WO1993024147A1

WO1993024147A1 - Lecithin adjuvanted modified live virus vaccines

Info

Publication number: WO1993024147A1
Application number: PCT/US1993/005088
Authority: WO
Inventors: Jay Dean Gerber
Original assignee: Smithkline Beecham Corporation
Priority date: 1992-05-29
Filing date: 1993-05-28
Publication date: 1993-12-09

Abstract

This invention provides a modified live virus vaccine in combination with a lecithin adjuvant. The adjuvant comprises about 5.0 % by weight to about 10 % by weight of lecithin and from about 90 % by volume to about 95 % by volume of an oil carrier.

Description

LECITHIN ADJUVANTED MODIFIED LIVE VIRUS VACCINES

This is a continuation-in-part of application Serial No. 07/890,903 filed on May 29, 1992.

Field of the Invention

This invention relates to the field of modified live vaccines. More particularly, the invention relates to the field of modified live vaccines adjuvanted with lecithin in combination with a carrier which is selected from the group consisting of edible and non-edible oils.

Background of the Invention

Modified live viruses (MLVs) are known to be less immunogenic than the virulent virus from which they derive. The goal of modification or attenuation can be to remove the virulence or reduce it such that the virus can be safely administered as a vaccine. These modified live virus vaccines are generally freeze-dried for stability and reconstituted with water prior to administration. In an effort to improve the serological and immune response in animals vaccinated with modified live vaccines, U.S. Patent 4,650,677 teaches that the use of an oil-in-water emulsion as the solvent to rehydrate the modified live virus has the effect of improving the serological and immune response of the vaccinated animals. U.S. Patent 4,650,677 teaches that the quantity of oil in the emulsion is usually between 15 and 50% by volume.

Summary of the Invention

There is a need in the art for modified live vaccines which are adjuvanted to enhance virus neutralizing titer and to reduce the virus shed to noninfected animals yet contain minimum amounts of oil to reduce or eliminate tissue damage at the injection site that is commonly associated with oil adjuvants containing higher amounts of oil. See e.g., Straw et al., AVMA, Vol. 106, No. 4, February 15, 1990. Further, there is a need for modified live vaccines capable of circumventing the vaccine neutralizing effects of maternal antibodies in the vaccinated animal.

Accordingly, this invention provides a novel modified live virus vaccine in combination with an adjuvant diluent. The adjuvant diluent comprises about 5.0% by weight to about 10% by weight of lecithin and from about 90% by volume to about 97% by volume of an oil carrier. Further provided by this invention is a novel method of immunizing animals with a modified live virus vaccine, said animals having maternal antibody derived serum neutralizing titers to the virus, comprising vaccinating the animal with a vaccine of this invention.

Brief Description of the Drawings

Figure 1. Override of maternal antibody following vaccination with an adjuvanted PR- Vac® (MLV) of this invention. Seropositive piglets were vaccinated at 4 weeks (Group 1, solid line), 7 weeks (Group 2, dashed line), 10 weeks (Group 3, dotted line), or not vaccinated (Group 4, long dashed line). Vaccinated animals were given an intravenous boost with nonadjuvanted inactivated antigen 3 weeks following vaccination (arrows).

Detailed Description of the Invention In one aspect of this invention there is provided a modified live virus vaccine in combination with a lecithin adjuvant in a carrier which is selected from the group consisting of edible and non-edible oils.

The lecithin adjuvant and carrier are substantially as described in United States Patent Number 5,084,269 which patent is incorporated by reference as if fully set forth herein. Briefly, the lecithin adjuvant comprises 5.0% by weight to 10% by weight of lecithin and from 90% by weight to 95% by weight of an oil carrier. The lecithin and oil carrier usually is from about 5% to about 50% by volume of the vaccine as taught in 5,0284,269. Currently, the preferred lecithin adjuvant diluent comprises about 5.0% by weight to about 10% by weight of lecithin and from about 90% by volume to about 97% by volume of an oil carrier.

