CaniLeish for Dogs

By | 2012-01-29

Scientific discussion

This medicine is approved for use in the European Union

CaniLeish is a lyophilisate and solvent for suspension for injection, intended for the active immunisation of Leishmania negative dogs from 6 months of age to reduce the risk to develop an active infection and clinical disease after contact with Leishmania infantum.

The active substance of CaniLeish is Leishmania infantum excreted secreted proteins (ESP).

CaniLeish was eligible for the submission of a dossier for granting of a Community marketing authorisation via the centralised procedure under Article 3 (2) (a) of Regulation (EC) No 726/2004 which refers to medicinal products intended for use in animals containing a new active substance which was not authorised in the Community. The Committee also confirmed that the requirements for veterinary products intended for Minor Use or Minor Markets (MUMS) were met and therefore the provisions of the relevant guideline were applicable for this application.

No specific inspection was considered necessary with regard to CaniLeish. The presented pharmacovigilance system was considered satisfactory.

The benefits of CaniLeish are the stimulation of active immunity in Leishmania negative dogs from 6 months of age to reduce the risk to develop an active infection and clinical disease after contact with Leishmania infantum. The onset of immunity is 4 weeks after the primary vaccination course and the duration of immunity is 1 year after the last (re-)vaccination.

The most common side effects are moderate and transient local reactions that may occur after injection such as swelling, nodule, pain on palpation or erythema. These reactions resolve spontaneously within 2 to 15 days. Other transient signs commonly seen following vaccination may be observed such as hyperthermia, apathy and digestive disorders lasting 1 to 6 days. Allergic-type reactions are uncommon and appropriate symptomatic treatment should then be administered.

Canine leishmaniosis is a widespread infectious disease in endemic areas of the Mediterranean basin, Asia and America. This is a zoonosis considered as a serious veterinary problem with an increasing impact on public health. The disease is due to the development and multiplication in the macrophages and mononuclear cells of a protozoan parasite – Leishmania infantum. The infected dogs constitute the main domestic reservoir and play a central role in the accidental transmission of parasites to humans. The parasite is transmitted from an infected dog to a non-infected dog by the bites of sandflies of the genus Phlebotomus. The outcome of the infection is highly variable. Infected dogs may develop symptomatic infection resulting in death if not treated or develop only one or many mild symptoms but a high percentage of infected animals remain asymptomatic.

List of main abbreviations used frequently through the scientific discussion

BSA: Bovine Serum Albumin

Cv: coefficient pf variation (validation of methods)

CMLA: canine macrophage leishmanicidal activity

Con A: Concanavalin A

DTH: Delayed Type Hypersensitivity

ESP: Excreted Secreted Proteins

IFA: indirect fluorescent antibody test

IgG: Immunoglobulin G

LIP: Leishmania infantum promastigotes

IFN-y: Interferon gamma

IL: Interleukin

LLT: Lymphoblastic Transformation test

NNN medium: Novy – Nicolle – Mac Neal medium

Ph. Eur: European Pharmacopeia

PSA: Promastigote Surface Antigens

SC: Subcutaneous

Sd: Standard deviation (validation of methods)

SLA: Soluble leishmania antigens

SPC: Summary of Product Characteristics

Thl: Type 1 T helper cell (lymphocyte)

TSE: Transmissible spongiform encephalopathy

CaniLeish: Quality assessment

Qualitative and quantitative particulars of the constituents

Each dose of 1 ml of vaccine contains the following:

Freeze-dried fraction:

Substances Quantity per dose
Active ingredient Excreted-Secreted proteins (ESP) Not less than 100 µq
Adjuvant Quillaja saponaria purified extract
Excipients mannitol
sucrose
Trometamol

Liquid fraction: for 1 ml of solvent

Substances Quantity per dose
Excipients Sodium chloride Water for injection 9 mg Qs 1 ml

The active substance Excreted-Secreted Proteins are constituted of parasitic proteins that are characterised by a defined protein pattern. The quantitative formulation of the vaccine relies on a quantification of the total protein content by a non-specific test. Among these proteins, some antigens have a major role for induction of immunity.

