WO2014027083A1

WO2014027083A1 - An immunogenic composition of killed leptospira bacteria

Info

Publication number: WO2014027083A1
Application number: PCT/EP2013/067132
Authority: WO
Inventors: Henricus Leo Bernardus Maria Klaasen; Peter LOEFFEN; Eric Onno Rijke
Original assignee: Intervet International B.V.; Intervet Inc.
Priority date: 2012-08-17
Filing date: 2013-08-16
Publication date: 2014-02-20
Also published as: DK2884996T3; JP6502849B2; US20150216976A1; CN104582719A; AU2013304048A1; JP2015530372A; US9375480B2; RU2015109124A; BR112015002984A2; RU2667642C2; AU2013304048B2; EP2884996A1; ES2772451T3; CN104582719B; PT2884996T; EP2884996B1

Abstract

The present invention pertains to a composition containing an immunogenic cell preparation of killed Leptospira bacteria in a citric acid solution. It also pertains to a vaccine to protect an animal against an infection with Leptospira bacteria, wherein the vaccine comprises this composition, and to the use of citric acid to stabilise an immunogenic preparation of killed Leptospira bacteria in a liquid carrier, by dissolving the citric acid in the carrier.

Description

AN IMMUNOGENIC COMPOSITION OF KILLED LEPTOSPIRA BACTERIA

Field of the invention

The present invention pertains to a composition containing an immunogenic cell preparation of killed Leptospira bacteria, a vaccine to protect an animal against an infection with leptospira bacteria, and the use of an acid acid to stabilise an

immunogenic preparation of killed Leptospira bacteria.

Background art Leptospirosis is a world-wide zoonosis caused by infection with any of the many pathogenic serovars of Leptospira. The genus of the organisms is tremendously variable and contains in excess of 250 serovars. The prevalence of individual pathogenic serovars varies in different locales, probably influenced by such factors as climate, indigenous fauna and agricultural practices. Leptospirosis affects virtually all mammalian species. Human leptospirosis also is important and occurs endemically in the tropics and as epidemics in temperate climates. However, unlike other species, humans are not important in the maintenance of the disease in nature, and human-to- human transmission is rare.

Human vaccination against leptospirosis is a goal of the research community but progress has been extremely slow. Given the fact that canine leptospirosis is an important disease and that dogs are a source of leptospirosis, many vaccines are available against the canine disease. Commercial canine leptospirosis vaccines traditionally contain inactivated antigens of serogroups Canicola and

Icterohaemorrhagiae (Ictero), and have been available for more than fifty years. Recent recommendations for Europe are to continue inclusion in the vaccine of antigen of serogroups Canicola and Ictero, plus the inclusion of antigen of serogroups

Grippotyphosa (Grippo) and Australis. Whereas in the USA currently four canine vaccines with antigens of four serogroups (Canicola, ictero, Grippo and Pomona) are available, in Europe a number of traditional bivalent vaccines (Canicola, Ictero) and only one trivalent vaccine (Canicola, Ictero and Grippo) are currently available. Most commercially available leptospirosis vaccines, if not all, comprise an immunogenic cell preparation of killed Leptospira bacteria, such as for example formaline killed whole cells. These vaccines are known to be very stable at a storage temperature well below room temperature, typically between 2°C and 8°C. However, especially given the fact that leptospirosis is endemic in the tropics, it would be an important advantage of having vaccine stability at a temperature above room temperature, since this would allow storage under ambient conditions, not needing any cooling equipment. In the past, it has been tested whether denaturating any proteins that are present in such killed whole cell vaccines (for example by applying a heat shock treatment that does not affect the leptospira antigens), since such proteins might be responsible for the de-stability of the leptospira antigens, might lead to a significant improvement of the stability. The result however was negative.

Object of the invention There is a need for significantly improving the stability of compositions comprising an immunogenic cell preparation of killed Leptospira bacteria, in particular to arrive at a composition which still is effective in evoking an immune response, even after at least a three months storage at a temperature above room temperature. In particular, there is a need for a leptospirosis vaccine, which remains effective, at least in aiding in preventing an infection with Leptospira bacteria, even after a three month storage of the vaccine at 37°C, in particular after a nine month storage at this temperature.

