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WO2025132735A1 - A freeze-dried composition containing live attenuated pathogens, a process for preparing a freeze-dried composition, a vaccine, and a method of vaccinating a host animal - Google Patents

A freeze-dried composition containing live attenuated pathogens, a process for preparing a freeze-dried composition, a vaccine, and a method of vaccinating a host animal Download PDF

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Publication number
WO2025132735A1
WO2025132735A1 PCT/EP2024/087327 EP2024087327W WO2025132735A1 WO 2025132735 A1 WO2025132735 A1 WO 2025132735A1 EP 2024087327 W EP2024087327 W EP 2024087327W WO 2025132735 A1 WO2025132735 A1 WO 2025132735A1
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Prior art keywords
freeze
vaccine
composition
vol
drying
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French (fr)
Inventor
Alyssa ANDERSON
Savannah HARVEY
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Intervet International BV
Intervet Inc
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Intervet International BV
Intervet Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/102—Pasteurellales, e.g. Actinobacillus, Pasteurella; Haemophilus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells avirulent or attenuated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine

Definitions

  • a freeze-dried composition containing live attenuated pathogens a process for preparing a freeze-dried composition, a vaccine, and a method of vaccinating a host animal
  • the present invention relates to the field of vaccinology. More specifically, the present invention relates to a freeze-dried composition containing live attenuated pathogens. The invention furthermore relates to a vaccine comprising such a composition, and a method to vaccinate a host animal by administration of a vaccine comprising such a composition.
  • Vaccines are widely used to protect both human and animals against many different diseases.
  • a vaccine composition typically contains an agent that resembles a diseasecausing microorganism or component thereof, such as weakened or killed forms of the microorganism, a toxin, or (one of) its surface proteins.
  • the agent stimulates the body's immune system to recognize the agent as a threat and destroy it, and to recognize and destroy any of the microorganisms associated with that agent that it may encounter in the future.
  • Vaccines are commonly administered as an intramuscular or subcutaneous injection, as an intranasal spray or in the form of an orally administered liquid.
  • Bovine respiratory disease also known as shipping fever pneumonia or undifferentiated fever
  • BRD Bovine respiratory disease
  • Symptoms of BRD include fever, depression and increase of the respiration frequency.
  • BRD is characterized by lung lesions such as necrosis, thrombosis and exudation.
  • the most important causative bacteria of BRD are Mannheimia haemolytica, Pasteurella multocida, Mycoplasma bovis, and Histophilus somni. Prior to 2003, Histophilus somni was considered to be divided in three different species, i.e. Haemophilus somnus, Histophilus ovis, and Histophilus agni.
  • Several vaccines have been developed to mitigate or prevent BRD.
  • Commercial vaccines comprise for example antigens of Mannheimia haemolytica and/or Pasteurella multocida, such as in the form of bacterins or toxoids (de-toxified toxins).
  • US 2019/0381161 A1 describes an oral vaccine against respiratory disease in ruminants comprising live attenuated Mannheimia haemolytica and Pasteurella multocida bacteria.
  • the vaccine comprises 0.8 wt/vol% Polyvinylpyrrolidone (PVP) K-12 originating from the culturing media of the bacteria, and a bulking agent containing sucrose and PVP K-60 to a final concentration of 0.5 wt/vol% PVP K-60 and 6% sucrose.
  • PVP Polyvinylpyrrolidone
  • Vaccines are often administered in liquid form.
  • vaccines formulated as liquids can be subject to chemical degradation, e.g., aggregation, denaturation, hydrolysis, or oxidation between the time of production and the time of administration of the agent or excipients. This can result in a reduced effectiveness of the vaccine due to e.g. (partial) inactivation of the agent.
  • Liquid vaccine compositions can also be sensitive to temperature: high temperatures can increase inactivation, and freezing temperatures can result in ice that can damage the agent present in the vaccine.
  • vaccines that are to be stored in a frozen state often comprise a cryoprotectant, such as glycerol, allowing storage at temperatures of -20°C or less for extended periods of time.
  • a cryoprotectant such as glycerol
  • freeze-drying of the vaccine composition Another way of extending the time a vaccine can be stably stored is freeze-drying of the vaccine composition. This has additional advantages over a liquid composition.
  • a freeze-dried composition is for example lighter and therefore more economical to transport.
  • freeze-dried vaccine compositions usually do not need to be stored frozen but can often be stored at a more economical temperature of 2-8 °C.
  • a disadvantage of freeze-drying is that the process of freeze-drying can negatively affect the activity of the agent present in the vaccine composition. Since part of the activity of the agent is lost during freeze-drying, the vaccine obtained by reconstituting the freeze-dried composition is less effective.
  • a mixture comprising N-Z amine, dextran, gelatin, and lactose can be added to the vaccine composition before freeze-drying. It is used to protect both viruses and bacteria, and is for example employed in the commercially available vaccines Bovilis® Nasalgen® 3, Bovilis® Nasalgen® 3-PMH, Bovilis® Once® PMH® SQ, Bovilis® Vista® Once SQ, and Bovilis® Coronavirus.
  • this protective mixture a portion of the activity of the agent is still lost during freeze-drying. Therefore, there remains a need of compositions comprising pathogens which lose a lower amount of activity during freeze-drying.
  • composition comprising pathogens which composition retains a relatively high antigenic activity during freeze- drying.
  • Freeze-drying of a composition comprising pathogens and at least 1 wt/vol% PVP K-12, based on the composition before freeze-drying, and sucrose resulted in a relatively low pathogen titer log loss.
  • the pathogens remain relatively stable during freeze-drying in the presence of 1 wt/vol% PVP K-12 and sucrose. Since a relatively large portion of the freeze-dried pathogens remains active, the freeze-dried composition has a relatively high antigenic activity. Thus, the process of preparing a freeze-dried composition comprising the pathogen is relatively efficient.
  • PVP K-12 is known as a growth medium compound for bacteria.
  • the antigenic activity in a standard volume of vaccine, e.g. 2 mL, prepared from the freeze-dried composition is relatively high.
  • the vaccine prepared from the freeze-dried composition can be used to vaccinate an animal.
  • the invention relates to a method of vaccinating an animal by parenteral administration of vaccine comprising live attenuated pathogens, a bulking agent that comprises in combination (i) polyvinylpyrrolidone with a weight- averaged molecular weight between 1 ,000 and 7,000 Da in an amount of at least 1 wt/vol%, based on the composition before freeze-drying, and (ii) a sugar, and a pharmaceutically acceptable carrier.
  • the pathogen titer and therefore the antigenic titer of the vaccine prepared from the freeze-dried composition is relatively high, a relatively small amount of live attenuated pathogens providing the antigen is sufficient for an effective vaccine.
  • the vaccine is relatively cheap to produce.
  • Live in the sense of this invention means that the pathogen is replicative in a host and/or in vitro.
  • Adjuvants enhances the immune response to a vaccine.
  • Adjuvants are for example aluminum salts, such as aluminum hydroxide, aluminum phosphate and aluminum sulphate, mineral oils such as paraffin oil and squalene, and adjuvant compositions such as Freund’s complete adjuvant and Freund’s incomplete adjuvant.
  • the “low-temperature glass transition temperature” (Tg’) is the temperature at which an amorphous material transitions from a viscous or “rubbery” state to a brittle “glass” state.
  • the “collapse temperature” (Tc) is the temperature at which an amorphous material softens to the point that it can no longer support its own structure.
  • the present invention relates to a freeze-dried composition
  • a freeze-dried composition comprising live attenuated pathogens, and a bulking agent that comprises in combination (i) polyvinylpyrrolidone (PVP) with a weight-averaged molecular weight between 1 ,000 Da and 7,000 Da in an amount of at least 1 wt/vol%, based on the composition before freeze- drying, and (ii) a sugar.
  • PVP polyvinylpyrrolidone
  • the freeze-dried composition is obtained by freeze-drying a composition comprising live attenuated pathogens, PVP with a weight-averaged molecular weight between 1 ,000 Da and 7,000 Da, and a sugar.
  • the freeze-dried composition typically is a freeze-dried cake which may adopt various forms, such as a layer in a bottle, a tablet or a spherical object such as a lyosphere.
  • Live attenuated pathogens, or modified-live pathogens have long been known to be useful for vaccination.
  • Stable attenuation can be achieved by introducing a mutation that induces a loss in replicative or infective capacity of the pathogen, such as a mutation to external organelles, coat or capsule, to the expression of a virulence factor, or to the internal organization.
  • Attenuated pathogens can be obtained in a variety of ways, such as through a method of direct or aspecific induced mutation, e.g. by consecutive in vivo or in vitro passaging, use of mutagens such as chemicals or ionizing radiation, or recombinant DNA technology.
  • a pathogen may be considered attenuated with respect to the infection of a particular host, while it is fully pathogenic with respect to another host.
  • pathogens are well known in the art. Generally, the pathogens are grown in a suitable growth medium and under suitable growth conditions to allow the multiplication of the pathogen. After a period of growth, typically between 24 and 72 hours at a temperature of between 30 and 40°C, the pathogens are harvested and optionally processed. Finally, the pathogens are incorporated into a composition for freeze-drying.
  • the pathogens are preferably bacteria.
  • bovine respiratory disease is economically important, since it is the most common and costly disease affecting beef cattle in the world. Therefore, the pathogens are more preferably bacteria associated with bovine respiratory disease.
  • the bacteria are selected from the bacterial species which are the most important cause of BRD, i.e. selected from the species of Mannheimia haemolytica, Pasteurella multocida, Mycoplasma bovis, Histophilus somni, and a combination of two or more thereof.
  • the freeze-dried composition comprises the species Histophilus somni. It is herein understood that that species Histophilus somni includes the species Haemophilus somnus, Histophilus ovis, and Histophilus agni, as they were known prior to 2003.
  • PVP having a relatively high weight-averaged molecular weight such as PVP of the K-60 class
  • PVP K-60 may be unsafe for subcutaneous administration. Due to the relatively large size, PVP K-60 cannot be excreted by the kidneys. This may result in accumulation of PVP K- 60 in the body of the vaccinated animal.
  • the relatively small PVP K-12 is known as a medium component, but was found to reduce the antigenic titer of the pathogen culture grown in the presence of PVP K-12. It was therefore very surprising that it was found that when live attenuated pathogens were combined with PVP K-12 and sucrose after harvesting of the pathogens, relatively little pathogenic titer was lost during freeze-drying.
  • the freeze-dried composition comprises PVP with a weight-averaged molecular weight of between 1 ,000 Da and 7,000 Da.
  • the PVP has a weight-averaged molecular weight of between 2,000 and 6,500 Da. More preferably, the PVP has a weight- averaged molecular weight of between 3,000 and 6,000 Da, and most preferably between 4,000 and 5,500 Da. Too little PVP will likely be less effective in protecting the pathogens against inactivation during freeze-drying. Therefore, the freeze-dried composition comprises at least 1 wt/vol% PVP, based on the composition before freeze-drying.
