COMPOSITIONS FOR IMPROVING VACCINE SAFETY AND EFFICACY AND METHODS OF USE THEREOF

Abstract

The present disclosure provides methods and compositions for reducing the incidence, severity, and/or duration of at least one sign of respiratory infection. The methods include the steps of administering a composition comprising gastrointestinal microbiota and an immunogenic composition to an animal in need thereof.

Claims

1. A method for reducing the severity, incidence, or duration of at least one clinical sign of respiratory infection in a pig or group of pigs or increasing the efficacy of an immunogenic composition comprising the steps of: administering a composition comprising one or more microorganisms that are found in the gastrointestinal microbiome of an animal to each pig; and administering at least one immunogenic composition effective for reducing the severity, incidence, or duration of at least one clinical sign of at least one respiratory infection in a pig to each pig, wherein the severity, incidence, or duration of at least one clinical sign of respiratory infection is reduced in comparison to a pig or group of pigs that received the administration of the immunogenic composition but did not receive the administration of the composition, or wherein the efficacy of the immunogenic composition is increased in comparison to a pig or group of pigs that received the administration of the immunogenic composition but did not receive the administration of the composition.

2. The method of claim 1, wherein the immunogenic composition is effective against a pathogen selected from the group consisting of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), porcine circovirus (PCV), swine influenza virus, classical swine fever, pseudorabies virus, Salmonella, Haemophilus parasuis, Bordetella bronchiseptica, Pasteurella, Actinobacillus pleuropneumoniae, and any combination thereof.

3. The method of claim 1, wherein the severity, incidence, or duration of at least one clinical sign of respiratory infection is reduced at least 10% in comparison to a pig or group of pigs that received the administration of immunogenic composition but did not receive the administration of the composition.

4. The method of claim 1, wherein the composition is administered more than one time.

5. The method of claim 1, wherein the composition is administered at least one time before and at least one time after the administration of the immunogenic composition.

6. (canceled)

7. The method of claim 1, wherein the microorganisms in the composition are from an animal that was healthy.

8. (canceled)

9. The method of claim 1, wherein the diversity of the microbiome of animals receiving the composition is higher than in animals not receiving the composition.

10. The method of claim 1, wherein comprises at least 2 log CFU/ml/dose of microorganism when the microorganism is a bacteria, or at least 2 TCID50/ml/dose when the microorganism is a virus.

11. A method of slowing the increase in viremia in a pig or group of pigs after challenge or infection by a virulent pathogen comprising the steps of: administering a composition comprising one or more microorganisms that are found in the gastrointestinal microbiome of an animal to each pig; and administering at least one immunogenic composition effective for reducing the severity, incidence, or duration of at least one clinical sign of at least one respiratory infection in a pig to each pig, wherein the viremia of the virulent pathogen is reduced in comparison to a pig or group of pigs that received the administration of the immunogenic composition but did not receive the administration of the composition.

12. The method of claim 11, wherein the immunogenic composition is effective against a pathogen selected from the group consisting of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), porcine circovirus (PCV), swine influenza virus, classical swine fever, pseudorabies virus, Salmonella, Haemophilus parasuis, Bordetella bronchiseptica, Pasteurella, Actinobacillus pleuropneumoniae, and any combination thereof.

13. The method of claim 11, wherein the viremia is reduced at least 10% in comparison to a pig or group of pigs that received the administration of immunogenic composition but did not receive the administration of the composition.

14. The method of claim 11, wherein the composition is administered more than one time.

15. The method of claim 11, wherein the composition is administered at least one time before and at least one time after the administration of the immunogenic composition.

16. (canceled)

17. The method of claim 11, wherein the microorganisms in the composition are from an animal that was healthy.

18. (canceled)

19. The method of claim 11, wherein comprises at least 2 log CFU/ml/dose of microorganism when the microorganism is a bacteria, or at least 2 TCID50/ml/dose when the microorganism is a virus.

20. (canceled)

21. (canceled)

22. (canceled)

23. (canceled)

24. (canceled)

25. (canceled)

26. (canceled)

27. (canceled)

28. (canceled)

29. A kit comprising a composition comprising: one or more microorganisms that are found in the gastrointestinal microbiome of an animal; at least one immunogenic composition effective for reducing the severity, incidence, or duration of at least one clinical sign of at least one respiratory infection in a pig to each pig; and instructions for the administration of both the immunogenic composition and the one or more microorganisms.