Summary of the invention

In order to meet the object of the invention, a composition according to the preamble has been devised, wherein the immunogenic cell preparation of killed Leptospira bacteria is present in a citric acid solution (wherein the term "acid" includes the conjugated base of the acid). It has been shown that by having the antigens present in this solution, the stability of the antigens is markedly improved. A vaccine according to the present invention stored for at least three months at 37°C retaining an antigenic mass level sufficient to provide almost full protection against an infection with Leptospira bacteria. Definitions

A vaccine in the sense of this invention is a constitution suitable for application to an animal (including humans), comprising one or more antigens, typically combined with a pharmaceutically acceptable carrier such as a liquid containing water, optionally comprising immune stimulating agents (adjuvants), salts, buffers, preservatives, viscosity modifiers etc., which upon administration to the animal induces an immune response for treating a disease or disorder, i.e. aiding in preventing, ameliorating or curing the disease or disorder.

A carrier is a means for carrying the antigens in order to be able and present them in a practical form. Typical carriers are water or other hydrophilic liquids, but solid particles are also commonly used (for example for antigens administered via a dry powder nebuliser).

A cell preparation of killed bacteria is a preparation in essence containing killed bacterial cells, optionally further treated (which may include at least partly lysing the cells) for example to arrive at more stable or more immunogenic preparation. A cell preparation is opposed to a preparation that comprises an extract of bacterial cells such as extracted surface proteins and/or polysaccharides.

Embodiments of the invention

In an embodiment the solution comprises 20 mmols of citric acid per litre. In another embodiment the solution is buffered at a pH between 5 and 6.

In yet another embodiment the Leptospira bacteria are chosen from Leptospira interrogans serogroup Canicola serovar Portland-Vere, Leptospira interrogans serogroup Australis serovar Bratislava, Leptospira interrogans serogroup

lcterohaemorrhagiae serovar Copenhagen! and Leptospira kirschneri serogroup

Grippotyphosa serovar Dadas, in particular the Leptospira bacteria are Leptospira interrogans serogroup Australis serovar Bratislava. The use of citric acid to stabilise an immunogenic preparation of killed Leptospira bacteria in a liquid carrier, by dissolving the citric acid in the carrier, is in particular embodied in a use wherein the preparation retains at least 25% of its antigenic mass (measured in ELISA units, when compared to the same preparation stored at a temperature of 2-8°C) during a 3 month storage at a temperature above 18°C, in particular at 37°C.

Examples

The invention will now be further explained based on the following examples.

Example 1 : Determining the efficacy of killed leptospira antigens as present in a commercially available vaccine in dogs

Example 2: Determining the stability of leptospira antigens when stored at 37°C.

Example 3: Determining the stability of leptospira antigens formulated in citric acid when stored at 37°C.

Example 1

In this first example the efficacy of killed leptospira antigens as present in a

commercially available vaccine was determined in conventional dogs, the antigens being formulated according to the label of the vaccine (100%), or diluted four times (25%). Defined concentrations of chemically inactivated Leptospira bacteria of the same type and serovars as present in the commercially available vaccine Nobivac L4

(available from MSD Animal Health, Boxmeer, The Netherlands; see also the 21 May 2012 publication of the Summary of Opinion of the CVMP of the European Medicines Agency regarding Nobivac L4), viz. Leptospira interrogans serogroup Canicola serovar Portland-Vere (Leptospira canicola; 5300 ELISA Units/ml), Leptosira interrogans serogroup Australis serovar Bratislava (Leptospira Bratislava; 1000 ELISA Units/ml ), Leptosipra interrogans serogroup lcterohaemorrhagiae serovar Copenhagen!

(Leptospira icterohaemorrhagiae; 540 ELISA Units/ml) and Leptosipra kirschneri serogroup Grippotyphosa serovar Dadas (Leptospira grippothyphosa; 1000 ELISA Units/ml) were formulated in 10 mM PBS buffer at pH 7.2 (the buffer contained per litre water 8.9 gr NaCI, 0.2 gr KCI, 1.44 gr Na₂HP0₄ and 0.2 gr KH₂P0₄). The final formulation was filled into 3 ml vials, capped with rubber stoppers and sealed with aluminium crimp caps. This vaccine was stored at 2-8°C (stable for up to 39

months). Next to this vaccine, a diluted vaccine was made by diluting the antigen in the same PBS buffer to reach a final concentration that contains 25% of the amount of Elisa Units per ml. The vaccines were tested as follows.