  • the freeze-dried composition comprises at least 1.1 wt/vol% PVP, such as at least 1.2 vol/wt% PVP, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or even at least 2 vol/wt% PVP, such as at least 2.1 wt/vol%, at least 2.2 wt/vol%, at least 2.3 wt/vol%, at least 2.4 wt/vol%, at least 2.5 wt/vol%, at least 2.6 wt/vol%, at least 2.7 wt/vol%, at least 2.8 wt/vol.% or at least 2.9 wt/vol%,
  • the freeze-dried composition also comprises a sugar. It was shown that the sugar in particular contributes to a relatively high low-temperature glass transition temperature (Tg’) and collapse temperature (Tc). Thus, the Tg’ and Tc are reached relatively quickly, and the freeze-drying cycle can be relatively short. This contributes to a more economical freeze- drying process.
  • the sugar is a disaccharide. More preferably, the sugar is chosen from the group of sucrose, lactose, maltose, trehalose, and a combination of two or more therefore, even more preferably from the group of maltose and trehalose, and a combination thereof. Most preferably the sugar comprises sucrose.
  • the freeze-dried composition comprises at least 1 wt/vol% sugar, more preferably at least 2.5 wt/vol%, even more preferably at least 5 wt/vol.%, and most preferably at least 7.5 wt/vol% sugar, based on the composition before freeze-drying.
  • the freeze-dried composition preferably has a Tc of at most -40°C, preferably at most -37.5°C, more preferably at most -35°C, and most preferably at most -32.5°C, and/or a Tg’ of at most -40°C, preferably at most -37.5°C, more preferably at most -35°C, and most preferably at most -32.5°C.
  • the live attenuated pathogen is the active component of the freeze- dried composition providing the antigen. Incorporating a relatively high amount of sugar leaves less room for the pathogens. Therefore, the freeze-dried composition preferably comprises at most 20 wt/vol% sugar, more preferably at most 18 wt/vol%, and most preferably at most 16 wt/vol% sugar, based on the composition before freeze-drying.
  • freeze-dried composition comprises one or more further components such as a further pathogen, an adjuvant, or a preservative.
  • the freeze-dried composition comprises gelatin. It was found that the additional presence of gelatin in the bulking agent protects the pathogens in the composition even more against inactivation during freeze-drying. Therefore, the freeze-dried composition preferably comprises at least 0.1 wt/vol% gelatin, based on the composition before freeze- drying. More preferably, the freeze-dried composition comprises at least 0.2 wt/vol% gelatin, even more preferably at least 0.4 wt/vol%, and most preferably at least 0.6 wt/vol%, based on the composition before freeze-drying.
  • the live attenuated pathogen is the active component of the freeze- dried composition providing the antigen. Incorporating a relatively high amount of gelatin leaves less room for the pathogens. Therefore, the freeze-dried composition preferably comprises at most 2 wt/vol% gelatin, more preferably at most 1 .5 wt/vol%, and most preferably at most 1 wt/vol%, based on the composition before freeze-drying.
  • the PVP is added to the composition after harvesting of the pathogens and before freeze-drying.
  • the invention relates to a process of preparing a freeze-dried composition comprising a live attenuated pathogen and a bulking agent comprising polyvinylpyrrolidone with a weight-averaged molecular weight between 1 ,000 and 7,000 Da and a sugar, comprising the steps of: culturing live attenuated pathogens, harvesting and optionally processing the live attenuated pathogens to yield a first composition comprising live attenuated pathogens, combining the composition comprising live attenuated pathogens with polyvinylpyrrolidone with a weight-averaged molecular weight between 1000 and 7000 Da in an amount of at least 1 wt/vol%, based on the composition before freeze drying, and a sugar, to yield a second composition comprising live attenuated pathogens, and freeze-drying the second composition comprising live attenuated pathogens to provide the freeze-dried composition.
  • the obtained freeze-dried composition has a relatively high pathogen titer, and therefore a relatively high antigenic titer. Furthermore, the freeze-dried composition is convenient for storage and transport. However, in order to be suitable for administration to an animal, the freeze-dried composition is reconstituted in order to prepare a vaccine ready for administration.
  • the invention relates to a vaccine comprising a freeze-dried composition comprising live attenuated pathogens, a bulking agent that comprises in combination (i) polyvinylpyrrolidone (PVP) with a weight-averaged molecular weight between 1 ,000 Da and 7,000 Da in an amount of at least 1 wt/vol%, based on the composition before freeze-drying, and (ii) a sugar, and a pharmaceutically acceptable carrier.
  • PVP polyvinylpyrrolidone
  • the pharmaceutically acceptable carrier provides liquid to rehydrate the freeze-dried composition.
  • the pathogens present in the freeze-dried composition are reactivated, and a vaccine in the form of a suspension of live attenuated pathogens is provided.
  • the pharmaceutically acceptable carrier is sterile water or a sterile physiological saline solution.
  • the invention relates to a method of vaccinating a host animal by administration of a vaccine comprising a freeze-dried composition comprising live attenuated pathogens, a bulking agent that comprises in combination (i) polyvinylpyrrolidone (PVP) with a weight- averaged molecular weight between 1 ,000 Da and 7,000 Da in an amount of at least 1 wt/vol%, based on the composition before freeze-drying, and (ii) a sugar, and a pharmaceutically acceptable carrier.
  • PVP polyvinylpyrrolidone
  • the vaccine may be administered in any way suitable for a particular host animal. It may for example be administered enterally, such as via the oral or rectal route. Preferably, the vaccine is administered parenterally, such as intramuscularly, intradermally, or intravenously. In particular subcutaneous vaccination is a convenient way of vaccinating animals, especially in case of relatively large animals. Therefore, the vaccine is most preferably administered subcutaneously.
  • a dose of the vaccine is preferably administered to a host animal of between 2 and 6 weeks of age, such as to a host animal of 3, 4, or 5 weeks of age.
  • the vaccine may be administered in any suitable regimen. It may for example be administered as a single dose, or as multiple doses over the course of several weeks or months.
  • the vaccine is for example administered as a first prime dose and a second booster dose. If the vaccine is administered as multiple doses, the second dose is preferably administered at least 2 weeks and at most 8 weeks after the first dose. More preferably, the second dose is administered at least 4 weeks and at most 6 weeks after the first dose. In other words, the second dose is preferably administered when the host animal is between 4 and 14 weeks of age, such as when the host animal is 6, 8, 10 or 12 weeks of age.
  • Ruminants are a particularly large group of animals comprising roughly 200 domestic and wild species.
  • the host animal that is vaccinated is preferably a ruminant, such as a goat, sheep, or cervine.
  • bovine animals including cattle are of special economic importance in e.g. the dairy and meat industry.
  • the host animal is therefore more preferably a bovine animal.
  • the host animal is selected from the group of cattle, such as a cow, buffalo or bison.
  • bovine respiratory disease is the most common and costly disease affecting beef cattle in the world. Therefore, the host animal is preferably vaccinated against bovine respiratory disease.
  • Histophilus somni was cultured in Tryptic Soy broth. The whole culture was harvested, and attenuated live whole bacterial cells were used as antigen for the preparation of a vaccine composition.
  • the composition for freeze-drying was prepared by combining harvested Histophilus somni culture with a bulking agent in the volume ratio indicated in Table 1 .
  • the various prepared compositions comprised bulking agent components in a final amount as indicated in Table 1 .
  • compositions for freeze-drying Culture volume and bulking agent volume are indicated as vol/vol% based on the composition before freeze-drying.
  • Bulking agent components are indicated as wt/vo!% based on the composition before freeze-drying.
  • the titer of Histophilus somni of the compositions was determined both before and after freeze-drying using a standard titer determination protocol.
  • compositions comprising Histophilus somni and a bulking agent as indicated in Table 1 were used before the compositions were subjected to freeze-drying.
  • sterile water was added to the freeze-dried composition and the composition was allowed to rehydrate for at least 10 minutes.
  • the colonies of the plates containing 30-300 colonies were counted and averaged.
  • the average counts were converted to CFU per mL by multiplying by the reciprocal of the dilution, and subsequently dividing the result by 0.1 to convert to milliliters.
  • Tg low- temperature glass transition temperature
  • Pathogen titer log loss of Histophilus somni after freeze-drying of a composition comprising Histophilus somni and the bulking agent composition as indicated.
  • Culture volume and bulking agent volume are indicated as vol/vol% based on the composition before freeze-drying.
  • Bulking agent components are indicated as wt/vol% based on the composition before freeze-drying.
  • BRSV after freeze-drying.
  • Culture volume and bulking agent volume are indicated as vol/vol% based on the composition before freeze-drying.
  • Bulking agent components are as indicated in table 2 (rows in Table 2a correspond to rows in Table 2), based on the composition before freeze-drying.
  • the effect on improving the titer loss is independent of the type of pathogen.
  • the presence of a relatively high amount of sucrose increases the Tc and Tg’ of the freeze-dried composition as compared to a relatively low amount of sucrose. Also in the additional presence of gelatin, the Tc and Tg’ are relatively high, as compared to compositions comprising relatively low amounts of sucrose.
  • a bulking agent comprising at least 1 wt/vol% PVP K-12, based on the composition before freeze- drying, and a relatively high amount of sucrose and optionally gelatin also has a relatively high Tc and Tg’.
  • freeze-drying of a composition comprising at least 1 wt/vol% PVP K- 12, based on the composition before freeze-drying, and a relatively high amount of sucrose and optionally gelatin can be performed relatively economically since the Tc and Tg’ can be reached relatively quickly and the dry-freezing cycle can be relatively short.
  • Standard growth medium for Histophilus somni with or without 1 wt/vol% PVP K-12 was inoculated with x amount of Histophilus somni.
  • the condition including 1 wt/vol% PVP K- 12 was performed in duplicate. 8 hours after inoculation, the optical density (OD) of the cultures was measured at 540 nm to determine the total amount of bacteria present in the culture at this time point. Furthermore, the titer of Histophilus somni corresponding to the amount of live bacteria was measured as described for Example 1 .
  • a vaccine composition comprising 2.25 wt/vol% PVP K-12, 8.4 wt/vol% sucrose and 0.75 wt/vol% gelatin was prepared by combining harvested Histophilus somni and a bulking agent comprising PVP K-12, sucrose and gelatin as described in Example 1 in a volume ratio of 85:15.
  • the vaccine composition contained Histophilus somni at a titer of about 1x10 8 CFU/mL.
  • the vaccine composition was freeze-dried.
  • the vaccine composition was reconstituted with 2 mL sterile water before vaccination, and injected subcutaneously.
  • All calves from the vaccine groups received a subcutaneous dose at about four weeks of age.
  • the calves receiving a double dose received a second subcutaneous dose about two weeks after the first dose.
  • the average lung lesion score was strongly reduced in calves that were vaccinated with a vaccine obtained from reconstituting a freeze-dried vaccine composition comprising live attenuated Histophilus somni, 2.25 wt/vol% PVP K-12 and 0.75 wt/vol% gelatin, both when the calves received a single dose and when the calves received a double dose.
  • the vaccine obtained from reconstituting a freeze-dried vaccine composition comprising live attenuated Histophilus somni, 2.25 wt/vol% PVP K-12, 8.4 wt/vol% sucrose, and 0.75 wt/vol% gelatin protects against a challenge with virulent Histophilus somni compared to unvaccinated control animals.

