Tables 21-3 and 21-4 in [“Vaccines.” 5th édition. S. Plotkin, et al, suprci\). Examples of suitable diluents are PBS or saline. Preservative may optionally be présent, e.g. phenoxyethanol, thimerosal or parabens. If a preservative is présent, it is preferably présent at low levels. In case a combination vaccine comprises IPV, the use of thimerosal is preferably avoided, since thimerosal may lead to loss of potency of the

IPV component (see e.g. Sawyer LA, 1994, Vaccine 12 : 851-856; EP 2066344). Further components that may optionally be présent as trace constituents are polysorbate-80, gelatin and remnants from chemical toxoidation (e.g., if PT is chemically toxoided) such as glutaraldehyde, formaldéhyde.

Preferably the vaccines according to the invention are stored between 2-8°C.

In certain embodiments the compositions of the invention comprise further one or more adjuvants. Adjuvants are known in the art to further increase the immune response to an applied antigenic déterminant (for a review on adjuvants, see e.g. Montomoli, 2011, Expert Rev Vaccines 10: 1053-1061). Examples of suitable adjuvants include aluminium salts such as aluminium hydroxide and/or aluminium phosphate; oil-emulsion compositions (or oil-in-water compositions), including squalene-water émulsions, such as MF59 (see e.g. WO 90/14837); saponin formulations, such as for example QS21 and Immunostimulating Complexes (ISCOMS) (see e.g. US 5,057,540; WO 90/03184, WO 96/11711, WO 2004/004762,

WO 2005/002620); Toll-like receptor (TLR) agonists, e.g. a TLR7 agonist (see e.g. WO 2012/117377, page 15-18 for examples), e.g. in combination with an aluminium sait, e.g. aluminium hydroxide to which the TLR agonist may be adsorbed; bacterial or microbial dérivatives, examples of which are monophosphoryl lipid A (MPL), 3-0deacylated MPL (3dMPL), CpG-motif containing oligonucleotides, ADP-ribosylating bacterial toxins or mutants thereof, such as E. coli heat labile enterotoxin LT, choiera toxin CT, and the like. For example PT and tetanus toxoid also hâve adjuvant properties of their own. In certain embodiments the compositions of the invention comprise aluminium as an adjuvant, e.g. in the form of aluminium hydroxide, aluminium phosphate, aluminium potassium phosphate, or combinations thereof, in concentrations of 0.05 - 5 mg, e.g. from 0.075-1.0 mg, of aluminium content per dose.

In other embodiments, the compositions used in the invention do not comprise further adjuvants.

Preferably, the vaccine compositions according to the invention comprise an adjuvant. In certain preferred embodiments, the adjuvant is an aluminium sait such as aluminium phosphate, aluminium hydroxide or a combination thereof. Preferably, one, more or ail of the aP antigens are adsorbed onto an aluminium sait. Also the other antigens may be adsorbed onto an aluminium sait. In certain embodiments, one, more or ail of the aP antigens (PT, FPIA, FIM, PRN if présent) are adsorbed onto aluminium hydroxide. In certain embodiments, one, more or ail of the aP antigens (PT, FHA, FIM, PRN if présent) are adsorbed onto aluminium phosphate. Formulation of aP vaccines and aP combination vaccines with aluminium salts is for instance described in [Denoël, 2002, Vaccine 20: 2551-2555]. Typically, the individual components are individually adsorbed onto the aluminium sait, and the components are thereafter mixed to form the vaccine formulation. This also allows to préparé vaccines in which certain components are adsorbed onto a first alumimium sait [e.g. A1(PO)4], while other components are adsorbed onto a second aluminium sait [e.g. A1(OH)3]. Also the other components of a combination vaccine may be adsorbed onto an aluminium sait, e.g. DT and TT may be adsorbed onto aluminium hydroxide or aluminium phosphate, or a combination of these. The DT and TT components may be adsorbed to the same or to a different aluminium sait as the aP components. Further components of combination vaccines of increasing valency may also be adsorbed onto aluminium salts, e.g. HBsAg, Hib and/or IPV may or may not be adsorbed onto aluminium salts. In certain preferred embodiments wherein a combination vaccine comprises HBsAg, the HBsAg is adsorbed onto aluminium phosphate (see e.g. WO 93/24148). If Hib is included in the combination vaccine and certain other components such as one or more of DT, TT or aP are adsorbed onto aluminium hydroxide, the risk of interférence (réduction of efficacy of the Hib component) can be reduced for instance by adsorbing Hib onto aluminium phosphate or use Hib that is not adsorbed onto an alumium adjuvant, and combine this with the other components by either contemporaneously (i.e. just prior to administration) adding the Hib, or by mixing with the other components that hâve been adsorbed onto aluminium hydroxide adjuvant in such a manner that the aluminium hydroxide adjuvant has been pre-saturated, as described in detail in WO 99/48525. The skilled person thus is aware of various ways of formulating combination vaccines according to the invention in a suitable manner.