Husbandry of the test animals

Six-week-old conventional beagle dogs without detectable agglutinating serum antibodies against each of the leptospira serogroups mentioned here above were provided by a commercial supplier. In each of the studies, treatment groups (n=8) consisted of pups of both sexes and pups derived from different litters in order to prevent gender and litter effects interfering with treatment effects. The selected dogs were free of clinical abnormalities or disease prior to inclusion in these studies.

Husbandry was the same in each study

Vaccination

In all studies the dogs were vaccinated twice subcutaneously, at the ages of 6 and 10 weeks with 1 ml of the respective vaccine.

Challenge

Challenge strains used were Leptospira interrogans serogroup Canicola serovar Canicola strain Moulton, Leptospira interrogans serogroup Icterohaemorrhagiae serovar Copenhagen! strain CF1 (both strains received from the National Leptospirosis

Reference Centre, KIT Biomedical Research, Amsterdam, The Netherlands),

Leptospira kirschneri serogroup Grippotyphosa serovar Bananal/Liangguang strain 1 1808, and Leptospira interrogans serogroup Australis serovar Bratislava strain As-05- 101 (both received from the United States Department of Agriculture, National Animal Diseases Center). For each of the four strains the following procedure was followed. The actual challenge material was prepared from a culture stored in liquid nitrogen, and passaged twice in Ellinghausen-McCullough medium modified by Johnson and Harris (Faine 1994; Clinical leptospirosis in humans, and clinical leptospirosis in animals; in Leptospira and Leptospirosis, CRC Press, 1994; pp 1 -13, 199-225). The cultures stored in liquid nitrogen were re-isolates from experimentally infected animals: strains CF1 and 1 1808 were hamster kidney re-isolates, strain Moulton was a dog kidney re-isolate, and strain As-05-101 was a dog urine re-isolate. In all studies challenge was performed three weeks after the second vaccination using a combination of two challenge routes: intraperitoneal (IP) injection (2 ml) and conjunctival instillation (0.25 ml into the ventral conjunctival sac of each eye). Bacterial cell concentrations (cells/ml) in the challenge materials were determined by total direct microscopic count under dark-field microscopy and were between 0.5 and 10x10⁹ cells/ml.

Examination

The dogs were examined regularly throughout each experiment, including measurement of body temperature after challenge using subcutaneous transponders. Blood and serum samples were taken by jugular venepuncture at regular intervals during the experiment for thrombocyte count, bacterial culture, PCR and leptospiral antibody assay. The serum samples were analysed by the microscopic agglutination test (MAT) against serogroups Canicola, lctero, Grippo and Australis (Faine 1994). The

microscopic agglutination titres were expressed as the reciprocals of the highest serum dilutions that induced 50 % agglutination. Blood samples using EDTA as anticoagulant were used for assessment of thrombocyte counts after challenge using a Cell-Dyn 3500 cell counter (Abbott Laboratories, Abbott Park, Illinois, USA).

EDTA or heparinised blood was used for re-isolation of challenge organisms from blood until day 21 post-challenge. For the same purpose urine samples were collected at regular intervals by cystocentesis, and kidney tissue taken at necropsy 4 weeks post- challenge was sampled and processed as described previously (Klaasen HLBM, Molkenboer MJCH, Vrijenhoek MP et al. Duration of immunity in dogs vaccinated against leptospirosis with a bivalent inactivated vaccine. Vet ^'crofc>/o/ 2003;95:121 - 132). Samples of blood, urine and kidney tissue homogenate were inoculated into EMJH medium and cultures were incubated and examined as described by Klaasen. Serum samples from days 0, 3 and 7 post-challenge were examined for the presence of DNA from challenge organisms with a real-time PCR targeting the secY gene. This PCR is a validated assay and is suitable for detection of DNA from all pathogenic Leptospira species (Ahmed A, Engelberts MFM, Boer KR et al. Development and validation of a real-time PCR for detection of pathogenic Leptospira species in clinical materials. PLoS ONE 2009;4(9):e7093).The PCR was performed by the WHO/FAO/OIE and National Leptospirosis Reference Centre, KIT Biomedical Research, Amsterdam, The

Netherlands. The PCR results were scored as positive, suspect or negative.