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Abstract

In order to provide a composition which has retained a relatively high antigenic activity during freeze-drying, a freeze-dried composition is provided comprising live attenuated pathogens, and a bulking agent that comprises in combination (i) polyvinylpyrrolidone with a weight-averaged molecular weight between 1000 and 7000 Da in an amount of at least 1 wt/vol%, based on the composition before freeze drying, and (ii) a sugar.

Description

A freeze-dried composition containing live attenuated pathogens, a process for preparing a freeze-dried composition, a vaccine, and a method of vaccinating a host animal
TECHNICAL FIELD
The present invention relates to the field of vaccinology. More specifically, the present invention relates to a freeze-dried composition containing live attenuated pathogens. The invention furthermore relates to a vaccine comprising such a composition, and a method to vaccinate a host animal by administration of a vaccine comprising such a composition.
BACKGROUND
Vaccines are widely used to protect both human and animals against many different diseases. A vaccine composition typically contains an agent that resembles a diseasecausing microorganism or component thereof, such as weakened or killed forms of the microorganism, a toxin, or (one of) its surface proteins. The agent stimulates the body's immune system to recognize the agent as a threat and destroy it, and to recognize and destroy any of the microorganisms associated with that agent that it may encounter in the future. Vaccines are commonly administered as an intramuscular or subcutaneous injection, as an intranasal spray or in the form of an orally administered liquid.
Bovine respiratory disease (BRD), also known as shipping fever pneumonia or undifferentiated fever, is a respiratory disease of cattle and is the most common and costly disease affecting beef cattle in the world. It is a multifactorial syndrome and develops as a result of complex interactions between environmental factors, host factors, and pathogens. It commonly arises when environmental stressors, such as handling, transport or weaning, and an initial pathogen weaken the immune system of the host animal. The causative bacteria can then colonize the lower respiratory tract of the animal. Symptoms of BRD include fever, depression and increase of the respiration frequency. At a histological level BRD is characterized by lung lesions such as necrosis, thrombosis and exudation. The most important causative bacteria of BRD are Mannheimia haemolytica, Pasteurella multocida, Mycoplasma bovis, and Histophilus somni. Prior to 2003, Histophilus somni was considered to be divided in three different species, i.e. Haemophilus somnus, Histophilus ovis, and Histophilus agni. Several vaccines have been developed to mitigate or prevent BRD. Commercial vaccines comprise for example antigens of Mannheimia haemolytica and/or Pasteurella multocida, such as in the form of bacterins or toxoids (de-toxified toxins).
US 2019/0381161 A1 describes an oral vaccine against respiratory disease in ruminants comprising live attenuated Mannheimia haemolytica and Pasteurella multocida bacteria. The vaccine comprises 0.8 wt/vol% Polyvinylpyrrolidone (PVP) K-12 originating from the culturing media of the bacteria, and a bulking agent containing sucrose and PVP K-60 to a final concentration of 0.5 wt/vol% PVP K-60 and 6% sucrose.
Vaccines are often administered in liquid form. However, vaccines formulated as liquids can be subject to chemical degradation, e.g., aggregation, denaturation, hydrolysis, or oxidation between the time of production and the time of administration of the agent or excipients. This can result in a reduced effectiveness of the vaccine due to e.g. (partial) inactivation of the agent. Liquid vaccine compositions can also be sensitive to temperature: high temperatures can increase inactivation, and freezing temperatures can result in ice that can damage the agent present in the vaccine.
To prevent the vaccine from damaging due to freezing, vaccines that are to be stored in a frozen state often comprise a cryoprotectant, such as glycerol, allowing storage at temperatures of -20°C or less for extended periods of time. Another way of extending the time a vaccine can be stably stored is freeze-drying of the vaccine composition. This has additional advantages over a liquid composition. A freeze-dried composition is for example lighter and therefore more economical to transport. Furthermore, freeze-dried vaccine compositions usually do not need to be stored frozen but can often be stored at a more economical temperature of 2-8 °C.
A disadvantage of freeze-drying is that the process of freeze-drying can negatively affect the activity of the agent present in the vaccine composition. Since part of the activity of the agent is lost during freeze-drying, the vaccine obtained by reconstituting the freeze-dried composition is less effective. In order to better protect the agent from inactivation due to freeze-drying, a mixture comprising N-Z amine, dextran, gelatin, and lactose can be added to the vaccine composition before freeze-drying. It is used to protect both viruses and bacteria, and is for example employed in the commercially available vaccines Bovilis® Nasalgen® 3, Bovilis® Nasalgen® 3-PMH, Bovilis® Once® PMH® SQ, Bovilis® Vista® Once SQ, and Bovilis® Coronavirus. However, even in the presence of this protective mixture, a portion of the activity of the agent is still lost during freeze-drying. Therefore, there remains a need of compositions comprising pathogens which lose a lower amount of activity during freeze-drying.
Thus, it is an object of the present invention to provide a composition comprising pathogens which composition retains a relatively high antigenic activity during freeze- drying.
SUMMARY OF INVENTION
Surprisingly, it was found that the activity of a pathogen is less affected when a composition comprising the pathogen is freeze-dried in the presence of polyvinylpyrrolidone (PVP) with a weight-averaged molecular weight between 1 ,000 and 7,000 Da in an amount of at least 1 wt/vol%, based on the composition before freeze- drying, and a sugar.
Thus, in a first aspect, the invention relates to a freeze-dried composition comprising live attenuated pathogens (of the same or different kind), and a bulking agent that comprises in combination (i) polyvinylpyrrolidone with a weight-averaged molecular weight between 1 ,000 and 7,000 Da in an amount of at least 1 wt/vol%, based on the composition before freeze-drying, and (ii) a sugar.
Freeze-drying of a composition comprising pathogens and at least 1 wt/vol% PVP K-12, based on the composition before freeze-drying, and sucrose resulted in a relatively low pathogen titer log loss. In other words, the pathogens remain relatively stable during freeze-drying in the presence of 1 wt/vol% PVP K-12 and sucrose. Since a relatively large portion of the freeze-dried pathogens remains active, the freeze-dried composition has a relatively high antigenic activity. Thus, the process of preparing a freeze-dried composition comprising the pathogen is relatively efficient. PVP K-12 is known as a growth medium compound for bacteria. However, it was found that growing certain pathogens, such as Histophilus somni, in the presence of PVP in the growth medium, resulted in a strongly reduced pathogenic titer in the culture. Therefore, it was even more surprising that the PVP added to the composition reduced a loss of titer.
Thus, in a second aspect, the invention relates to a process of preparing a freeze-dried composition comprising a live attenuated pathogen and a bulking agent comprising polyvinylpyrrolidone with a weight-averaged molecular weight between 1 ,000 and 7,000 Da and a sugar, comprising the steps of: culturing live attenuated pathogens, harvesting and optionally processing the live attenuated pathogens to yield a first composition comprising live attenuated pathogens, combining the composition comprising live attenuated pathogens with polyvinylpyrrolidone with a weight-averaged molecular weight between 1000 and 7000 Da in an amount of at least 1 wt/vol%, based on the composition before freeze drying, and a sugar, to yield a second composition comprising live attenuated pathogens, and freeze-drying the second composition comprising live attenuated pathogens to provide the freeze-dried composition.
In this way, a freeze-dried composition is obtained with a relatively high pathogen titer, and therefore a relatively high antigenic titer.
The freeze-dried composition can be reconstituted in a pharmaceutically acceptable carrier to form a vaccine composition suitable for administration to an animal. Therefore, in a third aspect, the invention relates to a vaccine comprising a pharmaceutically acceptable carrier and a freeze-dried composition comprising live attenuated pathogens, and a bulking agent that comprises in combination (i) polyvinylpyrrolidone with a weight-averaged molecular weight between 1 ,000 and 7,000 Da in an amount of at least 1 wt/vol%, based on the composition before freeze-drying, and (ii) a sugar.