In certain embodiments, a vaccine composition according to the invention comprises PT, FHA, FIM2/3, and aluminium hydroxide, and optionally PRN. In certain embodiments, a vaccine composition according to the invention comprises PT, FHA, FIM2/3, and aluminium phosphate, and optionally PRN. In certain embodiments, a vaccine composition according to the invention comprises PT, FHA, FIM2/^Z3, aluminium hydroxide and aluminium phosphate, and optionally PRN. In certain embodiments, a vaccine composition according to the invention comprises DTaP5*, and aluminium hydroxide. In certain embodiments, a vaccine composition according to the invention comprises DTaP5*, and aluminium phosphate. In certain embodiments, a vaccine composition according to the invention comprises DTaP5*, aluminium hydroxide and aluminium phosphate. In certain embodiments, a vaccine composition according to the invention comprises aP5*-IPV, and aluminium hydroxide or aluminium phosphate or aluminium hydroxide and aluminium phosphate. In certain embodiments, a vaccine composition according to the invention comprises DTaP5*-IPV, and aluminium hydroxide or aluminium phosphate or aluminium hydroxide and aluminium phosphate. In certain embodiments, a vaccine composition according to the invention comprises DTaP5*-Hib, and aluminium hydroxide or aluminium phosphate or aluminium hydroxide and aluminium phosphate. In certain embodiments, a vaccine composition according to the invention comprises DTaP5*-HepB, and aluminium hydroxide or aluminium phosphate or aluminium hydroxide and aluminium phosphate. In certain embodiments, a vaccine composition according to the invention comprises DTaP5*-Hib-HepB, and aluminium hydroxide or aluminium phosphate or aluminium hydroxide and aluminium phosphate. In certain embodiments, a vaccine composition according to the invention comprises DTaP5*-Hib-IPV, and aluminium hydroxide or aluminium phosphate or aluminium hydroxide and aluminium phosphate. In certain embodiments, a vaccine composition according to the invention comprises DTaP5*-HepB-IPV, and aluminium hydroxide or aluminium phosphate or aluminium hydroxide and aluminium phosphate. In certain embodiments, a vaccine composition according to the invention comprises DTaP5*-Hib-HepB-IPV, and aluminium hydroxide or aluminium phosphate or aluminium hydroxide and aluminium phosphate.

Administration of the compositions according to the invention can be performed using standard routes of administration. Non-limiting embodiments include parentéral administration, such as by injection e.g. intradermal, intramuscular, transcutaneous, intranasal, etc. In one embodiment the vaccine is administered by intramuscular injection into the thigh or into the deltoid muscle. The skilled person knows the various possibilities to administer a vaccine according to the invention, in order to induce an immune response to at least one of the antigens in the vaccine. Generally, the standard dose of pertussis vaccine is 0.5 mL given intramuscularly in the antérolatéral thigh or, if necessary, the deltoid. However, the amount of the components in the compositions provided to a patient during one administration can be varied as is known to the skilled practitioner. Also the adjuvant, if used, can be adapted to the delivery system.