Lecithin is available from a variety of commercial sources such as Central Soya, Chemurgy Division, Fort Wayne, IN.

The adjuvant can be used at varying levels with different modified live viruses. The amount of lecithin in an oil carrier can readily be determined by individual testing with each modified live virus. Additional adjuvants and additives can be added to the adjuvant diluent of the invention where necessary. For example, a final concentration of 12% by volume aluminum hydroxide has been added to the adjuvant diluent of the invention successfully. The amounts of lecithin and oil remain as taught herein. Although not wishing to be bound by any theory, it is believed lecithin associated with an oil emulsion carrier works as an efficient means of delivering the vaccine antigen to cells of the immune system. The lecithin may be layered on the surface of the oil droplets in the emulsion or occur as a lipid vesicle or liposomes in association with the oil carrier.

An example of a suitable modified live virus is a pseudorabies virus (PrV) such as PrV based on the Bucharest strain. Other suitable modified live viruses expected to be useful in this invention include bovine herpes virus, bovine respiratory syncytial virus, bovine viral diarrhea virus, parainfluenza virus type 3, porcine parvovirus, feline panleukopenia virus, feline herpes virus, feline calici virus, equine herpesvirus, canine distemper virus, infectious bronchitis virus and any other animal modified live virus vaccine. In the vaccine of the present invention, the modified live virus is present in an amount sufficient to provide the desired immune response. One skilled in the art will recognize that a sufficient amount will vary depending upon the virus employed as well as a variety of other factors. Suitable modified live viruses for use in the vaccine of this invention are commercially available such as Pr-Vac®. In another aspect of this invention there is provided a method of immunizing an animal comprising vaccinating the animal with an adjuvanted vaccine of this invention, whereby virus shed is reduced and virus neutralizing titer is increased over vaccination with a modified live virus vaccine administered with water.

A method of immunizing animals, such as swine, having maternal antibody derived serum neutralizing titers to the virus, such as pseudorabies virus, is also provided by the invention. For example, swine having maternal antibodies to pseudorabies virus are vaccinated usually at about four to ten weeks of age with a vaccine comprising a modified live pseudorabies virus, e.g. based on the Bucharest strain, and an adjuvant comprising 10% by weight lecithin in a 90% by weight mineral oil carrier. The adjuvant and carrier preferably comprise 5% of the vaccine by volume.

It is within the scope of this invention to combine the vaccine with killed viral preparations which may contain separate adjuvants such as saponin and aluminum hydrogel. For example, a modified live-killed combination vaccine contemplated by this invention might contain modified live pseudorabies virus and killed Actinobacillus pleuropneumoniae, Mycoplasma hyopneumoniae, Bordetella bronchiseptica, Haemophilus parasuis and Erysipelothrix rh siopathiae. EXAMPLES

Materials and Methods

Preparation Of Emulsion

A stock emulsion is prepared in the following manner with composition per liter:

Lecithin/Light Mineral Oil Solution (Pennzoil)

(10% by wt. lecithin in 366ml mineral oil) 400 ml Polyoxyethylene sorbitan monooleate (Tween 80) 14 ml

Sorbitan monooleate (Span 80) 6 ml

Phosphate Buffered Saline (PBS) 580 ml ml

The lecithin mineral oil Solution is sterilized by filtration. Tween 80 and

Span 80 are each sterilized by autoclaving. Methyl-paraben, propylparaben, and butyl-paraben, at a total concentration not greater than 3.34 grams per liter of emulsion are added as preservative. These ingredients are mixed together using a valex mixer or equivalent for 16 to 24 hours at 37°C. The mixture is then cooled to between 25 and 30°C, and emulsified with the PBS, pH 7.2 ±0.2, in a Ross emulsifier (model ME401) or equivalent. Emulsification is continued by recirculation until all of the adjuvant is added into the saline. The emulsion is then passed once through a Gaulin homogenizer or equivalent. The emulsion may be stored at 4°C. Assembly of the adjuvant lecithin in combination with a carrier which is selected from the group consisting of edible and non-edible oils