In addition to the quantitative test, other tests (based on proteomic analyses) were developed to appreciate the quality of the parasitic proteins. These tests confirmed the representativeness of the protein patterns obtained at the end of the production process.

Containers

Freeze-dried fraction: A 3 ml insulin type vial made of neutral borosilicate type I glass is used (Ph. Eur. 3.2.1.) and sealed with a buthyl elastomer rubber lyophilisation stopper and an aluminium cap.

Liquid fraction (solvent): A 3 ml insulin type vial made of neutral borosilicate type I glass is used (Ph. Eur 3.2.1.) and sealed with a buthyl elastomer rubber stopper and an aluminium cap.

Treatment:

Vials: dry heat sterilization for sterilization and depyrogenation is implemented (according to Ph. Eur. 5.1.1)

Stoppers: autoclaving takes place.

Filling and stopping are conducted under a class A environment.

The certificates of controls conducted were provided and were acceptable.

Development Pharmaceutics

Canine leishmaniosis is a widespread infectious disease in endemic areas of the Mediterranean basin, Asia and America and is a zoonosis considered as a serious veterinary problem with an increasing impact on public health. The disease is due to a protozoan parasite – Leishmania infantum ( = Leishmania chagasi in South America). The infected dogs constitute the main domestic reservoir and play a central role in the transmission of parasites to humans. The parasite is transmitted from an infected dog to a non-infected dog by the bites of sandflies of the genus Phlebotomus.

In the sandfly (vector), the parasites exist as multiplicative procyclic promastigotes and infective metacyclic promastigotes (after differentiation in the digestive tract). After transmission to the mammalian host, through the bite of infected sandflies, the parasites enter into macrophages. They persist as intracellular amastigotes living predominantly in the phagolysosome of macrophages. After initial infection, amastigotes may replicate some time before triggering an inflammatory and adaptative immune response.

Method of manufacture

Flow charts of the production processes and steps were provided.

Lyophilisate

In the first phase the excreted secreted proteins active substance is produced after culture of the Leishmania infantum. In the second phase the final product is formulated / manufactured. The formulation is based on fixed antigen content per dose and a fixed amount of adjuvant. Constituents of the excipients and adjuvant are weighed, dissolved in water and sterilised. The L. infantum ESP active substance is added under stirring and sterile conditions. The pH of the excipient and adjuvant fraction is adjusted if necessary. After formulation, the product is filled, freeze-dried and packaged. The sealed vials are stored at 5+/-3 °C in a cold room until controls and release.

Liquid fraction (solvent)

This phase starts with the preparation of the vials and stoppers for liquid preparation, the production of the diluent, then sodium chloride powder is weighed, dissolved in water for injection, and sterilised. Then diluents bottles are filled, stopped and sealed and the sealed vials are autoclaved.

Control of starting materials

Starting materials listed in a pharmacopoeia

Dimethyl sulfoxide, hydrochloric acid concentrated mannitol, sodium chloride, sodium hydrogen carbonate, sodium hydroxide, sucrose, trometamol, highly purified water and water for injection. For all the above materials certificate of analyses were provided and found acceptable.

Starting materials not listed in a pharmacopoeia

Starting materials of biological origin

Description of starting materials of biological origin

Active substance: Leishmania infantum – origin and history

Origin: isolated from a man in Morocco in 1967.

History: the strain was adapted to aseric medium and parasites were selected and cloned using defined media. The resulting parasite strain thus originates from the Leishmania infantum reference strain and was used for the construction of the seed lot system.

Master seed

Three amplifications were performed on defined and aseric medium. A cryopreservant was added on the last harvest before storage in liquid nitrogen.

Working seed

The working seed comprises the master seed undergone a few passages. The amplifications were performed on defined and aseric medium. A cryopreservant was added on the last harvest before storage in liquid nitrogen.