30. The kit of claim 29, wherein the immunogenic composition is effective against a pathogen selected from the group consisting of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), porcine circovirus (PCV), swine influenza virus, classical swine fever, pseudorabies virus, Salmonella, Haemophilus parasuis, Bordetella bronchiseptica, Pasteurella, Actinobacillus pleuropneumoniae, and any combination thereof.

31. The kit of claim 29, wherein the instructions instruct a user to administer the composition more than one time.

32. The kit of claim 29, wherein the instructions in composition is administered at least one time before and at least one time after the administration of the immunogenic composition.

33. (canceled)

34. (canceled)

35. (canceled)

36. The kit of claim 29, wherein the immunogenic composition comprises at least 2 log CFU/ml/dose of microorganism when the microorganism is a bacteria, or at least 2 TCID50/ml/dose when the microorganism is a virus.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0042] FIG. 1 is a schematic representation of the experimental design to investigate the effects of FMT on PRRSV challenge in PRRS-vaccinated and nonvaccinated pigs;

[0043] FIG. 2A is a graph illustrating PRRS viremia in nonvaccinated pigs transplanted with fecal microbiota or saline with 10% glycerol. Data is shown as the mean±standard deviation log.sub.10 copies/PCR reaction for each group. Statistical significance or trends (*p<0.05; ‡p≤0.1) are shown for each day based on unpaired t-test analysis;

[0044] FIG. 2B is a graph illustrating PRRS viremia in vaccinated pigs transplanted with fecal microbiota or saline with 10% glycerol. Data is shown as the mean±standard deviation log.sub.10 copies/PCR reaction for each group. Statistical significance or trends (*p<0.05; ‡p≤0.1) are shown for each day based on unpaired t-test analysis;

[0045] FIG. 3A is a graph illustrating microscopic lung lesion scores in nonvaccinated pigs with or without fecal microbiota transplantation at 42 days post-challenge with PRRSV. Lung lesions are scored by a blinded board certified pathologist reviewing histopathology slides of sections from each lung lobe. Scores include 0: no lesions, 1: mild and multifocal interstitial pneumonia with <50% lobe involvement, 2: mild to moderate and multifocal interstitial pneumonia with 50-75% lobe involvement, 3: moderate to severe and multifocal interstitial pneumonia with 50-75% lobe involvement, and 4: severe diffuse interstitial pneumonia with >75% lobe involvement. No significant difference was detected between nonvaccinated pigs with and without FMT (p=0.7403; Mann Whitney U test);

[0046] FIG. 3B is a graph illustrating microscopic lung lesion scores in vaccinated pigs with and without fecal microbiota transplantation at 42 days post-challenge with PRRSV. Lung lesions are scored by a blinded board certified pathologist reviewing histopathology slides of sections from each lung lobe. Scores include 0: no lesions, 1: mild and multifocal interstitial pneumonia with <50% lobe involvement, 2: mild to moderate and multifocal interstitial pneumonia with 50-75% lobe involvement, 3: moderate to severe and multifocal interstitial pneumonia with 50-75% lobe involvement, and 4: severe diffuse interstitial pneumonia with >75% lobe involvement. No significant difference was detected between vaccinated pigs with and without FMT (p=0.2245; Mann Whitney U test).

[0047] FIG. 4A is a photograph showing no microscopic lung lesions in pigs 42 days after challenge with virulent PRRSV;

[0048] FIG. 4B is a photograph showing mild and multifocal interstitial pneumonia with <50% lobe involvement in pigs 42 days after challenge with virulent PRRSV;

[0049] FIG. 4C is a photograph showing mild to moderate and multifocal interstitial pneumonia with 50-75% lobe involvement in pigs 42 days after challenge with virulent PRRSV;

[0050] FIG. 4D is a photograph showing moderate to severe and multifocal interstitial pneumonia with 50-75% lobe in pigs 42 days after challenge with virulent PRRSV;

[0051] FIG. 5A is a graph illustrating weight gain in FMT and control pigs without PRRS MLV vaccination prior to virulent PRRSV challenge. Data is shown as mean absolute weights±standard deviation as measured weekly throughout the 28 day post-vaccination and 42 day post-challenge periods. A trend towards significance (‡p=0.086, unpaired t-test) was detected on 14 days post-challenge in the nonvaccinated group with transplanted pigs having higher absolute mean weights; and

[0052] FIG. 5B is a graph illustrating weight gain in FMT and control pigs with PRRS MLV vaccination prior to virulent PRRSV challenge. Data is shown as mean absolute weights±standard deviation as measured weekly throughout the 28 day post-vaccination and 42 day post-challenge periods. No significant difference in weight was detected in vaccinated pigs which received the FMT when compared to those which received saline.