Although it is preferred to hâve a single composition for vaccinating against pertussis, the skilled person will be aware that the effect of the invention as described herein can also be obtained by vaccinating with the components of the aP vaccine, i.e. PT, FHA, FIM at a human dose of 12-100 pg, and optionally PRN, wherein the components are not necessarily ail in the same composition, e.g. wherein (part of) the FIM is in a separate composition. For instance, a commercially available aP vaccine (having FIM at a dose of 0-5 pg per human dose) could be complemented by coadministration of FIM as a separate component to a total dose administered of 12-100 pg, e.g. 15-80 pg, 20-60 pg, 20-50 pg, or 20-25 pg FIM, e.g. by injecting a first aP vaccine composition comprising PT, FHA, optionally a dose of 5 pg FIM, and optionally PRN and injecting a separate composition comprising the (remainder of the) FIM to supplément to a total dosage of 12-100 pg, e.g. 15-80 pg, 20-60 pg, 20-50 pg or 20-25 pg FIM. In such embodiments, co-administration means that the separate compositions are administered (e.g. injected) within one hour, preferably within a few minutes between the administrations, preferably they are administered essentially simultaneously (e.g. by co-injection or by consecutive injections). Altematively, compounds might be mixed just prior to administration, so that a single injection (with a composition that is a composition according to the invention) is sufficient. The invention hence also provides a method for vaccinating a human subject against Bordetella pertussis, optionally a PRN-negative strain of Bordetella pertussis, comprising administering to the subject,the following Bordetella pertussis antigens: pertussis toxoid (PT), filamentous hemagglutinin (FHA), and fîmbriae types 2 and 3 (FIM), and optionally pertactin (PRN), wherein FIM is administered in an amount of ' 20

12-100 pg. In preferred embodiments, this is done by administering a single composition according to the invention.

The invention is further explained in the following examples. The examples do not limit the invention in any way. They merely serve to clarify the invention.

EXAMPLES

Example 1. High dose FIM in aP5 vaccine improves protection against PRNnegative B. pertussis

Methods:

A validated mouse Bordetella pertussis lung challenge model, which correlates with clinical efficacy of aP vaccines, (Guiso, 1999, Vaccine 17; 2366-2376; Denoel, 2005, Vaccine 23:5333-5341; Godfroid, 2004, Int. J. Med. Microbiol. 294; 269-276) was used to test the efficacy of a licensed 5-component acellular pertussis vaccine (ADACEL™, Sanofi Pasteur; in this example further referred to as aP5) at a quarter of a human dose with or without the addition of 2 pg of purified FIM2/3 antigen (List Biological Laboratories Inc). Each 0.5 mL dose of ADACEL™ contains 5 Lf tetanus toxoid, 2 Lf diphtheria toxoid and 5 acellular pertussis antigens (2.5 pg detoxified pertussis toxin, 5 pg filamentous hemagglutinin, 3 pg pertactin and 5 pg fimbriae types 2 and 3). In brief, female Balb/c mice were vaccinated subcutaneously with aP5 with or without FIM2/3 antigen at 4 and 7 weeks of âge at % of the human dose. Therefore, animais received either 1.25 pg of FIM2/3 (aP5 group) or 3.25 pg of FIM2/3 (aP5 + FIM group), which equals 5 pg and 13 pg in humans, respectively. At 9 weeks of âge, the mice were challenged intranasally with ~10⁶ cfu of B. pertussis WHO 18323 (a pertactin positive strain), a pertactin-negative strain PRN-STOP and a pertactin-negative strain PRN-IS [Queenan, 2013, A Engl J Med. 368: 583-4], At 2 hours, 2 days, 5 days and 8 days post-challenge (n=5/group) lung clearance was determined by counting the B. pertussis colonies grown on Bordet-Gengou agar plates after plating serial dilutions of lung homogenate.

Results:

The treatment response-over time profile after vaccination with aP5 with or without the addition of FIM2/3 antigen is depicted in Fig. 1. To test for a statistically significant différence between mean loglO CFU counts on day 5 and day 8 between mice vaccinated with aP5 or aP5 + FIM, the Wilcoxon Exact Test was used. In addition, the treatment response over time (from day 0 to day 8) profile data was modeled by an analysis of covariance (Milliken GA et al. Analysis of Covariance, Dept of Statistics, Kansas State University, 1989.; SAS Institute Inc., SAS/STAT User’s Guide, Version 6, Fourth Edition, Volume 2. Cary, NC: SAS Institute Inc., 1989).