Controls

The following controls are preformed on the master and working seed: identity, purity, stability after passages and research for extraneous agents.

The absence of extraneous viruses was investigated in the seeds (Master seeds and working seeds) based on the Ph. Eur. monograph 062. All tests produced satisfactory results.

Hemin chloride

Use: growth factor in the culture media

Source/origin: porcine origin – animals from the Netherlands subject to ante and post-mortem examination.

Controls were considered adequate taking into account the suppliers documentation on origin/source, the gamma irradiation certificate, the tests of sterility, growing capacity, physical and chemical characteristics, assay. In order to further guarantee the absence of risk of transmission of extraneous agents through the use of porcin hemin on the vaccine production process, the applicant:

• implemented a systematic control of extraneous viruses in this raw material before irradiation treatment

• provided a validation of the irradiation method to inactivate viruses

• provided a validation of the viral clearance efficacy of the dissolution of hemin chloride in sodium hydroxide 1M for storage before its use in the vaccine production.

Based on the above data, the applicant conducted a risk assessment, which was acceptable and justified that the risk of transmission of extraneous agents through the use of hemin is close to nil.

Purified extract of Quillaja saponaria

Use: adjuvant

Source/origin: vegetal origin

Controls were considered adequate taking into account the supplier’s documentation and identification by liquid chromatography. Amongst the tests performed on adjuvant by the supplier, there is testing of the haemolytic activity and HPLC profile.

Viral risk assessment

A detailed risk assessment was presented in compliance with Ph. Eur. 5.2.5. and Ph. Eur. 5.1.7 requirements.

Considering:

• the extraneous agent testing performed on the seed lot, manufacturing process including dilutions in aseric and defined culture media,

• the use of only one starting material of biological origin (hemin chloride from porcine origin) which is carefully sourced and undergoes drastic production process as well as irradiation

• absence of use of cells or substrates that could propagate hypothetical viral infectivity,

It can be concluded that the risk of transmitting extraneous agents through the use of this vaccine is close to nil.

Starting materials of non biological origin

Starting material purchased from defined suppliers

These are powder media used as components of the Leishmania infantum culture medium. Detailed composition was provided. These media are composed of aminoacids, vitamins and other components (salts, sugars, nucleotides) all from vegetable, mineral, yeast or chemical origin.

In-house media

Leishmania infantum parasites (LIP) culture medium

• Freeze-drying excipient: Composition: sucrose , mannitol, trometamol, water for injection

Specific measures concerning the prevention of the transmission of animal spongiform encephalopathy

A detailed risk assessment was presented in accordance with Ph. Eur. monograph 1483 and existing guidance documents.

Considering:

• that seed materials are prepared in aseric and axenic media and knowing that TSE infectivity is recognised to be established and maintained in vitro with high difficulty and only with cells of neural origin,

• the absence of use of serum and starting material of TSE susceptible species for the manufacture of the vaccine,

• the indication of the vaccine for dogs, which are not susceptible to TSE by subcutaneous route It can be concluded that the risk of transmitting TSE agents through the use of this vaccine is nil.

On the basis of the above the CVMP concluded that the starting materials of animal origin used in the production of the final product comply with the current regulatory texts related to the TSE Note for Guidance (EMEA/410/01-Rev.2) and Commission Directive 1999/104/EEC.

Overall conclusion on quality

The quality part was adequately documented. The production process is relatively simple and relies on the culture of Leishmania infantum in aseric and defined media which allows the removing of Leishmania through adequate processing steps and the recovering of Excreted Secreted Proteins that constitute the active substance of the vaccine.

An overview of the production process and the controls performed during the production of the freeze-drying fraction containing the active substance and of the liquid fraction was presented. The nature of the raw materials, manufacturing process, controls and treatments applied enable to ensure sterility of the vaccine and absence of introduction of any extraneous agent, and to ensure consistency and homogeneity of the production. This is ensured by the controls performed on raw materials and vaccine products as well as process parameters investigated and recorded during the manufacture.

