STABLE EMULSIONS OF BACTERIAL ANTIGENS
20210378957 · 2021-12-09
Assignee
Inventors
Cpc classification
A61K9/0019
HUMAN NECESSITIES
A61K47/14
HUMAN NECESSITIES
A61K39/295
HUMAN NECESSITIES
A61K39/39
HUMAN NECESSITIES
A61K39/00
HUMAN NECESSITIES
International classification
A61K47/14
HUMAN NECESSITIES
Abstract
The present invention is based on the discovery of new and advantageous properties of a class of known polymeric emulsifiers. The emulsifier class was found to be resistant to degradation by crude bacterial antigen preparations, which degradation caused emulsion instability when using prior art emulsifiers. This allows the formulation of safe, stable, and effective vaccines based on these emulsions of oil and water comprising such bacterial antigens. The polymeric emulsifier is a block copolymer having a general formula A-B-A in which component B is the divalent residue of a water-soluble polyalkylene glycol and component A is the residue of an oil-soluble complex monocarboxylic acid. Preferred emulsifier is a PEG-30-di-(polyhydroxystearate).
Claims
1. An emulsion comprising an oil phase, an aqueous phase, an emulsifier and a non-live bacterial antigen, wherein the emulsifier is a polymeric emulsifier which is a block copolymer having a general formula A-B-A in which component B is the divalent residue of a water-soluble polyalkylene glycol and component A is the residue of an oil-soluble complex monocarboxylic acid.
2. The emulsion of claim 1, wherein the non-live bacterial antigen contains an esterase.
3. The emulsion of claim 1, wherein the non-live bacterial antigen comprises inactivated bacterial cells.
4. The emulsion of claim 1, wherein the emulsion is a water-in-oil (W/O) emulsion.
5. The W/O emulsion of claim 4, wherein components A and component B all have a molecular weight of at least 500 g/mol.
6. The W/O emulsion of claim 4, wherein component A is a polyhydroxystearic acid, and component B is a polyethylene glycol.
7. The W/O emulsion of claim 4, wherein the polymeric emulsifier is a PEG-30-di-(polyhydroxystearate).
8. A method for the manufacture of the W/O emulsion of claim 4, the method comprising the steps of: a. admixing the oil phase and the polymeric emulsifier, and b. emulsifying the mixture of step a. with the aqueous phase, whereby the aqueous phase comprises the non-live bacterial antigen.
9. A vaccine for the protection of a human or animal target against infection or disease caused by a bacterium comprising the emulsion of claim 1.
10. (canceled)
11. The vaccine of claim 9, wherein the vaccine is for the protection of an animal target.
12-14. (canceled)
15. A method of vaccinating a human or animal target against infection or disease caused by a bacterium, comprising administering to said target the vaccine of claim 9.
16. A method of vaccinating an animal target against infection or disease caused by a bacterium, comprising administering the vaccine of claim 11 to said animal target.
17. The vaccine of claim 11, wherein the animal target is a fish.
18. A method of vaccinating a fish against infection or disease caused by a bacterium, comprising administering the vaccine of claim 17 to said fish.
Description
EXAMPLES
1. Example 1: Analysis of Prior Art Emulsions
[0218] To analyse why the prior art emulsion vaccines were breaking, the stability was analysed of such emulsion vaccines comprising crude non-live bacterial antigens, with Polysorbate 80 and Sorbitan mono-oleate as emulsifiers. The test vaccines used comprised a 7 way combination of antigens, from viral and bacterial origins, comparable to prior art vaccines, and contained identitical quantities of the following inactivated antigens per dose (0.1 ml) of vaccine: [0219] Salmon pancreas disease virus (SPDV), strain F93-125, ≥75% RPP (1) [0220] Infectious pancreatic necrosis virus (IPNV), serotype Sp, ≥1.5 ELISA units (2) [0221] Aeromonas salmonicida subsp. salmonicida, ≥10.7 log 2 ELISA units (3) [0222] Vibrio salmonicida, ≥90% RPS (4) [0223] Vibrio anguillarum serotype O1, ≥75% RPS [0224] Vibrio anguillarum serotype O2a, ≥75% RPS [0225] Moritella viscosa, ≥5.8 log 2 ELISA units (3)
(1) RPP: relative percentage protection in a laboratory test in Atlantic salmon
(2) antigen amount measured in finished product
(3) serological response in Atlantic salmon
(4) RPS: relative percent survival in a laboratory test in Atlantic salmon
[0226] The test vaccines were formulated as water-in-oil emulsions, with light liquid paraffin oil as adjuvant, and contained as emulsifier either Polysorbate 80 and sorbitan mono-oleate, or contained 0.5% w/w Cithrol DPHS as emulsifier. Water:oil weight ratio of the vaccine was 45:55.