For the B. pertussis WHO18323 strain (a PRN positive strain, Fig. IA), a comparison of différence in loglO CFU counts at day 5 using a one-sided Wilcoxon’s exact nonparametric test, showed that mean loglO CFU counts after vaccination with aP5 + FIM were significantly lower (p<0.05) than those after vaccination with aP5 alone. Modelling of sample treatment response-time profile data showed no statistically significant différence in treatment response from day 0 to day 8 from a fitting by an analysis of covariance with day as a term for slope and day*day as a term for quadratic curvature.

After challenge with the B. pertussis PRN-STOP strain (Fig. IB), a comparison of différence in loglO CFU counts at day 5 using a one-sided Wilcoxon’s exact nonparametric test showed that mean loglO CFU counts after vaccination with aP5 + FIM were significantly lower (p<0.02) than those after vaccination with aP5 alone. Modeling of sample treatment response-time profile data showed no statistically significant différence in treatment response from day 0 to day 8 from a fitting by an analysis of covariance model.

For the B. pertussis PRN-IS strain (Fig. IC), a comparison of différence in loglO CFU counts at day 5 indicated no significant différences between vaccine treatments using a one-sided Wilcoxon’s exact nonparametric test. On day 8 there was il a trend that mean loglO CFU counts were lower after vaccination with aP5 + FIM compared with mean loglO CFU counts after vaccination with aP5 alone (P<0.08) using Wilcoxon’s exact nonparametric test. The asymptotic analog of the Wilcoxon’s exact test showed a significant vaccine différence on day 8 (p<0.03). Modeling of sample treatment response-time profile data suggested a différence in treatment response from day 0 to day 8 from fîtting by an analysis of covariance with day as a term for slope and day*day*trt as a term quadratic curvature.

To test for a différence in treatment effects, a linear contrast between treatment profiles from day 0 to day 8 indicated that mean loglO CFU counts after vaccination with aP5 + FIM were significantly lower (p<0.01) than those measured after vaccination with aP5 alone.

In summary, these results show that addition of 2pg of FIM2/3 to l/4^th of a human dose of Adacel vaccine significantly increases the efficacy of this vaccine in a mouse nasopharyngeal challenge model against a PRN-negative B. pertussis isolate. This finding is surprising since there hâve not been any previous suggestions that increasing the amount of FIM in a pertussis vaccine would increase its efficacy. This resuit is specifically relevant in the context of PRN-negative strains that are currently emerging and causing disease around the world.

Example 2. Increased vaccine efficacy after addition ofFIM in the mouse challenge model is dose dépendent

Methods:

To investigate whether an increased dose of FIM correlated with increased vaccine efficacy, which would suggest the effect to be FIM-specific, the validated mouse Bordetella pertussis lung challenge model as described above was used. Animais were vaccinated with a 1/10 human dose of a licensed 2-component aP vaccine (PENTAVAC®, Sanofi Pasteur MSD; vaccine hereinafter referred to as aP2) with or without the addition of FIM2/3 (List Biological Laboratoires Inc; material further referred to as FIM). Each human 0.5 mL dose of PENTAVAC® contains: (at least) 40 IU tetanus toxoid (TT); (at least) 30 IU diphtheria toxoid (DT); 40, 8 and 32 Dantigen units of inactivated poliovirus (IPV) types 1, 2 and 3 respectively; 10 pg H. Influenzae type B polysaccharide conjugated to TT (Hib-TT); and 2 acellular pertussis antigens (25 pg detoxified pertussis toxin (PT) and 25 pg filamentous hemagglutinin (FHA)). An amount of 0.5; 1.0; 1.5; 2.0; 2.5 or 5.0 pg FIM, corresponding to 5, 10, 15, 20, 25 or 50 pg FIM per human dose, respectively, was adsorbed to aluminium hydroxide and co-administered (as a separate injection) with the licensed aP2 vaccine (thus mimicking an aP vaccine with high dose of FIM according to the invention). At 9 weeks of âge, the mice were challenged intranasally with ~10⁶ cfu of a PRNnegativeB. pertussis strain (1195). At 2 hours (n= 5/group) and 5 days (n=10/group) lung clearance was determined by counting the B. pertussis colonies grown on Bordet-Gengou agar plates after plating serial dilutions of lung homogenate.