Many tests have been developed by the applicant which enable to:

• Specifically identify the active substance and thus specific recognition of a major protein by specific antibodies after its migration by electrophoresis is achieved allowing confirmation of the presence of this major antigen and its integrity.

• Quantify the active substance: ESP are the only proteins present in the vaccine. A non-specific protein assay allows quantification of the total amount of proteins. This quantification is used to formulate the vaccine on a fixed target. As no protein is added in the finished product, this amount can be controlled by a newly developed test performed on the final vaccine.

• Validate the purity of the active substance: An electrophoresis in defined conditions ensures the conformity of the protein pattern with the expected profile. Validation demonstrated that this kind of test allows detection of the presence of an extra-protein or the over-expression on one particular protein.

On the final product, in addition a potency test is performed which allows to test the activity of the vaccine and the ability to induce an immune response.

These tests provide clear specifications for the active substance and ensure the consistency and homogeneity of the vaccine production and hence the safety and the efficacy of the released batches.

CaniLeish: Safety assessment

CaniLeish is a freeze-dried vaccine containing Excreted Secreted Proteins of the Leishmania infantum parasite in the promastigote form, adjuvanted with a purified extract of Quillaja saponaria.

One vaccine dose is formulated with a fixed target of protein – 110 µg of ESP – adjuvanted with 60 µg of purified extract of Quillaja saponaria and reconstituted with one dose of diluent before use. The vaccine is intended for the immunisation of healthy dogs against Leishmania infantum infection. The regimen of vaccination recommends three subcutaneous injections of one dose of vaccine at 3 weeks intervals in dogs from 6 months of age onwards (primary vaccination). An annual booster immunisation with one dose of vaccine is recommended (re-vaccination scheme).

The adjuvant of the vaccine (purified extract of Quillaja saponaria) belongs to saponins and derivatives which are known to have haemolytic activity. This lytic action on erythrocytes membrane depends on the structure of the saponin itself and on the physicochemical properties of the cells. For this reason, the applicant tested the effect of CaniLeish on dog erythrocytes and haemolytical analysis including red blood cell counts were performed during safety studies.

Overall conclusion on safety assessment

The safety of the vaccination with CaniLeish was investigated primarily in Leishmania free Beagle dogs receiving 3 standard doses (as described in the SPC) or repeated administrations or an overdose (2 doses) of a vaccine formulated with an overage of antigen. Studies demonstrated that mild local reactions such as swellings associated or not with redness, pain or scabs and a weak hyperthermia may be observed after vaccination that will resolve spontaneously within few days. These reactions have been described in the SPC and are regarded as acceptable post-vaccination reactions for a canine vaccine.

The tolerance of the vaccine was good in the dogs that had been in contact with the parasite before the first injection in one of the conducted field studies and therefore considered a statement in section “Special precautions for use” was included:

“Injection of the vaccine to dogs already infected by Leishmania infantum did not show any specific adverse reactions other than those described in section 4.6”.

For the user there is a risk of self injection, which is however very low. In addition, appropriate warnings and advice on the SPC have been included. For the environment there is negligible risk that the vaccine components may cause unexpected effects to the environment. As the target species is dogs there was no requirement for residue studies.

CaniLeish: Efficacy assessment

The following information on the epidemiology, cycle of transmission, disease and immunity related to Leishmania infantum (dossier + bibliography) are considered important for better understanding the rationale followed in the laboratory and field trials presented. Therefore some important information on the disease is presented below.

Overall conclusions on efficacy

The vaccine is intended to be used in Leishmania free dogs from 6 months of age onwards to protect against Leishmania infantum after 3 vaccine injections as primo-vaccination and an annual single booster vaccination.

Studies presented in this dossier confirmed the difficulty to assess the efficacy of a vaccine against a parasitic disease with heterogeneous evolution and manifestation.