Because freeze-drying results in relatively little pathogen titer loss and therefore in relatively little antigenic activity loss, the antigenic activity in a standard volume of vaccine, e.g. 2 mL, prepared from the freeze-dried composition is relatively high. The vaccine prepared from the freeze-dried composition can be used to vaccinate an animal. Therefore, in a fourth aspect, the invention relates to a method of vaccinating an animal by parenteral administration of vaccine comprising live attenuated pathogens, a bulking agent that comprises in combination (i) polyvinylpyrrolidone with a weight- averaged molecular weight between 1 ,000 and 7,000 Da in an amount of at least 1 wt/vol%, based on the composition before freeze-drying, and (ii) a sugar, and a pharmaceutically acceptable carrier.
Because the pathogen titer and therefore the antigenic titer of the vaccine prepared from the freeze-dried composition is relatively high, a relatively small amount of live attenuated pathogens providing the antigen is sufficient for an effective vaccine. Thus, the vaccine is relatively cheap to produce.
DEFINITIONS
A “pathogen” is an organism that is able to cause a disease in a host. A pathogen can also be referred to as a germ or an infectious microorganism or agent. Pathogens can for example be viruses, bacteria, unicellular eukaryotes such as protozoa, and multicellular eukaryotes such as a fungus.
“Live” in the sense of this invention means that the pathogen is replicative in a host and/or in vitro.
“Attenuated” in the sense of this invention means that the pathogen has a reduced capacity to induce infection or disease in a particular host as compared to unattenuated versions of the pathogen. Relevant antigens are still displayed to the host’s immune system, but do not cause (serious) disease to the host. In this way, the host is effectively protected against (the consequences of) an infection with an unattenuated version of the pathogen.
A “bulking agent” as referred to in this application acts as a filler and as cryoprotectant for an active component during freezing of a composition comprising the active component. Known bulking agents are for example amino acids such as glycine or arginine, a specific protein such as bovine serum albumin or a hydrolysate (e.g. N-Z®-amine), polymers such as dextran or gelatin, and combinations thereof, such as the combination of N-Z-amine, dextran, gelatin and lactose.
“Polyvinylpyrrolidone” (PVP) is a synthetic polymer of 1-vinyl-2-pyrrolidone units which has been known since the 1930’s and is used in e.g. foodstuffs, technical products such as paint or glue, in cosmetics and in pharmaceuticals. PVP is also known under the synonyms polyvidone or povidone. PVP is available in weight-averaged polymer sizes ranging from about 2,000 Da to about 2 million Da. Different classes corresponding to different weight-averages polymer size ranges can be discriminated: K-12: about 1 ,000 Da to about 7,000 Da; K-15: about 8000 Da to about 12000 Da; K-17: about 10,000 Da to about 16,000 Da; K-25: about 30,000 Da to about 40,000 Da; K-30: about 45,000 Da to about 58,000 Da; K-60: about 270,000 to about 400,000 Da; K-90: about 1 ,000,000 Da to about 1 ,500,000 Da (Hefei Trendchem Co., LTD.).
A “sugar” is a carbohydrate according to the general formula Cn(H2O)n. Sugars include monosaccharides and disaccharides. Monosaccharides are for example glucose, fructose, and galactose. Disaccharides, also referred to as or compound sugars or double sugars, are molecules made of two bonded monosaccharides, such as sucrose (glucose + fructose), lactose (glucose + galactose), and maltose (two molecules of glucose).
“Gelatin” or “gelatine” is a soluble protein-peptide mixture from animal origin that is obtained by partially and irreversibly hydrolyzing collagen. The hydrolysis reduces proteins into smaller peptides. It is also referred to as hydrolyzed collagen, collagen hydrolysate, gelatin hydrolysate, hydrolyzed gelatin and collagen peptides. Suitable gelatin is for example commercially available as dehydrated Difco™ Gelatin from BD Biosciences.
An ’’adjuvant” enhances the immune response to a vaccine. Adjuvants are for example aluminum salts, such as aluminum hydroxide, aluminum phosphate and aluminum sulphate, mineral oils such as paraffin oil and squalene, and adjuvant compositions such as Freund’s complete adjuvant and Freund’s incomplete adjuvant.
“Freeze-drying” is also known as lyophilization or cryodesiccation. Freeze-drying involves freezing the product and subsequently lowering the pressure, thereby removing water in the form of ice by sublimation. This is in contrast to dehydration by most conventional methods that evaporate water using heat. A freeze-drying cycle starts with decreasing the temperature to freeze the product, and ends when substantially all water is sublimated.
An “antigen” is a molecule, moiety, foreign particulate matter or an allergen, such as pollen, that can bind to a specific antibody or T-cell receptor. The antigen can therefore trigger an immune response. An antigen can for example be a whole (live or non-live) pathogen such as a bacterial cell, or a protein, a peptide (amino acid chain), a polysaccharide (chain of sugars molecules), a lipid, or a nucleic acid.
“Antigenic activity” is the capacity of an antigen to bind to a specific antibody or T-cell receptor and subsequently trigger an immune response.
A “vaccine” or “vaccine composition” in the sense of this invention is a constitution suitable for application to an animal, comprising one or more antigens in an immunologically effective amount (i.e. capable of stimulating the immune system of the target animal sufficiently to at least reduce the negative effects of a post-vaccination challenge of the wild-type micro-organisms), typically combined with a pharmaceutically acceptable carrier such as a liquid containing water or other biocompatible liquid, optionally comprising adjuvants, stabilizers, 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. In general, a vaccine can be manufactured by using art-known methods that basically comprise admixing the antigens (or a composition containing the antigens) with the pharmaceutically acceptable carrier.
A “bacterin” is a specific type of vaccine, made from attenuated or inactivated (killed) bacteria. The inactivated bacteria may be whole or (partly) lysed.
A “pharmaceutically acceptable carrier” is a carrier that is useful in the preparation of a pharmaceutical composition such as a vaccine that is generally non-toxic and neither biologically nor otherwise undesirable with respect to a host animal. A pharmaceutically acceptable carrier is for example a liquid such as water, a physiological salt solution, or a phosphate buffered saline solution. It can also be a more complex carrier such as a buffer comprising further additives, such as stabilizers or preservatives.
“Reconstitution” in the sense of the invention means rehydration of a freeze-dried composition, e.g. by adding sterile water to the freeze dried composition, to provide a liquid composition.
The “low-temperature glass transition temperature” (Tg’) is the temperature at which an amorphous material transitions from a viscous or “rubbery” state to a brittle “glass” state.
The “collapse temperature” (Tc) is the temperature at which an amorphous material softens to the point that it can no longer support its own structure.
“Ruminant” refers to an animal assigned to the suborder Rumination. Ruminants make use of the process of rumination to digest feed. Ruminating mammals include cattle, all domesticated and wild bovines, goats, sheep, giraffes, deer, gazelles, and antelopes.
“Bovine” refers to ruminants from the subfamily Bovinae. Bovine animals include cattle, bison, African buffalo, water buffalos, and four-horned and spiral-horned antelopes.
DESCRIPTION OF EMBODIMENTS
In a first aspect, the present invention relates to a freeze-dried composition comprising live attenuated pathogens, and a bulking agent that comprises in combination (i) polyvinylpyrrolidone (PVP) with a weight-averaged molecular weight between 1 ,000 Da and 7,000 Da in an amount of at least 1 wt/vol%, based on the composition before freeze- drying, and (ii) a sugar.
The freeze-dried composition is obtained by freeze-drying a composition comprising live attenuated pathogens, PVP with a weight-averaged molecular weight between 1 ,000 Da and 7,000 Da, and a sugar.