Results:

A dose-dependent decrease in the number of B. pertussis colonies cultured from the lungs of vaccinated mice was observed with an increasing amount of FIM. Similarly, a dose-dependent decrease in the number of B. pertussis colonies cultured from the lungs of vaccinated mice was observed in mice vaccinated with a 1/25 dilution and increasing amounts of FIM. Figure 2 shows the loglO CFU counts obtained from the lungs of mice vaccinated with 1/10 human dose of aP2 in the presence of an increasing amount of FIM, 5 days post challenge with B. pertussis strain 1195. Table 1 shows mean LoglO CFU counts and p-values calculated using Mann-Whitney (GraphPad Prism) comparing mean loglO CFU counts after vaccination with aP2 at 1/10 human dose with or without FIM. After the addition of 2 pg or more of FIM to aP2 1/10 human dose (human dose équivalent of 20 pg), vaccine efficacy increased significantly.

Conclusions’

The dose dépendent effect of an increased FIM dose on vaccine efficacy demonstrates this effect is spécifie for FIM. In addition, vaccines including a high dose of FIM are more efficacious than a vaccine containing the maximum amount of FIM présent in currently commercially available aP5 vaccines (5 pg per human dose). This is in line with the findings in example 1 that showed that increasing the dose of FIM in aP vaccine above 10 pg per human dose improved the efficacy, and further extends the findings to a different aP vaccine and an additional PRN-negative pertussis strain.

Example 3. Increased efficacy of an aP5 vaccine with the addition ofFIMis observed against multiple B. pertussis strains

Methods:

The validated mouse Bordetella. pertussis lung challenge model as described above was used to investigate whether an increased dose of FIM improved vaccine efficacy against a variety of B. pertussis strains. A 1/10 human dose of ADACEL® (Sanofi Pasteur; containing 2.5 pg PT, 5 pg FHA, 3 pg PRN and 5 pg FIM2/3 per human dose, see also example 1 ; herein referred to as aP5) or BOOSTRIX® (GlaxoSmithKline Biologicals; containing 5 Lf of tetanus toxoid, 2.5 Lf of diphtheria toxoid, 8pg PT , 8pg FHA and 2.5pg PRN per human dose; herein referred to as aP3) was used, with or without the addition (as separate injection after adsorption to aluminium hydroxide) of 5 pg of FIM (List Biological Laboratoires Inc), corresponding to 50 pg per human dose. Animais that were vaccinated at 4 and 7 weeks of âge, were challenged at 9 weeks of âge with one of the following B. pertussis strains: WHO 18323 (PRN-positive), 24422 (PRN-negative), 24421 (PRNnegative) or 1195 (PRN-negative). At 2 hours (n= 5/group) and 5 days (n=10/group) lung clearance was determined by counting the B. pertussis colonies grown on Bordet-Gengou agar plates after plating serial dilutions of lung homogenate.

Results:

Addition of 5 pg of FIM to a 1/10 human dose of aP5 improved vaccine efficacy against ail B. pertussis strains, which reached statistical significance against 3 of the tested pertussis strains (WHO 18323 (p=0.04), PRN-24421(p=0.03) and PRN-I195 (p=0.001)) (Figure 3, Table 2).

This experiment was also performed using aP3 (1/10 human dose). For three of the challenge strains (WHO 18323, 24422 and 24421) a réduction after addition of FIM was not observed with this aP3 vaccine (BOOSTRIX®), although this might possibly partly be explained by the already high efficacy observed for these challenge strains with this particular aP3 when given without FIM. A significant réduction in loglO CFU counts was however observed with the addition of FIM to this aP3 when challenging with B. pertussis strain 1195.

Overall, these data indicate that a vaccine with a high dose of FIM would hâve an improved efficacy against a range of pertussis strains.