The above emulsions is then used to formulate the final adjuvant diluent with the following composition per liter:

The water is sterilized by autoclaving. All components are combined and stirred together for at least 30 minutes. The adjuvant is hereinafter referred to as adjuvant (a). vaccine

In these Examples, a live attenuated pseudorabies vaccine based on the Bucharest strain sold under the tradename PR- Vac® (SmithKline Beecham Animal Health, Exton, PA) was used.

EXAMPLE 1 ADJUVANT COMPARISON

Experimental Design

Three adjuvant formulations were compared for the ability to enhance PR- Vac® titers. Groups of ten 3 to 4 week old pigs were vaccinated with a normal field dose of PR- Vac® (Serial 20221110, 10⁵-⁵TCID5o/dose). The vaccine was rehydrated in one of the following adjuvants: (a) lecithin in an oil in water emulsion prepared as previously described.

(b) lecithin in an oil in water emulsion prepared as previously described further containing E. coli lyzate with approximately 50 micrograms of endotoxin per dose as the adjuvant;

(c) 5% aluminum hydroxide gel;

These three adjuvants were compared to PR- Vac® rehydrated in water diluent. Six additional pigs were not vaccinated, and served as sentinels to monitor for possible transmission of the vaccine virus.

All animals were bled at the time of vaccination, and then at three weeks post vaccination. A second vaccination was given three weeks after the first to determine if further immunity could be stimulated.

Serum neutralizations were run according to the procedure currently used by the state diagnostic laboratories, which includes use of the virulent Shope strain of PrV. This procedure results in lower titers than are obtained with the vaccine strain. Results

The serum neutralization titers obtained after first and second vaccination are shown in Table 1. Following the first vaccination, the only adjuvant groups that demonstrated a consistent neutralizing titer were the two groups (Groups 1 and 2) that received either adjuvant (a) or (b) (19 of 20 animals in these two groups had a measurable titer). Addition of endotoxin, adjuvant (b) did not increase the magnitude of the response. None of the pigs given PR- Vac® in water appeared to seroconvert following the first vaccination. Serum neutralization titers increased in all groups following a second vaccination, but the relative magnitude of the responses mirrored the results of first vaccination. PR- Vac® rehydrated in adjuvant (a) gave the highest geometric mean titer (GMT) of 1:37. PR- Vac® rehydrated in water and PR- Vac® rehydrated with aluminum hydroxide gave the lowest GMT of 1:6.

Discussion

The comparison of these different adjuvants has demonstrated that the immune response to PR- Vac® can be improved by the lecithin adjuvanted vaccine in an oil carrier according to this invention. The animals vaccinated with PR- Vac® rehydrated in adjuvant (a), PR- Vac® rehydrated in water, and the control group were challenged with virulent PrV and examined for protection based on clinical signs and amount of virulent virus shed. This challenge experiment is detailed in Example 2.

EXAMPLE 2

ADJUVANT COMPARISON: CHALLENGE OF IMMUNITY

Experimental Design Two pigs from some vaccine groups in Example 1 were picked at random and necropsied 21 days following the second vaccination. Injection sites were examined for possible lesions. Five additional pigs from each group were chosen at random for challenge. The groups were as follows: Group 1; PR- Vac® with adjuvant (a), Group 2; PR- Vac® rehydrated in water, and Group 3; nonvaccinated controls. Pigs were challenged with

of virulent pseudorabies virus (ISU 4892 provided by the National Veterinary Service Laboratory, Ames, Iowa). The pigs were approximately 10 weeks old at the time of challenge. Body temperature and clinical signs of disease were monitored daily, nasal swabs were collected daily to monitor virus shed, and the pigs were weighed at 0, 7, and 14 days post challenge. RESULTS Injection site reactions