The demonstration of efficacy of the vaccine was based on a key field trial of 2 years duration involving vaccinated and control dogs submitted to natural exposure to infection in zones with high infection pressure. After these 2 years, the vaccine was demonstrated to reduce the number of dogs developing an active infection in the vaccinated group and for a dog to significantly reduce the probability to become infected and to develop a clinical disease. The benefit of the vaccination was therefore estimated in zones with high infection pressure where it may decrease the risk to develop an active infection and a symptomatic disease after contact with the parasite for vaccinated animals. In conditions with weak prevalence of the disease, no clear benefit of the vaccination could be established. This may be linked to the high number of animals needed to demonstrate the benefit in lower prevalence zone.

Laboratory studies provide limited information despite many biological investigations including humoral (IgGland IgG2, against ESP and PSA, total IgG) and cellular (lymphoblastic transformation test, IFNy ELISPOT assay, canine macrophage leishmanicidal activity) immunity evaluation. No biomarker or immunological profile correlated with protection or infection could be defined. Nevertheless data on experimental challenges showed that infection can be detected from 4 to 9 months after the experimental infection and allowed a constant and homogenous response to vaccination. However such responses could not be clearly linked to protection and future response of the dogs to infection.

Considering the diversity of evolution of the infection and the variable incubation period that may last for months, it was difficult to define for this vaccine periods such as onset or duration of immunity and protection in a laboratory studies but a duration of immunity lasting a year after the last re-vaccination and an onset of immunity of 4 weeks were supported by the field data.

CaniLeish: Benefit risk assessment

CaniLeish is a vaccine intended to reduce the incidence of asymptomatic and symptomatic forms of Leishmania infection by induction of a specific cell-mediated immunity in vaccinated dogs. It is based on the role of the Excreted Secreted Proteins of L. infantum to induce cellular immune response. The vaccine is made of a freeze-dried pellet and a diluent. The adjuvant – purified extract of Quillaja saponaria – known to participate to the activation of the cellular immune response is included in the freeze-dried fraction.

The assessment of the application dossier took into account that this vaccine is intended for a limited market and some reductions in requirements according to the guideline on Data requirement for immunological veterinary products for minor use and minor species (EMEA/CVMP/IWP/123243/2006) were implemented.

Benefit assessment

Direct therapeutic benefits

The objective is to induce sufficient immunity to Leishmania free dogs from 6 months of age to reduce the risk to develop an active infection and clinical disease after contact with Leishmania infantum.

Field trials demonstrated that the product is capable of reducing the number of Leishmania free dogs developing an active infection and significantly reduce the probability to become infected and to develop a clinical disease after contact with Leishmania infantum. The benefit of the vaccination was estimated in zones with high infection pressure where it may decrease the risk to develop an active infection and a symptomatic disease after contact with the parasite for vaccinated animals.

Additional benefits

CaniLeish is the first vaccine to be authorised for the prophylaxis against Leishmania infantum in Europe. The duration of immunity for the vaccine has been shown to be 1 year after the last re-vaccination and the onset of immunity 4 weeks.

Vaccination has been shown to be safe for Leishmania infected animals. Risk assessment Main potential risks

a) There is a risk of moderate and transient local reactions may occur such as swelling, nodule, pain on palpation or erythema. These reactions resolve spontaneously within 2 to 15 days. Other transient signs commonly seen following vaccination may be observed such as hyperthermia, apathy and digestive disorders lasting 1 to 6 days. Allergic-type reactions are uncommon and appropriate symptomatic treatment should then be administered

b) For the user there is a very low risk of self injection. Appropriate warnings and advice on the SPC will serve to minimise this risk.

c) For the environment there is negligible risk that the vaccine components may cause unexpected effects to the environment.

Specific potential risks, according to product type and application

a) Efficacy results do not show complete protection of vaccinated dogs. Despite vaccination a percentage of dogs still became infected with Leishmania infantum and from those vaccinated dogs that became infected a percentage of animals also developed clinical signs of the disease.

b) The benefit of the vaccination was established in zones with high infection pressure, whereas no clear benefit could be established in areas of low infection pressure.

c) In dogs developing Leishmaniosis (active infection and/or disease) despite vaccination, proceeding with the vaccine injections showed no benefit.