Conclusions:

The increased efficacy observed after immunization with a high level of FIM in combination with a commercially available aP5 vaccine against a panel of B. pertussis strains indicates the benefit of a high FIM dose is observed also against different pertussis strains. Therefore vaccines including a high dose of FIM according to the invention could contribute to decreasing the rate of pertussis disease.

Example 4. Increased efficacy with the addition of FIMfor different currently commercially available aP vaccines

Methods:

The validated mouse Bordetella pertussis lung challenge model as described above was used to investigate whether an increased dose of FIM (List Biological Laboratories Inc) improved vaccine efficacy of ADACEL® (Sanofi Pasteur; containing 2.5 gg PT, 5 gg FHA, 3 gg PRN and 5 gg FIM 2+3 as aP components per human dose), PENTAVAC® (Sanofi Pasteur MSD; containing 25 pg PT and 25 gg FHA as aP components per human dose) or BOOSTRIX® (GlaxoSmithKline Biologicals; containing 8 gg PT , 8 gg FHA and 2.5 gg PRN as aP components per human dose).

Mice were vaccinated with 1/10 human dose at 4 and 7 weeks of âge with or without the addition of 5 gg FIM to the commercial vaccine (corresponding to 50 gg FIM added per human dose), challenged at 9 weeks of âge with B. pertussis pertactinnegative strain 1195 and lung clearance was determined 5 days post-challenge (n=10/group) by counting the B. pertussis colonies grown on Bordet-Gengou agar plates.

Results:

For ail three vaccines the addition of FIM resulted in significantly lower mean LoglO CFU counts compared to mean LoglO CFU counts after vaccination with the commercial vaccine alone (Table 3).

Conclusion'.

The vaccine efficacy of ail three vaccines improved significantly after the addition of FIM. This suggests the effect of FIM can be generalized to a range of commercial aP vaccines that contain different amounts of pertussis antigens and different amounts of FIM. Efficacy of vaccines that already include FIM (ADACEL®) or those that do not include FIM (PENTAVAC® or BOOSTRIX®) improves, showing that it is not just presence of FIM that contributes to vaccine efficacy, but that also the dose of FIM is important.

Example 5. Increased &nti-FIM antibody functionality after vaccination with a high dose ofFIM against a wide range of pertussis strains

Functional activity of antibodies against pertussis components hâve been identified as important additional parameters to consider, in particular when evaluating new formulations containing PT and FIM which are known to induce antibodies with functional activity such as toxin neutralization and bacterial agglutination respectively. Assays to measure whole-cell B. pertussis agglutinating antibodies hâve been established. Although there is no functional threshold that has been found to correlate directly with the protective efficacy of pertussis vaccines, they are nevertheless an important immune parameter to détermine as part of the overall comparison of new vaccine formulations to those proven to be safe and effective (from WHO draft Recommendations for aP vaccines, WHO/BS/2011.2158, section C.2.1.2). Thus, given the relevance of such assays for FIM containing aP vaccines, we used an agglutination assay to further test the vaccines of the invention against various B. pertussis strains.

Methods:

Sérum was collected from mice that were vaccinated at 4 and 7 weeks of âge with a 1/10 human dose of PENTAVAC® (Sanofi Pasteur MSD; containing as aP antigens 25 pg PT and 25 pg FHA per human dose; referred to herein as aP2) with or without the addition of FIM (List Biological Laboratories Inc). An amount of 0.5 or 2.0 pg FIM, corresponding to 5 or 20 pg FIM per human dose, respectively, was adsorbed to aluminium hydroxide and co-adminstered (as a separate injection) with the commercially available aP2 vaccine. At week 9, 5 animais per dosing group were sacrificed and sera isolated from terminal bleeds were pooled for investigation of antiFIM (functional) antibody levels.To evaluate functional antibody responses to FIM, an agglutination assay was perfoimed. In this assay, the presence of functional antibodies in test sérum leads to the formation of antigen/antibody complexes when t

mixed with B. pertussis. Positive agglutination is defined as the presence of an opaque solution in the well, due to the presence of antigen/antibody complexes. Négative agglutination is observed as a defined bacterial sédiment at the bottom of the well. In brief, 50 μΐ of test sérum was serially diluted in PBS and mixed with 50 μΐ of a B. pertussis suspension of an ODôoo of 1.0. This mixture was incubated over night and the next day the presence or absence of a bacterial sédiment was determined using an inverted mirror. The agglutination titer is defined as the highest dilution which results in complété agglutination.