DETAILED DESCRIPTION OF THE DISCLOSURE

[0053] This written description uses examples to disclose the invention, including the best mode, and also to enable any person skilled in the art to practice the invention, including making and using any devices or systems and performing any incorporated methods. The patentable scope of the invention is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal languages of the claims.

[0054] Vaccine Study: Fecal microbiota transplantation was performed in pigs prior to vaccination with a PRRS modified live virus (MLV) vaccine and subsequent challenge with virulent PRRSV. First, feces was collected from 2 high health donors as previously described in PCT/US2018/033910. The exact same material was utilized to assess the effects of FMT on PRRS-only challenge in vaccinated and nonvaccinated pigs in the current study. The experimental design is summarized in FIG. 1. Forty high-health commercial pigs (average age 18.6±0.5 days) were obtained from a single source herd negative for PRRSV. Four brother barrows were divided into the four experimental groups (n=10/group) and balanced by weight upon arrival. Mean weight across all four groups was 11.45 lbs. Each group was housed in an individual pen and all pens were housed in 1 environmentally controlled room under biosafety-level 2 conditions. Biosecurity protocols were implemented between pens to prevent cross-contamination of gut microbes and PRRS viruses. Pigs were given access to food and water ad libitum. Upon arrival, FMT Vaccinated and FMT Nonvaccinated pigs received the fecal transplant material daily for 7 days (average 3.5 ml/day) while the Control Vaccinated and Control Nonvaccinated pigs received sterile saline with 10% glycerol for 7 days (average 3.5 ml/day). The volume of FMT material administered in the current study was less than the volume administered in PCT/US2018/033910, which was 5 ml/pig/day. The FMT had been prepared from this previous study (PCT above) using feces collected from two older sows with specific high health characteristics, including high parity (≥9), large litters with high numbers of born alive piglets, low pre-weaning mortality, no history of fetal mummification, no fecal parasites, and no antibiotic treatment within the year prior to collection. The fecal microbiota were processed, concentrated, and stored at −80° C. using a protocol adapted from the human FMT literature. Prior to administration, the FMT is thawed for 2 hrs on ice. After 7 days of fecal transplantation or mock saline transplantation, all pigs in the Vaccinated groups received a 2 mL dose of a commercial PRRS MLV vaccine (Ingelvac PRRS MLV; Boehringer Ingelheim Animal Health) administered intramuscularly according to the vaccine label instructions. At 28 days post-vaccination, all pigs in all four groups were challenged with virulent PRRSV administered as a 2 mL dose containing 105 TCID50 PRRSV in MEM. The 2-mL dose was split, with 1 mL administered intranasally and the remaining 1 mL administered intramuscularly. Pigs were followed for 42 days post-challenge with PRRSV (FIG. 1). Pigs were evaluated by a veterinarian or veterinary assistant daily. Standardized health evaluation protocols were used to score clinical disease post-infection. Blood samples were collected from all pigs on −35, −28, 0, 4, 7, 11, 14, 21, 28, 35, and 42 dpi. Additionally, blood samples were collected from the vaccinated groups on −24, −21, −17, −14, and −7 dpi. PRRSV viremia was quantified using the EZ-PRRSV MPX 4.0 Real Time RT-PCR Target-Specific Reagents (Tetracore). Individual body weights were collected on −35, −28, −21, −14, −7, 0, 7, 14, 21, 28, 35, and 42 dpi. Average daily gain (ADG) was calculated as the change in weight over the change in time. All pigs were humanely euthanized at 42 dpi and complete necropsies were performed by a board certified pathologist. Microscopic lung lesion severity was estimated using 0 to 4 scoring systems as previously described.

[0055] Overall, clinical signs were mild throughout the study and no mortalities occurred post-challenge with virulent PRRSV. PRRS virus replication was compared between control and vaccinated groups with and without FMT (FIG. 2). In the vaccinated groups, replication of the PRRS MLV vaccine was measured between 0 to 28 days post-vaccination. Fecal transplantation seemed to reduce replication of the PRRS MLV vaccine. Specifically, on −21, −17 and −14 days post-infection, the transplanted group had numerically less PRRS virus detectable in the serum. The total vaccine virus replication during this period (7 to 14 days post-vaccination) was calculated as the area under the curve (Table 1).