[0227] Variations were tested in regard to the presence of the M. viscosa antigen and/or the A. salmonicida antigen: [0228] 1. test vaccine with heptavalent antigens, as a W/O emulsion, using Tween 80 and Span 80 emulsifiers [0229] 2. test vaccine 1, but without M. viscosa antigen [0230] 3. test vaccine 1, but without A. salmonicida antigen [0231] 4. test vaccine 1, but without M. viscosa and without A. salmonicida antigens
[0232] In corresponding accelerated stability tests, the vaccine no. 1 showed breaking of the emulsion already after 1 week at 37° C., vaccines 2 and 3 showed breaking after 2-3 weeks, and vaccine 4 remained stable for the full 3 weeks at 37° C.
[0233] The vaccines were then analysed for any increase in the level of free fatty acids during real time stability tests at 4° C. over several months. This increase could give an indication of the degradation of the emulsifiers (Polysorbate 80 and sorbitan mono-oleate) over time. At the different time points samples were drawn and analysed using solid phase extraction (SPE) followed by size exclusion chromatography (SEC). In short: the various samples were loaded on a silica SPE cartridge, and the fatty acids were eluted using methanol. Subsequently, size exclusion separation was done on this eluate using an HPLC system (Agilent™ 1200) with Oligopore™ gel permeation columns at 35° C. and at 50 bar. An Isocratic mobile phase of tetrahydrofuran/acetic acid was used, and detection was by refractive index. For calibration, samples of oleic acid at 1 or 2 mg/ml were used. Positive control was a sample containing Polysorbate 80 and sorbitan mono-oleate at the same concentrations as in the test vaccine emulsions.
[0234] Overlay chromatograms comparing the fatty acid peak patterns of the samples tested in stability assay are provided in
[0235] 1.1. Results:
[0236] The overlay chromatogram in
[0237]
[0238] For test vaccine 4, the specific peak pattern corresponding to the prior art emulsifiers can clearly be seen between 15 and 22 minutes, and no increase of the oleic acid peak at 20 minutes was detected. Consequently in this emulsion vaccine the emulsifiers were not degraded, and this corresponded with the observed continued stability of this vaccine.
[0239] The sample of test vaccine 1 did still show some pattern of the emulsifiers, but at reduced height, and in addition showed a strongly increased peak at 20 minutes. This demonstrated the generation of oleic acid, resulting from the degradation of the emulsifiers, which correlated with the observed instability of the full heptavalent vaccine-emulsion over time.
[0240] A similar result is presented by
[0241] 1.2. Conclusions
[0242] In the emulsion vaccines tested, a release of free fatty acids over time could be observed, at the expense of a degradation of the emulsifiers. This correlated with the observed breaking of the emulsion in stability testing. Responsible for the degradation of the emulsifiers was the presence of non-live bacterial antigens from the bacteria M. viscose or A. salmonicida.
[0243] Consequently, it was demonstrated that instability of emulsion vaccines comprising an oil phase and an aqueous phase can be caused by the presence of non-live bacterial antigens, which caused a degradation of the emulsifiers Polysorbate 80.
2. Example 2: Efficacy Trials
[0244] 2.1. Introduction
[0245] The following experiment was performed to test if there were consequences for the vaccine's efficacy from the replacement of the prior art emulsifiers Tween 80 and Span 80, by Cithrol DPHS as emulsifier. The experimental vaccines tested comprised several antigens, mineral oil as adjuvant, and were formulated as water-in-oil emulsions.
[0246] Aspects of the safety of these emulsion vaccines were also studied, but these are only reported here very briefly.
[0247] 2.2. Materials and Methods
[0248] 2.2.1. Experimental Design
[0249] For assessing the serological response after vaccination, Atlantic salmon parr (of approximately 35 grams) were treated one week before vaccination with an increase of their water temperature from 12° C. to 17° C., by adjusting +2° C. every second day. Next the fish were intraperitoneally vaccinated with vaccines Hepta-P (heptavalent antigen containing emulsion, with polysorbate and sorbitan-oleate as emulsifiers), or Hepta-C (similar vaccine, comprising Cithrol as emulsifier), as test groups. A control group was also included that was injected with saline. The three groups, each consisting of 50 fish, were kept in separate tanks at 17° C. during the nine weeks immunization period. The fish were observed daily.
[0250] At 9 weeks pv, blood was sampled from 35 fish from each group. Immune response against Aeromonas salmonicida and Moritella viscosa were evaluated by performing ELISA's on individual serum samples.
[0251] 2.2.2. Test Vaccines
[0252] Both test vaccines Hepta-P and Hepta-C contained the same 7 antigens as also comprised in the test vaccine 1 described in Example 1 above. Mock vaccine was sterile saline (0.9% NaCl).
[0253] The vaccine bottles were incubated overnight at ambient temperature (15° C.) and hand-shaken prior to use.
[0254] 2.2.3. Test Animals Atlantic salmon, strain: Stofnfiskur, Iceland, of mixed sex, and mean weight at vaccination was 33.5 grams (n=20).
[0255] Test animals were given 7 days of acclimatisation at experimental conditions.