Two pigs from each of the vaccinated groups were examined for possible injection site reactions 21 days following the second vaccination. No detectable gross lesions were observed at the injection site from either of the animals that received the vaccine rehydrated in adjuvant (a). One of the two pigs (#467) that received vaccine rehydrated with water had a small (2 to 4 mm diameter) focus of fibrous tissue at or near the injection site. This area was considered as a possible residual lesion associated with the previous vaccination. A sample was taken for histopathological examination. However, upon examination, no evidence of present or past inflammation or reaction in the muscle or adjacent tissues was found. Weight gain

The body weight of each pig was determined at 0, 6, and 14 days post challenge (Table 2). One animal (Pig #486) in Group 1 (adjuvant (a)) developed a severe middle ear infection of bacterial origin. This animal lost 15 lbs. in the two weeks following challenge. All other vaccinated animals gained weight by the end of the two week challenge period. The ercent daily weight gain in Group 1 (excluding Pig #486) was 0.6 percent, while the mean percent of gain for Group 2 was 1J percent per day. All control pigs had died by day 7. Clinical signs of disease

All pigs were observed daily for clinical signs of disease following challenge (Table 3). All nonvaccinated animals exhibited signs of pseudorabies infection that included depression, labored breathing and central nervous system (CNS) disfunction. Four of the five con^->ls died within 5 days of challenge, and the fifth was euthanized on day 7 for huπ_^ane reasons. One pig in the adjuvant (a) group (Pig #486) exhibited a head tilt and some rasping of breath. As noted above, this animal was found to have a severe bacterial infection of the middle ear that would account for the head tilt. No histological evidence of pseudorabies infection was found. The remaining four pigs in the adjuvant (a) group remained normal, as did all five pigs in the group that received the vaccine rehydrated in water. Virus shed Shed of virulent virus was monitored in nasal secretions (Table 4). All vaccinated animals shed virus from 1 day post challenge to 4 days post challenge. By 6 days post challenge, virus shed was not detected in any animals from Group 1 (adjuvant (a)), while two animals from Group 2 (water diluent) were still shedding detectable amounts of virus. All vaccinated animals were negative by day 8. In addition to shedding for a shorter period of time, pigs in Group 1 shed less virus. Geometric mean virus titers of pigs in Group 1 were at least one and a half logs lower than those pigs in the water diluent group between days 1 and 4 post challenge. As expected, the control pigs shed much greater amounts of virus than either of the vaccinated groups. The mean titers on day 3 and day 4 post challenge were above 7 logs, and all pigs were still shedding large amounts of virus when they died. Discussion

The immune response of swine vaccinated with PR- Vac® was increased with the use of lecithin in combination with a carrier which is selected from the group consisting of edible and non-edible oils prepared as earlier described. Adjuvant (a) was not viricidal when used in place of sterile water to rehydrate the lyophilized product. Serum neutralizing titers were consistently increased over those produced by the conventional vaccine following a single vaccination. The animals were given a second immunization to mimic field conditions. In most eradication plans, breeding animals are double vaccinated with a modified live product to maintain immunity at the highest possible level in an attempt to prevent virus spread. An anamnestic response was generated by a second immunization with both vaccines, however higher titers were again produced by the adjuvant (a) than could be achieved by vaccine rehydrated in water. Thus it is believed that the lecithin adjuvanted vaccine has engendered a stronger secondary (memory) response than the normal vaccine without adjuvant.

Following challenge with a large dose of the highly virulent field isolate ISU 4892 p5 of pseudorabies virus, all vaccinated pigs were protected from clinical signs of disease. One animal in Group 1 did develop a severe middle ear infection that was probably exacerbated by the pseudorabies challenge. In contrast, all five control animals developed severe symptoms of pseudorabies, and either died or were euthanized for humane reasons by day 6 post challenge.