To investigate whether anti-FIM antibodies induced by vaccination of mice were functional against a panel of B. pertussis strains it was tested whether the sera had the capacity to agglutinate to 10 different FIM expressing B. pertussis strains. From a panel of 30 recent clinical B. pertussis isolâtes (kindly provided by Dr. Alan Evangelista, St. Christopher’s Hospital for Children in Philadelphia), 24 isolâtes showed clear agglutination readouts with a positive control commercial anti-FIM monoclonal antibody (06/128, NIBSC, UK), confirming these strains express the FIM antigen. From this panel of 24 strains, 5 PRN-negative and 5 PRN-positive strains were selected for testing the mouse sérum.

Results:

Vaccination in the presence of FIM induced functional antibody titers. Positive agglutination was observed with ail the 6 mouse sérum pools of mice vaccinated in the presence of FIM against ail 10 B. pertussis strains. The unvaccinated control group and the group receiving aP2 at 1/10 human dose alone, which does not include FIM, did not show any agglutination (Table 4). Although a high dose of FIM was shown to be more efficacious than a low dose of FIM in validated mouse Bordetella pertussis lung challenge model (example 2), there was no clear FIM dose-response corrélation (data not shown for other FIM doses), which is likely due to the limitation of this WHO standardized assay, which is not sensitive enough to detect small différences in agglutination.

Conclusion'.

Vaccination with a commercial vaccine in the presence of FIM results in induction of functional antibodies against a wide panel of B. pertussis strains, either PRN-negative or PRN-positive strains. This finding indicates that a vaccine including a high dose of

FIM would be effective in reducing pertussis disease caused by a wide range of B. pertussis strains.

Table 1. Effect on protection of adding increasing doses of FIM to aP2 vaccine

	Total amount FIM	FIM équivalent Human Dose	Mean LoglO CFU	P-value vs aP2 alone
Control	-	-	7.2	0.0002
aP2	-	-	2.9	-
aP2 + 0.5 gg FIM	0.5 gg	5 gg	3.5	0.3418
aP2 +1.0 gg FIM	1-0 gg	10 gg	2.1	0.0514
aP2 +1.5 gg FIM	1.5 gg	15 gg	2.5	0.5390
aP2 + 2.0 gg FIM	2.0 gg	20 gg	2.0	0.0164
aP2 + 2.5 gg FIM	2.5 gg	25 gg	1.6	0.0013
aP2 + 5.0 gg FIM	5.0 gg	50 gg	1.4	0.0001

Table 1: P-values as determined using Mann-Whitney, comparing the différence in mean LoglO CFU counts from the lung in mice challenged with PRN-negative B. Pertussis strain 1195 at week 9 after vaccination with aP2 at 1/10 human dose with the addition of FIM versus aP2 alone given in a 1/10 human dose at 4 and 7 weeks of âge. For details see example 2.

Table 2. Effect of adding FIM to aP5 vaccine to protection against different strains

	aP5	aP5 + FIM
strain	Total amount FIM (μ§)	FIM équivalent Human Dose (pg)	Mean LoglO CFU	Total amount FIM (pg)	FIM équivalent Human Dose (pg)	Mean LoglO CFU	P-value
WHO 18323	0.5	5	4.0	5.5	55	3.3	0.04
24422	0.5	5	4.6	5.5	55	3.6	0.08
24421	0.5	5	4.5	5.5	55	3.8	0.03
1195	0.5	5	5.3	5.5	55	4.0	0.001

Table 2: Mean LoglO CFU counts from the lung in mice 5 days post challenge with 4 different B. pertussis strains at week 9 after vaccination with aP5 at 1/10 human dose with or without the addition of 5 pg of FIM at 4 and 7 weeks of âge. P-values determined using Mann-Whitney. For details see example 3.