Procedures for freeze-drying or lyophilizing a composition such as a vaccine composition are well known in the art, and equipment to perform freeze-drying are widely commercially available. Generally, a sample is first frozen. Next, the sample is dried by applying a vacuum. In this step, the temperature of the sample is gradually increased to allow water present in the sample to sublimate without melting the composition. The drying step can be subdivided in a primary drying step in which free water is removed, and a secondary drying step in which chemically bound water is removed.
The process of freeze-drying removes a substantial amount of the water from the composition subjected to freeze-drying. Preferably, the freeze-dried composition comprises less than 10 wt.% water, more preferably less than 5 wt.%, even more preferably less than 2.5 wt.% and most preferably at most 1 wt.%, based on the weight of the freeze-dried composition.
The freeze-dried composition typically is a freeze-dried cake which may adopt various forms, such as a layer in a bottle, a tablet or a spherical object such as a lyosphere.
Live attenuated pathogens, or modified-live pathogens, have long been known to be useful for vaccination. Stable attenuation can be achieved by introducing a mutation that induces a loss in replicative or infective capacity of the pathogen, such as a mutation to external organelles, coat or capsule, to the expression of a virulence factor, or to the internal organization. Attenuated pathogens can be obtained in a variety of ways, such as through a method of direct or aspecific induced mutation, e.g. by consecutive in vivo or in vitro passaging, use of mutagens such as chemicals or ionizing radiation, or recombinant DNA technology. A pathogen may be considered attenuated with respect to the infection of a particular host, while it is fully pathogenic with respect to another host.
The cultivation of pathogens is well known in the art. Generally, the pathogens are grown in a suitable growth medium and under suitable growth conditions to allow the multiplication of the pathogen. After a period of growth, typically between 24 and 72 hours at a temperature of between 30 and 40°C, the pathogens are harvested and optionally processed. Finally, the pathogens are incorporated into a composition for freeze-drying.
The pathogens provide the antigen which stimulates the immune system upon vaccination. Therefore, the freeze-dried composition comprises preferably at least 1x106 colony forming units per milliliter (CFU/mL), based on the composition before freeze-drying. In this way, administration of a vaccine prepared from the freeze-dried composition contains a sufficient amount of pathogens and antigens to induce an immune response in the host animal. To induce an even stronger immune response in the host animal, the freeze-dried composition comprises more preferably at least 5x106 CFU/mL, even more preferably at least 1x107 CFU/mL, even more preferably at least 5x107 CFU/mL, and most preferably at least 1x108 CFU/mL, based on the composition before freeze-drying.
It is believed that the advantages in freeze-drying as achieved by the bulking agent comprising polyvinylpyrrolidone (PVP) with a weight-averaged molecular weight between 1 ,000 and 7,000 Da in an amount of at least 1 wt/vol%, based on the composition before freeze-drying, and a sugar are not dependent on the nature of the pathogen. In other words, the pathogens can for example be viruses, unicellular eukaryotes such as protozoa, and multicellular eukaryotes such as a fungi.
However, bacterins are commonly used as vaccines. A freeze-dried composition comprising attenuated bacteria is therefore of particular interest. Thus, the pathogens are preferably bacteria. As explained previously, bovine respiratory disease is economically important, since it is the most common and costly disease affecting beef cattle in the world. Therefore, the pathogens are more preferably bacteria associated with bovine respiratory disease. Even more preferably, the bacteria are selected from the bacterial species which are the most important cause of BRD, i.e. selected from the species of Mannheimia haemolytica, Pasteurella multocida, Mycoplasma bovis, Histophilus somni, and a combination of two or more thereof. Most preferably, the freeze-dried composition comprises the species Histophilus somni. It is herein understood that that species Histophilus somni includes the species Haemophilus somnus, Histophilus ovis, and Histophilus agni, as they were known prior to 2003.
PVP having a relatively high weight-averaged molecular weight, such as PVP of the K-60 class, may be unsafe for subcutaneous administration. Due to the relatively large size, PVP K-60 cannot be excreted by the kidneys. This may result in accumulation of PVP K- 60 in the body of the vaccinated animal. The relatively small PVP K-12 is known as a medium component, but was found to reduce the antigenic titer of the pathogen culture grown in the presence of PVP K-12. It was therefore very surprising that it was found that when live attenuated pathogens were combined with PVP K-12 and sucrose after harvesting of the pathogens, relatively little pathogenic titer was lost during freeze-drying. Therefore, the freeze-dried composition comprises PVP with a weight-averaged molecular weight of between 1 ,000 Da and 7,000 Da. Preferably, the PVP has a weight-averaged molecular weight of between 2,000 and 6,500 Da. More preferably, the PVP has a weight- averaged molecular weight of between 3,000 and 6,000 Da, and most preferably between 4,000 and 5,500 Da. Too little PVP will likely be less effective in protecting the pathogens against inactivation during freeze-drying. Therefore, the freeze-dried composition comprises at least 1 wt/vol% PVP, based on the composition before freeze-drying. Preferably, the freeze-dried composition comprises at least 1.1 wt/vol% PVP, such as at least 1.2 vol/wt% PVP, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or even at least 2 vol/wt% PVP, such as at least 2.1 wt/vol%, at least 2.2 wt/vol%, at least 2.3 wt/vol%, at least 2.4 wt/vol%, at least 2.5 wt/vol%, at least 2.6 wt/vol%, at least 2.7 wt/vol%, at least 2.8 wt/vol.% or at least 2.9 wt/vol%,
On the other hand, the live attenuated pathogen is the active component of the freeze- dried composition providing the antigen. Incorporating a relatively high amount of PVP leaves less room for the pathogens. Therefore, the freeze-dried composition preferably comprises at most 5 wt/vol%, based on the composition before freeze-drying. More preferably, the freeze-dried composition comprises at most 4.5 wt/vol% PVP, even more preferably at most 4 wt/vol% PVP, even more preferably at most 3.5 wt/vol% PVP, in particular at most 3 wt/vol% PVP.
The freeze-dried composition also comprises a sugar. It was shown that the sugar in particular contributes to a relatively high low-temperature glass transition temperature (Tg’) and collapse temperature (Tc). Thus, the Tg’ and Tc are reached relatively quickly, and the freeze-drying cycle can be relatively short. This contributes to a more economical freeze- drying process. Preferably, the sugar is a disaccharide. More preferably, the sugar is chosen from the group of sucrose, lactose, maltose, trehalose, and a combination of two or more therefore, even more preferably from the group of maltose and trehalose, and a combination thereof. Most preferably the sugar comprises sucrose.
Preferably, the freeze-dried composition comprises at least 1 wt/vol% sugar, more preferably at least 2.5 wt/vol%, even more preferably at least 5 wt/vol.%, and most preferably at least 7.5 wt/vol% sugar, based on the composition before freeze-drying.
To allow for a relatively short freeze-drying cycle, the freeze-dried composition preferably has a Tc of at most -40°C, preferably at most -37.5°C, more preferably at most -35°C, and most preferably at most -32.5°C, and/or a Tg’ of at most -40°C, preferably at most -37.5°C, more preferably at most -35°C, and most preferably at most -32.5°C. As described above, the live attenuated pathogen is the active component of the freeze- dried composition providing the antigen. Incorporating a relatively high amount of sugar leaves less room for the pathogens. Therefore, the freeze-dried composition preferably comprises at most 20 wt/vol% sugar, more preferably at most 18 wt/vol%, and most preferably at most 16 wt/vol% sugar, based on the composition before freeze-drying.
Optionally the freeze-dried composition comprises one or more further components such as a further pathogen, an adjuvant, or a preservative.
Preferably, the freeze-dried composition comprises gelatin. It was found that the additional presence of gelatin in the bulking agent protects the pathogens in the composition even more against inactivation during freeze-drying. Therefore, the freeze-dried composition preferably comprises at least 0.1 wt/vol% gelatin, based on the composition before freeze- drying. More preferably, the freeze-dried composition comprises at least 0.2 wt/vol% gelatin, even more preferably at least 0.4 wt/vol%, and most preferably at least 0.6 wt/vol%, based on the composition before freeze-drying.