The amount of virus shed from the nasal cavity of infected pigs was reduced by use of the adjuvant (a) diluent. Pigs that received the vaccine adjuvanted with (a) shed an average of at least fifty-fold less virus than pigs vaccinated with the normal vaccine. This figure may have been even higher, since the titers from some animals in Group 2 (water diluent) did not reach an endpoint on days 3 and 4 (Table 4). This reduction in viral shed will be a key factor in controlling virus spread in an infected herd.

This Example demonstrates that the performance of a normal field dose of PR- Vac® could be substantially improved by rehydration with a diluent consisting of lecithin in an oil carrier in accordance with this invention. TABLE 1

PR- Vac® Adjuvant Comparison Study Serum Neutralization Titers Following Vaccination

G.M.T. <1:2 1:2.4 1:32

G.M.T. <1:2 1:2.4 1:37

478 <2 <2 <2 479 480 481 482 493 500 511 512

G.M.T. <1:2 1:1.6 >1:5.5

G.M.T. <1:2 <1:2 1:5.7

G.M.T. <1:2 <1:2 <1:2

TABLE 2

Adjuvant Comparison Study Weight in Lbs. and Percent Daily Weight Gain

Pigs # Days Post Challenge Percent Daily Weight Gain

Group 1 (adjuvant 0 6 14 0 to 6 6 to 14 0 to 14

(a))

Average 87.8 83.2 91.0 -0.9 0.4 0.3

Average without #486 88.5 85.8 96.3 -0.5 1.1 0.6

Group 2 (Water)

469 69.0 69.0 76.0 0.0 1.3 0.7

470 87.0 93.0 104.0 1.1 2.4 1.4

471 101.0 107.0 120.0 1.0 2.4 1.3

472 75.0 79.0 84.0 0.9 1.5 0.9

473 79.0 81.0 92.0 0.4 2.1 1.2

Average 78.6 57.0 -2.5

* Pig #486 developed a severe middle ear infection of bacterial origin. No pseudorabies related brain lesions were seen. TABLE 3

Clinical signs of pseudorabies following challenge with

Clinical signs or Death

- Pig 486 was found to have a severe middle ear infection that would account for the head tilt. No evidence of PrV infection was found by histological examination of the brain. TABLE 4

Shed of virulent pseudorabies following challenge with ISU 4892 (10⁷-⁶TCID5o)¹

A swab of the nasal cavity was suspended in 2 ml of transport media (Hal's + 5% fetal bovine serum + high antibiotics) and stored frozen at -70°C until analysis. Results are expressed as the mean of two separate titrations.

2 The symbol > designates that titers did not reach endpoint. 3 No endpoint titer was obtained due to toxicity or bacterial contamination at the low dilutions.

4 No swab sample available. 5 No sample obtained due to pig mortality. EXAMPLE 3 OVERIDE OF MATERNAL ANTIBODIES

Experimental Design The goal of this Example was to determine the appropriate age at which seropositive piglets could be vaccinated with PR- Vac® (MLV) adjuvanted with lecithin in combination with a carrier which is selected from the group consisting of edible and non-edible oils, i.e. adjuvant (a).

Piglets born to sows vaccinated with two doses of PR-Vac®-Killed were divided into 3 groups. Each group consisted of 7 to 8 piglets, and groups were chosen so that the mean serum neutralization titer of each group would be approximately equal. Two additional piglets were used as sentinel controls. Animals were vaccinated with vaccine containing the minimum end of dating titer (10³-°TCID5ø/dose). fhe vaccination schedule was as follows: Group 1; vaccinated at 4 weeks of age, Group 2; vaccinated at 7 weeks of age, and Group 3; vaccinated at 10 weeks of age. All animals were given an intravenous injection boost with inactivated pseudorabies virus culture fluids (non-adjuvanted) 3 weeks following the vaccination. All pigs were bled weekly for assay by serum neutralization (SN). The Bucharest strain was used for the virus challenge in the serum neutralization assay. Results