Dogs with severe clinical signs after challenge were humanely euthanised. Post-mortem examination including histopathological examination with special attention given to the detection of interstitial nephritis [Klaasen 2003] was undertaken in these cases.

The definition of a dog positive for infection was a dog with at least two positive samples of blood (by culture) or serum (by PCR) or urine/kidney (by culture) on different days or a dog with challenge-induced nephritis or clinical or haematological evidence

(thrombocytopenia) for leptospirosis. The definition of a dog positive for renal infection (carrier animal, persistent shedder) was a dog with at least one positive sample of urine/kidney from day 14 post-challenge onwards or challenge-induced nephritis (demonstrated by histopathological examination).

Results

Below are the results for the four vaccines (undiluted and diluted). It shows that the vaccines, even at 25% dilution, still aid in protecting the dogs against an infection and protect almost all of the challenged animals against renal infection.

Table 1 Challenge strain Canicola

Vaccine Dogs positive for infection Dogs positive for renal infection

100% canicola 2/8 0/8

25% canicola 1/8 1/8

control 8/8 8/8

Table 2 Challenge strain lcterohaemorrhagiae

Vaccine Dogs positive for infection Dogs positive for renal infection

100% ictero 0/7 0/7

25% ictero 3/7 0/7

Control 7/7 7/7 Table 3 Challenge strain Grippotyphosa

Example 2

The killed leptospira bacteria are stable at 2-8°C for at least 39 months (label of commercially available vaccine Nobivac L4). In this experiment it was determined whether the four antigens, formulated as indicated under Example 1 (100% vaccines) keep a level of at least 25% of ELISA units when stored at 37°C during three months. The results are indicated here-beneath. The resulting antigenic mass (mean value of three different batches) is calculated as a percentage of the resulting antigenic mass of the same vaccines stored at 2-8°C.

Table 5 Percentage AG mass after storage at 37°C, compared to storage at 2-8°C

It shows that the killed whole cell antigens of the serogroups lcterohaemorrhagiae, Canicola and Grippotyphosa are relatively stable at 37°C, but that the antigens of the serogroup Australis, serovar Bratislava are highly unstable and do not reach the at least 25% level, set as a desired criterion for effectiveness as a vaccine. Although at room temperature, typically 20-25°C the remaining level of antigenic mass may be higher, it is clear that stability, in particular for the Bratislava antigens, but also for the

Grippotyphosa and Canicola antigens may be improved.

Further tests to improve stability were conducted first with the Bratislava antigens only, expecting that any significant improvement will also improve the stability of the other antigens.

Example 3

Defined concentrations (1000 U/ml) of the same Bratislava antigens as described in Example 1 were formulated with citric acid, by dissolving sodium citrate in formulations with different pH. A first buffer was used leading to a pH of 5 (4.1 grams sodium citrate dehydrate and 1.3 grams citric acid monohydrate per litre), and a second one leading to a pH of 6 (5.4 grams of sodium citrate dehydrate and 0.33 grams citric acid

monohydrate per litre). Also, sodium citrate as such was dissolved, giving a pH of 7. As a control, the antigens were put in a PBS buffer

as mentioned in Example 1 (pH 7.2), and a 20 mM acetate buffer (pH 5; 1 .99 grams sodium acetate trihydrate and 0.32 grams acetic acid). Final concentrations as indicated in table 6 below were reached. These formulations were filled into 3 ml glass vials, capped with rubber stoppers and sealed with aluminium crimp caps.

Table 6 Formulations used for stability experiments of Bratislava antigens

Half of the vials were incubated at 2-8°C, while the other half were incubated at 37°C. After 3 and 9 months vials were analysed using an antigenic mass ELISA test for Leptospira bratislava antigens. The antigenic mass was determined against the antigenic mass of a reference standard. The results are indicated here-beneath in Table 7. The resulting antigenic mass (mean value of three different vials) is calculated as a percentage of the resulting antigenic mass of the same vaccines stored at 2-8°C.