As described above, the live attenuated pathogen is the active component of the freeze- dried composition providing the antigen. Incorporating a relatively high amount of gelatin leaves less room for the pathogens. Therefore, the freeze-dried composition preferably comprises at most 2 wt/vol% gelatin, more preferably at most 1 .5 wt/vol%, and most preferably at most 1 wt/vol%, based on the composition before freeze-drying.
It was found that growing certain pathogens, such as Histophilus somni, in the presence of PVP in the growth medium, resulted in a strongly reduced antigen titer in the culture. Therefore, it is preferred that the PVP is added to the composition after harvesting of the pathogens and before freeze-drying. Thus, in a second aspect, the invention relates to a process of preparing a freeze-dried composition comprising a live attenuated pathogen and a bulking agent comprising polyvinylpyrrolidone with a weight-averaged molecular weight between 1 ,000 and 7,000 Da and a sugar, comprising the steps of: culturing live attenuated pathogens, harvesting and optionally processing the live attenuated pathogens to yield a first composition comprising live attenuated pathogens, combining the composition comprising live attenuated pathogens with polyvinylpyrrolidone with a weight-averaged molecular weight between 1000 and 7000 Da in an amount of at least 1 wt/vol%, based on the composition before freeze drying, and a sugar, to yield a second composition comprising live attenuated pathogens, and freeze-drying the second composition comprising live attenuated pathogens to provide the freeze-dried composition.
The obtained freeze-dried composition has a relatively high pathogen titer, and therefore a relatively high antigenic titer. Furthermore, the freeze-dried composition is convenient for storage and transport. However, in order to be suitable for administration to an animal, the freeze-dried composition is reconstituted in order to prepare a vaccine ready for administration. Thus, in a third aspect, the invention relates to a vaccine comprising a freeze-dried composition comprising live attenuated pathogens, a bulking agent that comprises in combination (i) polyvinylpyrrolidone (PVP) with a weight-averaged molecular weight between 1 ,000 Da and 7,000 Da in an amount of at least 1 wt/vol%, based on the composition before freeze-drying, and (ii) a sugar, and a pharmaceutically acceptable carrier.
The pharmaceutically acceptable carrier provides liquid to rehydrate the freeze-dried composition. Thus, the pathogens present in the freeze-dried composition are reactivated, and a vaccine in the form of a suspension of live attenuated pathogens is provided. Preferably, the pharmaceutically acceptable carrier is sterile water or a sterile physiological saline solution.
The reconstituted vaccine can then be used to vaccinate a host animal. Thus, in a fourth aspect, the invention relates to a method of vaccinating a host animal by administration of a vaccine comprising a freeze-dried composition comprising live attenuated pathogens, a bulking agent that comprises in combination (i) polyvinylpyrrolidone (PVP) with a weight- averaged molecular weight between 1 ,000 Da and 7,000 Da in an amount of at least 1 wt/vol%, based on the composition before freeze-drying, and (ii) a sugar, and a pharmaceutically acceptable carrier.
The vaccine may be administered in any way suitable for a particular host animal. It may for example be administered enterally, such as via the oral or rectal route. Preferably, the vaccine is administered parenterally, such as intramuscularly, intradermally, or intravenously. In particular subcutaneous vaccination is a convenient way of vaccinating animals, especially in case of relatively large animals. Therefore, the vaccine is most preferably administered subcutaneously.
Because environmental factors such as weaning are known to contribute to diseases such as BRD, a dose of the vaccine is preferably administered to a host animal of between 2 and 6 weeks of age, such as to a host animal of 3, 4, or 5 weeks of age. The vaccine may be administered in any suitable regimen. It may for example be administered as a single dose, or as multiple doses over the course of several weeks or months. The vaccine is for example administered as a first prime dose and a second booster dose. If the vaccine is administered as multiple doses, the second dose is preferably administered at least 2 weeks and at most 8 weeks after the first dose. More preferably, the second dose is administered at least 4 weeks and at most 6 weeks after the first dose. In other words, the second dose is preferably administered when the host animal is between 4 and 14 weeks of age, such as when the host animal is 6, 8, 10 or 12 weeks of age.
Ruminants are a particularly large group of animals comprising roughly 200 domestic and wild species. Thus, the host animal that is vaccinated is preferably a ruminant, such as a goat, sheep, or cervine. Of the ruminants, bovine animals including cattle are of special economic importance in e.g. the dairy and meat industry. The host animal is therefore more preferably a bovine animal. Most preferably, the host animal is selected from the group of cattle, such as a cow, buffalo or bison.
As described before, bovine respiratory disease is the most common and costly disease affecting beef cattle in the world. Therefore, the host animal is preferably vaccinated against bovine respiratory disease.
It will be clear to the skilled person that features and preferences described for one aspect of the invention may also apply to another aspect of the invention, unless they are mutually exclusive.
EXAMPLES
Example 1.
Effect of polyvinylpyrrolidone and sucrose on freeze-dried bacteria Methods
Preparation of the composition for freeze-drying
Histophilus somni was cultured in Tryptic Soy broth. The whole culture was harvested, and attenuated live whole bacterial cells were used as antigen for the preparation of a vaccine composition. The composition for freeze-drying was prepared by combining harvested Histophilus somni culture with a bulking agent in the volume ratio indicated in Table 1 . The various prepared compositions comprised bulking agent components in a final amount as indicated in Table 1 .
Figure imgf000016_0001
*Table 1. Compositions for freeze-drying. Culture volume and bulking agent volume are indicated as vol/vol% based on the composition before freeze-drying. Bulking agent components are indicated as wt/vo!% based on the composition before freeze-drying.
Determination of pathogen titer
The titer of Histophilus somni of the compositions was determined both before and after freeze-drying using a standard titer determination protocol. For determination before freeze-drying, compositions comprising Histophilus somni and a bulking agent as indicated in Table 1 were used before the compositions were subjected to freeze-drying. For determination after freeze-drying, sterile water was added to the freeze-dried composition and the composition was allowed to rehydrate for at least 10 minutes.
In short, a decimal dilution series for each composition and a positive control were prepared. 3 chocolate agar plates were inoculated with 100 pL of each dilution that was expected to result in 30-300 colony forming units (CFU) per plate. The plates were incubated at 36 ± 2 °C (5% CO2) for 48-72 hours.
The colonies of the plates containing 30-300 colonies were counted and averaged. The average counts were converted to CFU per mL by multiplying by the reciprocal of the dilution, and subsequently dividing the result by 0.1 to convert to milliliters.
To calculate the log loss after freeze-drying, the following formula was used:
Log loss = Log (titer before freeze-drying (CFU/mL) * composition volume) I (titer after freeze-drying (CFU/mL) * reconstituted volume).
Generally, a lower log loss is considered advantageous since more antigenic activity is preserved.
Freeze-drying microscopy
A standard protocol for freeze-drying microscopy (FDM) was used to determine the collapse temperature (Tc) of the compositions.
In short, 4 pL sample of a composition prepared as described above was placed in a freeze-drying microscope. The temperature was decreased to -45°C over the course of an hour. Subsequently, a vacuum was applied, resulting in the formation of a sublimation front. Next, the temperature was slowly increased at a rate of 2°C/min and the temperature at which the sample could no longer support its own structure and collapsed was determined to be the Tc.
Generally, a higher Tc is considered advantageous since this allows for a faster and therefore more efficient freeze-drying cycle.
Differential scanning calorimetry
A standard protocol for differential scanning calorimetry was used to determine the low- temperature glass transition temperature (Tg’).