Risk management or mitigation measures

a) Appropriate warnings have been placed in the SPC to warn of the potential risks to the target animal, end user and environment.

b) Appropriate warnings have been placed in the SPC to clarify the limitations of the indication, the limitations of benefit in low infection areas and the lack of benefit in continuing the vaccination in vaccinated dogs that have developed the disease.

Evaluation of the benefit risk balance

Leishmaniosis is an important disease in dogs that is endemic in the Mediterranean countries of Europe, the Middle East and many subtropical areas of the world. In the past decade, an increased incidence of canine leishmaniosis in endemic zones as well as spread of the infection to non-endemic areas of Europe has been observed. Canines are the main reservoir for the parasites and play a relevant role in transmission to humans. The aetiological agent – Leishmania infantum – is transmitted by sandflies of the genus Phlebotomus.

In endemic areas dogs become exposed immediately. Evolution of the infection in dogs is then complex and unpredictable. Some will develop protective immunity, some remain asymptomatic after infection and may relapse later and others develop a clinical disease. It is considered that establishment of infection and development of the disease both depend on the host’s immunological response and that once the parasite escapes immunity and is able to multiply, no clearance is possible anymore. Infection may evolve over a period of a few weeks to several months toward disease patterns that can be extremely variable and polymorphic, which makes it difficult to classify dogs within specific categories.

Along with typical clinical signs and history of exposure, the diagnosis is based on microscopic identification of the parasite or PCR testing on bone marrow samples. Serological techniques may reveal active infection.

The management of dogs infected with Leishmania is currently based on sanitary and/or medical prophylaxis but up to now, both showed limited capacity to fulfil eradication, or even control of canine leishmaniosis.

Sanitary measures are based on preventing physical contact of dogs with vector, reducing the microhabitats to sandflies, and employing insecticide (environmental or topical). Despite implementation of all these measures, canine Leishmaniosis could not be reduced efficiently. Moreover culling of seropositive dogs has not proved to be efficient; although this solution was adopted in Brazil it currently has failed to prevent the number of human cases to increase.

If applied medical treatment in diseased dogs consists of symptomatic treatments associated to leishmanicidal molecules (meglumine antimoniate, aminosidine, miltefosine) which reduce or eliminate clinical symptoms but do not achieve parasitological cure. The epidemiological risk persists and dogs that respond to chemotherapy can nevertheless experience clinical relapse after the cessation of treatment or during it. Besides, these medicines have shown to have a number of disadvantages such as price, repeated injections, hepato- and nephrotoxicity, which can make compliance to treatment quite difficult to achieve.

On the basis of the above and although efficacy results have not shown complete protection of vaccinated dogs, it can be concluded that vaccination against Leishmaniosis can become a valuable and/or complementary alternative to the existing tools, despite the limits of the vaccine. Despite the fact that complete protection against Leishmaniosis or eradication of the disease cannot be achieved, this vaccine is able to reduce the risk for developing active infection and disease at the individual scale and to participate to reduction of incidence of the disease at the level of a dog population. Additionally and although the epidemiological impact of the vaccination cannot be estimated from the provided data of this application, it is nevertheless expected that improvement of the situation in dogs with regard to Leishmaniosis will also have a positive impact on human health. Finally, no risk has been linked to the use of this vaccine in dogs (included infected ones with Leishmaniosis).

Hence, the benefit-risk assessment of this vaccine appears favourable for this vaccine, within the limits highlighted in the SPC.

Conclusion on benefit risk balance

The information provided in the dossier and in response to points raised is sufficient to confirm an overall positive benefit risk balance.

Conclusion

Based on the original and complementary data presented the Committee for Medicinal Products for Veterinary Use (CVMP) concluded that the overall benefit-risk balance was considered favourable for authorisation.