Seropositive piglets with maternal antibody derived SN titers that ranged from 1:64 to 1:16 were vaccinated at 4 weeks of age (Group 1, see Figure 1). These pigs did not exhibit a rise in titer following vaccination. Titers declined between 4 and 7 weeks of age in a fashion that was similar to nonvaccinated animals. However, the immune system of these pigs was primed by vaccination, as demonstrated by a sharp rise in titer following administration of non-adjuvanted inactivated PRV antigen at 7 weeks of age. In contrast, administration of the non- adjuvanted pseudorabies virus antigens in control animals did not elicit a rise in antibody titer. (Group 4)

The piglets in Group 2 were vaccinated at 7 weeks of age, when serum neutralization titers ranged from 1:23 to 1:6. A small increase in SN titer was seen following vaccination, which resulted in titers that remained steady, in contrast to the declining titers seen in the nonvaccinated control pigs (Group 4). A strong secondary response was seen following the intravenous injection boost at 10 weeks of age, indicating good priming of the immune system. The best primary response was seen in animals vaccinated at 10 weeks of age. Titers at the time of vaccination ranged from 1:11 to 1:3 (GMT=1:5). At two weeks post vaccination, the mean titer had increased to 1:17, which was eight-fold higher than the titer remaining in the nonvaccinated pigs. Discussion

This Example demonstrated that PR-Vac®(MLV) with lecithin in combination with a carrier which is selected from the group consisting of edible and non-edible oils can be used successfully to prime seropositive piglets against PrV at weaning. An earlier trial designed to determine the appropriate age for vaccination of seropositive piglets with PR-Vac®-Killed showed that the inactivated product did not prime animals vaccinated at 3 or 4 weeks of age. The earliest priming effect was seen at 6 weeks of age, followed by a second vaccination at 9 weeks of age. This gap between weaning and production of active immunity leaves a window of opportunity for PRV infection to occur.

Current field use of PR- Vac® (MLV) usually involves vaccination at 8 to 12 weeks of age. Earlier vaccination has generally not been effective, and in many cases even vaccination at 8 to 12 weeks has failed to provide active immunity as measured by detection of an SN titer at market weight. Administration of PR- Vac® in combination with adjuvant (a) to seropositive pigs at four weeks of age will actively prime the immune response to the virus. A second vaccination at seven to ten weeks of age would then result in the high levels of active immunity that are required to prevent clinical disease following exposure to virulent pseudorabies virus.

Claims

1. A modified live virus vaccine in combination with an adjuvant divalent, said adjuvant diluent comprising about 5.0% by weight to about 10% by weight of lecithin and from about 90% by volume to about 97% by volume of an oil carrier.

2. The vaccine of claim 1 wherein the modified live virus is a pseudorabies virus.

3. The vaccine of claim 2 wherein the pseudorabies virus is based on the Bucharest strain.

4. The vaccine of claim 1 wherein the adjuvant diluent is from about 5% to about 50% v/v of the vaccine.

5. The vaccine of claim 1 wherein the oil is mineral oil.

6. The vaccine of claim 1 wherein the adjuvant diluent is 10% by volume lecithin and 90% by weight mineral oil and comprises 5% by volume of the vaccine.

7. A vaccine comprising a modified live pseudorabies virus based on the Bucharest strain in an adjuvant diluent comprising about 4.6% by volume mineral oil and about 0.5% by weight lecithin.

8. A method of immunizing an animal comprising vaccinating the mammal with the vaccine of claim 1.

9. A method of immunizing a swine against pseudorabies comprising vaccinating the mammal with the vaccine of claim 2.

10. A method of immunizing animals with a modified live virus vaccine, said animals having maternal antibody derived serum neutralizing titers to the virus, comprising vaccinating the animal with the vaccine of claim 1.

11. The method of claim 10 wherein the virus is pseudorabies virus.