TABLE-US-00002 TABLE 1 Total vaccine virus replication between 7 to 14 days post-vaccination in pigs immunized with a PRRS MLV vaccine with and without fecal microbiota transplantation* Control Vaccinated FMT Vaccinated Pig AUC Pig AUC 30 18.69 1 15.63 18 20.02 39 18.21 9 21.95 13 20.34 22 22.71 21 20.90 37 23.98 17 21.43 2 24.79 27 21.68 26 25.28 32 21.74 15 26.14 31 21.79 6 27.57 8 23.71 35 27.67 7 24.21 Mean‡ 23.88 Mean‡ 20.96 SEM 0.9593 SEM 0.7925 *Total virus replication calculated as the area under the curve (AUC, log.sub.10 copies/PCR reaction) for individual pigs between 7, 11, and 14 days post-vaccination ‡Significant difference between means, p = 0.0308 (student's unpaired t test)

[0056] For control pigs, mean AUC was 23.88±0.9593 log 10 copies/PCR reaction with a range between 18.69 and 27.67. For transplanted pigs, mean AUC was 20.96±0.7925 log 10 copies/PCR reaction with a range between 15.63 and 24.21. The difference between the two groups with regards to total vaccine virus replication over this 1 week period was statistically significant (p=0.0308, unpaired t-test). Additionally, there was a trend towards significance with the FMT pigs having lower total vaccine virus replication as measured by the area under the curve between 4 and 21 days post-vaccination (p=0.0798, student's unpaired t-test). When comparing individual days, on −17 dpi, transplanted pigs had significantly lower PRRS viremia (p=0.04) and on −14 dpi, transplanted pigs had a trend towards reduced vaccine virus replication (p=0.1). Overall, a reduction in the replication of the PRRS MLV vaccine increases the safety of the vaccine and reduces vaccine virus shedding. This would be beneficial due to the risk of shedding vaccine virus to nonvaccinated pigs and the potential effects of vaccine virus replication on production parameters (i.e., morbidity and growth).

[0057] Post-challenge with virulent PRRSV, the vaccinated FMT pigs had a slower increase in PRRSV viremia followed by a more prolonged clearance. On 11 dpi, FMT vaccinated pigs had a trend towards increased virulent PRRS virus detection in the blood (p=0.08). In nonvaccinated pigs, no PRRSV was detected in transplanted or control pigs prior to virulent PRRSV challenge, confirming that no exposure to the PRRS MLV vaccine virus occurred and biosecurity was maintained. Post-challenge with virulent PRRSV, the nonvaccinated FMT pigs had a lower peak followed by more prolonged viral clearance as shown by a significant increase in PRRSV detection in the blood at 11 dpi (p=0.04).

[0058] At the conclusion of the 42-day post infection period, gross necropsies were completed on all pigs by a blinded board-certified veterinary pathologist. Lung sections were collected from each lung lobe and fixed in formalin. H&E stained histopathology slides of lung tissue were reviewed and scored for severity of interstitial pneumonia (FIG. 3). Scores include 0: no lesions, 1: mild and multifocal interstitial pneumonia with <50% lobe involvement, 2: mild to moderate and multifocal interstitial pneumonia with 50-75% lobe involvement, 3: moderate to severe and multifocal interstitial pneumonia with 50-75% lobe involvement, and 4: severe diffuse interstitial pneumonia with >75% lobe involvement. Examples of lung lesions representing the 4 scores that were detected in the current study are shown in FIG. 4. The majority of pigs had some degree of interstitial pneumonia; however, no significant difference was detected between the transplanted and control pigs which received PRRS MLV vaccination (p=0.2245; Mann Whitney U test) or between the transplanted and control pigs without vaccination (p=0.7403; Mann Whitney U test).

[0059] Overall, weight gain was similar between the FMT and control pigs in both the vaccinated and nonvaccinated groups (FIG. 5). On 14 dpi, there was a trend towards a significant difference between the absolute weights in the nonvaccinated FMT and control groups. Control pigs had a mean weight of 33.5±3.3 kg compared to a mean weight of 37.2±5.3 kg in the FMT group (p=0.086, student's unpaired t test). No other significant differences or trends towards significance were detected in absolute weights between the four groups.