Table 3. Effect of addition of FIM to different commercial aP vaccines

	Total amount of FIM (pg)	FIM Equivalent Human Dose (pg)	Mean LoglO CFU	P-value
ADACEL®	0.5	5	5.3
ADACEL® + FIM	5.5	55	3.94	0.001

PENTAVAC®	0	0	2.89
PENTAVAC® + FIM	5	50	1.38	0.0001

BOOSTRIX®	0	0	4.1
BOOSTRIX® + FIM	5	50	2.87	0.004

Table 3: Mean LoglO CFU counts from the lung in mice, 5 days post challenge with B. pertussis strain 1195 at week 9 after vaccination with 3 different licensed aP vaccines at 1/10 human dose with or without the addition of 5 pg of FIM at 4 and 7 weeks of âge. P-values determined using Mann-Whitney. For details see example 4.

Table 4. Functional activity of antibodies against various strains by addition of FIM to aP2 vaccine

	Total amount FIM (pg)	FIM équivalent Human Dose (pg)	FIM Antibody level	Agglutionation against PRN- Négative Strains	Agglutionation against PRN-Positive Strains
Strain #				1	2	3	4	5	6	7	8	9	10
Control	-	-	<	<	<	<	<	<	<	<	<	<	<
aP2	-	A	<	<	<	<	<	<	<	<	<	<	<
aP2 + 0.5 pg FIM	0.5	5.0	193	480	480	480	480	320	320	200	480	320	320
aP2 + 2.0 pg FIM	2.0	20	285	960	960	960	960	960	960	640	960	640	800

Table 4: Anti-FIM antibody levels and agglutination titers against a panel of B. pertussis strains as determined in sérum pools collected from 5 mice per group, vaccinated at 4 and 7 weeks of âge with a 1/10 human dose of aP2 with or without the addition of FIM, See example 5 for details <: below lower limit of détection (LLOD)

Claims

1. An acellular pertussis (aP) vaccine composition comprising Bordetella pertussis antigens pertussis toxoid (PT), filamentous hemagglutinin (FHA), and

5 fimbriae types 2 and 3 (FIM), wherein FIM is présent in an amount of 12-100 pg per human dose.

2. An aP composition according to claim 1, wherein FIM is présent in an amount of 15-60 pg per human dose.

3. An aP composition according to any one of the preceding claims, wherein FIM is présent in an amount of 20-60 pg per human dose.

4. An aP composition according to any one of the preceding claims,

15 wherein FIM is présent in an amount of 20-50 pg per human dose.

5. An aP composition according to any one of the preceding claims, wherein FIM is présent in an amount of 20-25 pg per human dose.

20

6. An aP vaccine composition according to any one of the preceding claims, further comprising pertactin (PRN).

7. An aP vaccine composition according to any one of the preceding claims, wherein PT is genetically detoxified.

8. A composition according to any one of the preceding claims, further comprising antigens from one or more pathogens other than B.pertussis.

9. A composition according to claim 8, comprising

30 tetanus toxoid and diphtheria toxoid.

10. A composition according to claim 8 or 9, comprising one or more of:

a) Heamophilus influenzae (Hib) oligosaccharide or polysaccharide conjugate;

b) hepatitis B virus surface antigen (HBsAg); and

c) inactivated polio virus (IPV).

11. A composition according to any one of the preceding daims, further comprising an adjuvant.

12. A composition according to claim 11, wherein the adjuvant comprises aluminium hydroxide, aluminium phosphate, or a combination thereof.

13. Use of a a composition according to any one of daims 1-12 in the manufacture of a vaccination against Bordetella pertussis.

14. Use of a composition according to any one of daims 1-12 in the manufacture of a médicament for protecting a subject from whooping cough that is caused by infection with a PRN-negative strain of Bordetella pertussis.

15. Use of the following Bordetellapertussis antigens: pertussis toxoid (PT), filamentous hemagglutinin (FHA), and fimbriae types 2 and 3 (FIM), and optionally pertactin (PRN), wherein FIM is administered in an amount of 12-100 pg in the manufacture of a vaccination against Bordetella pertussis, optionally a PRNnegative strain of Bordetella pertussis.