In short, 15 pL sample was placed in triplicates in a Perkin Elmer 8500 Differential Scanning Calorimeter. Briefly, the temperature was decreased to -70°C to allow the sample to freeze. The temperature was then increased at a rate of 20°C/min. This temperature oscillation was then repeated. The temperature at which the phase transition occurred was determined to be the Tg’. Results
Pathogen titer log loss after freeze-drying
As can be seen from Table 2, freeze-drying Histophilus somni in the presence of a bulking agent comprising 1 .5 wt/vol% N-Z Amine AS, 0.3 wt/vol% dextran, 0.15 wt/vol% gelatin, and 1 wt/vol% lactose (NDGL) results in a log loss of pathogen titer of 1 .20.
However, when Histophilus somni is freeze-dried in the presence of 1 .24 wt/vol% PVP K- 12 and 6.6 wt/vol% sucrose, based on the composition before freeze-drying, the bacterial log loss is reduced to 0.88. The antigen log loss is further reduced to 0.70 when the amount of PVP K-12 is increased to 2.81 wt/vol% and the amount of sucrose is increased to 9 wt/vol%. When the bulking agent comprised 0.75 wt/vol% gelatin in addition to 2.25 wt/vol% PVP K-12 and 8.4 wt/vol% sucrose, based on the composition before freeze- drying, the log loss was even further reduced to 0.53.
Figure imgf000018_0001
ble 2. Pathogen titer log loss of Histophilus somni after freeze-drying of a composition comprising Histophilus somni and the bulking agent composition as indicated. Culture volume and bulking agent volume are indicated as vol/vol% based on the composition before freeze-drying. Bulking agent components are indicated as wt/vol% based on the composition before freeze-drying.
This experiment was repeated for a two-way combination vaccine containing Mannheimia haemolytica bacteria and Pasteurella multocida bacteria, and a three-way vaccine containing in addition BRSV (Bovine respiratory syncytial virus), to assess whether or not the improvement in titer loss depends on a type of live pathogen or not. In the vaccines, the bacteria were formulated at a titer of around 5E08 CFU, and the BRSV at a titer of around 7.0 (Log TCID50). The results after freeze-drying are indicated in the below table 2a. The bulking agent components were the same as in the above table 2.
Figure imgf000019_0001
* Table 2a. Pathogen titer log loss of Mannheimia haemolytica, Pasteurella multocida and
BRSV after freeze-drying. Culture volume and bulking agent volume are indicated as vol/vol% based on the composition before freeze-drying. Bulking agent components are as indicated in table 2 (rows in Table 2a correspond to rows in Table 2), based on the composition before freeze-drying.
As can be seen, for the stabilizing composition of the invention, the effect on improving the titer loss is independent of the type of pathogen.
Thermal analysis
As can be seen from Table 3, the presence of a relatively high amount of sucrose increases the Tc and Tg’ of the freeze-dried composition as compared to a relatively low amount of sucrose. Also in the additional presence of gelatin, the Tc and Tg’ are relatively high, as compared to compositions comprising relatively low amounts of sucrose.
Figure imgf000019_0002
* Table 3. Glass transition temperature (Tg’) and collapse temperature (Tc) of compositions comprising a bulking agent as indicated. Culture volume and bulking agent volume are indicated as vol/vol% based on the composition before freeze-drying. Bulking agent components are indicated as wt/vol% based on the composition before freeze- drying. ND = not determined. Conclusion
A bulking agent comprising at least 1 wt/vol% PVP K-12, based on the composition before freeze-drying, and sucrose protects the bacteria Histophilus somni better against inactivation during freeze-drying than a bulking agent comprising NDGL. The bacteria are even better protected when the bulking agent additionally comprises gelatin. A bulking agent comprising at least 1 wt/vol% PVP K-12, based on the composition before freeze- drying, and a relatively high amount of sucrose and optionally gelatin also has a relatively high Tc and Tg’. Thus freeze-drying of a composition comprising at least 1 wt/vol% PVP K- 12, based on the composition before freeze-drying, and a relatively high amount of sucrose and optionally gelatin can be performed relatively economically since the Tc and Tg’ can be reached relatively quickly and the dry-freezing cycle can be relatively short.
Example 2.
Effect of polyvinylpyrrolidone on Histophilus somni pathogen titer
Methodology
Standard growth medium for Histophilus somni with or without 1 wt/vol% PVP K-12 was inoculated with x amount of Histophilus somni. The condition including 1 wt/vol% PVP K- 12 was performed in duplicate. 8 hours after inoculation, the optical density (OD) of the cultures was measured at 540 nm to determine the total amount of bacteria present in the culture at this time point. Furthermore, the titer of Histophilus somni corresponding to the amount of live bacteria was measured as described for Example 1 .
Results
As shown in Table 4, the OD value reflecting the total amount of bacteria was similar 8 hours after inoculating a growth medium comprising 1 wt/vol% PVP K-12 as compared to control conditions.
However, the pathogen titer reflecting the live bacterial counts was reduced by an average of 80.3% in trial 1 , and an average of 68.8% in trial 2 when the bacteria were grown in the presence of 1 wt/vol% PVP K-12 as compared to control conditions.
Figure imgf000021_0001
* Table 4. Optical density measured at 540 nm and pathogen titer (CFU/mL) of Histophilus somni cultured in the absence (control) or presence (1% PVP K-12) of 1 wt/vol% PVP K- 12. The pathogen titer relative to control is calculated as a percentage of Histophilus somni pathogen titer cultured in the presence of PVP K-12 compared to the control.
Conclusion
The presence of 1 wt/vol% PVP K-12 in the growth medium of Histophilus somni bacteria strongly inhibited the pathogen titer of Histophilus somni compared to control conditions in which no PVP K-12 was present in the growth medium.
Example 3.
Animal study efficacy
Study design
In Trial 1 , calves approximately four weeks of age were randomized into a control group and an experimental vaccine group, each containing 8 calves. In Trial 2, calves approximately four weeks of age were randomized into a control group containing ten calves and two experimental vaccine groups each containing eleven calves.
A vaccine composition comprising 2.25 wt/vol% PVP K-12, 8.4 wt/vol% sucrose and 0.75 wt/vol% gelatin was prepared by combining harvested Histophilus somni and a bulking agent comprising PVP K-12, sucrose and gelatin as described in Example 1 in a volume ratio of 85:15. The vaccine composition contained Histophilus somni at a titer of about 1x108 CFU/mL. The vaccine composition was freeze-dried. The vaccine composition was reconstituted with 2 mL sterile water before vaccination, and injected subcutaneously.
All calves from the vaccine groups received a subcutaneous dose at about four weeks of age. The calves receiving a double dose received a second subcutaneous dose about two weeks after the first dose.
Four (trial 1 ) or five (trial 2) weeks after the last vaccination, the calves were challenged via endoscope at the bronchial junction with a virulent Histophilus somni strain. Seven days after challenge, the calves were euthanized, and necropsy was conducted with no identification of treatment groups. Lungs were harvested from the calves and the percentage of pneumonic versus normal lung tissue was determined. The affected lunglobe areas of isolated lungs (visible consolidation) were identified and noted down using a grid pattern, for both lungs, and for both the ventral and the dorsal side. Next, the extent of affection of lobes was counted from the number of grids, which was then multiplied by the proportion of the total lung normally represented by that lobe. All calculated values were added, the maximal score is 100.
Results
As can be seen in Table 5, the average lung lesion score was strongly reduced in calves that were vaccinated with a vaccine obtained from reconstituting a freeze-dried vaccine composition comprising live attenuated Histophilus somni, 2.25 wt/vol% PVP K-12 and 0.75 wt/vol% gelatin, both when the calves received a single dose and when the calves received a double dose.
Figure imgf000022_0001
* Table 5. Average lung lesion score in unvaccinated calves (control) or calves vaccinated with a vaccine reconstituted from a freeze-dried composition comprising live attenuated Histophilus somni, 2.25 wt/vol% PVP K-12, 8.4 wt/vol% sucrose and 0.75 wt/vol% gelatin, based on the composition before freeze-drying.
Conclusion
The vaccine obtained from reconstituting a freeze-dried vaccine composition comprising live attenuated Histophilus somni, 2.25 wt/vol% PVP K-12, 8.4 wt/vol% sucrose, and 0.75 wt/vol% gelatin protects against a challenge with virulent Histophilus somni compared to unvaccinated control animals.