Table 7 Percentage AG mass after storage at 37°C, compared to storage at 2-8°C

It shows that the stability of killed bacterial antigens of the serogroup Australis, serovar Bratislava is significantly increased by the addition of citric acid to the formulation. The desired criterion of at least 25% of the antigenic mass during a three months storage at 37° (and even considerably longer), compared to the vaccine stored under standard conditions has been met by at various pH levels, although a pH between 5 and 6 seems to lead to the best results. Using a pH of 5 as such, without the citric acid, does not lead to any usable result (formulation 5). A lower limit for the citric acid can easily be determined by routine experimentation: as long as the stability after three months storage at 37°C is 25% or higher, the desired stability criterion is met. An upper limit can be found the same way, although safety of the formulation might then also become a criterion. The required safety level can be set at any desired level and does not interfere with the present invention as such.

Given the high resemblance of antigens, it may reasonably be expected that by using the same measure (adding the Leptospira antigens to a citric acid solution), the stability of other killed bacterial leptospira antigens will be improved as well. This was tested and the results are indicated below with respect to Example 4.

Example 4

Defined concentrations of the same four Leptospira antigens as described in Example 1 were formulated with three different citric acid concentrations (10, 20 and 50 mmol/l) at pH 6. As a control, the antigens were put in a PBS buffer. The formulations were filled into 3 ml glass vials, capped with rubber stoppers and sealed with aluminium crimp caps. Half of the vials were incubated at 2-8°C, while the other half were incubated at 27°C. After 8 weeks and 9 months vials were analysed using an antigenic mass ELISA test for Leptospira antigens. The antigenic mass was determined against the antigenic mass of a reference standard. The results are indicated here-beneath in Tables 8, 9, 10 and 1 1 for the different types of antigens. The resulting antigenic mass (mean value of three different vials) is calculated as a percentage of the resulting antigenic mass of the same vaccines stored at 2-8°C.

Table 8 Percentage AG mass of Bratislava antigens after storage at 27°C, compared to storage at 2-8°C

Table 9 Percentage AG mass of Grippotyphosa antigens after storage at 27°C, compared to storage at 2-8°C

Citrate concentration [mM] 8 weeks storage 9 months storage

10 100 100

20 100 100

50 94 94

0 (control) 100 59 Table 10 Percentage AG mass of lcterohaemorrhagiae antigens after storage at 27°C, compared to storage at 2-8°C

Table 11 Percentage AG mass of Canicola antigens after storage at 27°C, compared to storage at 2-8°C

It shows that the stability of various types of killed bacterial leptospira antigens are significantly increased by the addition of citric acid to the formulation in line with the expectations based on the results with the Bratislava antigens. The desired criterion of at least 25% of the antigenic mass during a three months storage at 27° (and even considerably longer), compared to the vaccine stored under standard conditions has been met at various citric acid solutions.

Claims

1 . A composition containing an immunogenic cell preparation of killed Leptospira bacteria in a citric acid solution.

2. A composition according to claim 1 , characterised in that the solution comprises 20 mmols of citricacid per litre.

3. A composition according to any of the preceding claims, characterised in that the solution is buffered at a pH between 5 and 6.

4. A composition according to any of the preceding claims, characterised in that the Leptospira bacteria are chosen from Leptospira interrogans serogroup Canicola serovar Portland-Vere, Leptospira interrogans serogroup Australis serovar Bratislava,

Leptospira interrogans serogroup lcterohaemorrhagiae serovar Copenhagen! and Leptospira kirschneri serogroup Grippotyphosa serovar Dadas.

5. A composition according to claim 4, characterised in that the Leptospira bacteria are Leptosira interrogans serogroup Australis serovar Bratislava.

6. A vaccine to protect an animal against an infection with Leptospira bacteria, characterised in that the vaccine comprises a composition according to any of the preceding claims.

7. Use of citric acid to stabilise an immunogenic preparation of killed Leptospira bacteria in a liquid carrier, by dissolving the citric acid in the carrier.

8. A use according to claim 7, characterised in that the preparation retains at least 25% of its antigenic mass during a 3 month storage at a temperature above 18°C.

9. A use according to claim 7 or 8, characterised in that the preparation retains at least 25% of its antigenic mass during a 3 month storage at a temperature between 18°C and 37°C.

10. A use according to any of the claims 7-9, characterised in that the preparation retains at least 25% of its antigenic mass during a 3 month storage at 37°C.