Claims

1. A freeze-dried composition comprising live attenuated pathogens, and a bulking agent that comprises in combination
- polyvinylpyrrolidone with a weight-averaged molecular weight between 1000 and 7000 Da in an amount of at least 1 wt/vol%, based on the composition before freeze drying, and
- a sugar.
2. The freeze-dried composition according to claim 1 , wherein the polyvinylpyrrolidone has a weight-averaged molecular weight between 2,000 and 6,500 Da, preferably between 3,000 and 6,000 Da, more preferably between 4,000 and 5,500 Da.
3. The freeze-dried composition according to any one of the previous claims, wherein the vaccine comprises between 1 and 5 wt/vol% polyvinylpyrrolidone, based on the composition before freeze drying.
4. The freeze-dried composition according to any one of the previous claims, wherein the vaccine comprises between 1 and 20 wt/vol% sugar, based on the composition before freeze drying.
5. The freeze-dried composition according to any one of the previous claims, wherein the sugar is a disaccharide, preferably wherein the sugar is chosen from the group of sucrose, lactose, maltose, trehalose, and a combination of two or more therefore, more preferably wherein the sugar comprises sucrose.
6. The freeze-dried composition according to any one of the previous claims, wherein the bulking agent further comprises gelatin, preferably between 0.1 and 2 wt/vol% gelatin, based on the composition before freeze drying.
7. The freeze-dried composition according to any one of the previous claims, wherein the pathogens are bacteria.
8. The freeze-dried composition according to claim 7, wherein the bacteria are associated with bovine respiratory disease, preferably wherein the bacteria associated with bovine respiratory disease are selected from the species of Mannheimia haemolytica, Pasteurella multocida, Mycoplasma bovis, Histophilus somni, and a combination of two or more thereof, more preferably wherein the bacteria comprise the species Histophilus somni.
9. A process of preparing a freeze-dried composition according to any one of claims 1 to 8, comprising the steps of: culturing live attenuated pathogens, harvesting and optionally processing the live attenuated pathogens to yield a first composition comprising live attenuated pathogens, combining the composition comprising live attenuated pathogens with (i) polyvinylpyrrolidone with a weight-averaged molecular weight between 1 ,000 and 7,000 Da in an amount of at least 1 wt/vol%, based on the composition before freeze drying, and (ii) a sugar, to yield a second composition comprising live attenuated pathogens, and freeze-drying the second composition comprising live attenuated pathogens to provide the freeze-dried composition.
10. A vaccine comprising the freeze-dried composition according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier.
11 . A method to vaccinate a host animal, by parenteral administration of the vaccine according to claim 10, preferably by subcutaneous administration of the vaccine.
12. The method according to claim 11 , wherein the host animal is a ruminant, preferably a bovine animal.
13. The method according to any one of claims 11 or 12, wherein the host animal is vaccinated against bovine respiratory disease.
14. The method according to any one of claims 11 to 13, wherein a first dose of the vaccine is administered when the host animal is between 2 and 6 weeks of age, and preferably a second dose of the vaccine is administered at least 2 weeks and at most 8 weeks after the first dose.
15. A vaccine for use in vaccinating an animal, wherein a vaccine according to claim 10 is administered parenterally.
PCT/EP2024/087327 2023-12-20 2024-12-19 A freeze-dried composition containing live attenuated pathogens, a process for preparing a freeze-dried composition, a vaccine, and a method of vaccinating a host animal Pending WO2025132735A1 (en)

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Citations (2)

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Publication number Priority date Publication date Assignee Title
US20190381161A1 (en) 2016-12-12 2019-12-19 Intervet Inc. Oral vaccine against ruminant respiratory disease comprising polyvinylpyrrolidone
US20200390877A1 (en) * 2017-12-07 2020-12-17 Merck Sharp & Dohme Corp. Formulations of dengue virus vaccine compositions

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
US20190381161A1 (en) 2016-12-12 2019-12-19 Intervet Inc. Oral vaccine against ruminant respiratory disease comprising polyvinylpyrrolidone
US20200390877A1 (en) * 2017-12-07 2020-12-17 Merck Sharp & Dohme Corp. Formulations of dengue virus vaccine compositions

Non-Patent Citations (1)

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Title
CL�NET DIDIER ET AL: "A spray freeze dried micropellet based formulation proof-of-concept for a yellow fever vaccine candidate", EUROPEAN JOURNAL OF PHARMACEUTICS AND BIOPHARMACEUTICS, ELSEVIER SCIENCE PUBLISHERS B.V., AMSTERDAM, NL, vol. 142, 12 July 2019 (2019-07-12), pages 334 - 343, XP085764882, ISSN: 0939-6411, [retrieved on 20190712], DOI: 10.1016/J.EJPB.2019.07.008 *

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