[0256] Routine disease monitoring was performed on the experimental population by a veterinarian responsible for fish health. In addition, the batch of experimental fish used, tested negative for IPNV, SPDV and ISAV by PCR.
[0257] The vaccinated salmon were individually marked by maxillae clipping or adipose fin clipping; the salmon injected with the control substance remained unlabelled.
[0258] Test animals were kept in fresh water tanks, at 17° C.±2° C. with at least 85% oxygen, or at 12° C.±2° C. with at least 75% oxygen, pH=6.8-7.2.
[0259] Feed was commercial fish feed, available to appetite. Feeding and environmental controls were carried out daily. After vaccination, the fish were observed until they had properly recovered from anaesthesia.
[0260] 2.2.4. Vaccination
[0261] Prior to vaccination the experimental fish were starved for 36 hrs and anaesthetized. The test- and the control groups were given the vaccine or control substance by i.p. injection, at 0.1 ml/dose, using single use syringes of 0.5×4 mm.
[0262] 2.2.5. Monitoring of Results
[0263] Further serology data were collected from the fish kept at 17° C.: blood was collected at 9 w. pv. from 35 fish from each test group kept at 17° C., and of 12 fish from the control group. After overnight clotting, the sera were mixed 1:1 with 86% glycerol and stored at −20° C. until analysis by ELISA.
[0264] The Elisa methods applied are the standard tests for these antigens, and are well-known to be indicative of in vivo efficacy.
[0265] Antibodies against M. viscosa in serum were measured using a direct ELISA. In short, ELISA plates were coated with inactivated M. viscosa and test and control sera were added in serial two-fold dilutions to the plate. Bound antibodies were detected using rabbit anti-salmon IgM, followed by HRP-conjugated mouse anti-rabbit IgG. A colour reaction reflecting bound salmon antibodies was developed by adding a TMB substrate, and the colour measured using an ELISA reader. The antibody titre was expressed as Log 2 value of the maximum dilution of the sample that gave an OD-value equal to 3 times the mean found for a negative control serum measured on each plate.
[0266] Antibodies against A. salmonicida in serum were measured using a similar direct ELISA as described for M. viscosa, except that the ELISA plates were coated with inactivated A. salmonicida. The antibody titre was expressed as Log 2 value of the maximum dilution of the sample that gave an OD-value equal to 5 times the mean found for a negative control serum measured on each plate.
[0267] The antibody titres were calculated using the CBA™ program (Abend Vertical) and the titres were expressed in Log 2 values as the maximum dilution giving 5 times the mean background. Validity was based on the scores of test- and control samples being within certain value ranges.
[0268] 2.3. Results and Discussion
[0269] 2.3.1. Results of Tests for Safety and Serology at 9 w pv and 17° C.
[0270] 2.3.1.1. Antibody Response Against A. salmonicida at 9 w pv
[0271] Specific antibodies against M. viscosa and A. salmonicida were measured in the same sera.
[0272] The antibody titres against A. salmonicida induced by the Hepta-C vaccine were significantly higher than those induced by the Hepta-P vaccine (ANOVA, p<0.0001), while the antigens used and their amounts were the same. Both vaccines induced titres that were above the potency requirement (10.7 Log 2).
[0273] As is also evident from the smaller standard deviation, the antibody titres induced by the Hepta-C vaccine also showed less spread between fish than for the Hepta-P vaccinates.
[0274] The ELISA titres in the saline group were below the detection limit (6.6). Results are presented in Table 1.
[0275] Both vaccine groups induced significantly increased antibody titres as compared to the control group. Whereby the Hepta-C vaccine performed even better than the Hepta-P vaccine in regard to efficacy against A. salmonicida.
TABLE-US-00001 TABLE 1 Serology results against A. salmonicida at 9 w pv, and at 17° C. Log2 Ab titre against A. salmonicida Group mean stand. dev. Hepta-P (Tween + Span) 13.0 1.8 Hepta-C (Cithrol) 15.2 1.2 Saline ≤6.6 0.0
[0276] 2.3.1.2. Antibody Response Against M. viscosa at 9 w pv
[0277] For the induction of seroresponse against M. viscosa, a similar picture emerged as for A. salmonicida: the titres induced by the Hepta-C vaccine were significantly higher (ANOVA, p<0.0001) and with less spread, than the titres induced by the Hepta-P vaccine, even though both contained the same antigens and at the same amounts. Results are presented in Table 2. Both vaccines induced titres that were above the potency requirement (5.8 Log 2).
[0278] Both vaccine groups induced significantly increased antibody titres as compared to the control group.
[0279] Again, both vaccine groups induced significantly increased antibody titres as compared to the control group. Whereby the Hepta-C vaccine performed even better than the Hepta-P vaccine in regard to efficacy against M. viscosa.
TABLE-US-00002 TABLE 2 Serology results against M. viscosa at 9 w pv, and at 17° C. Log2 Ab titre against M. viscosa Group mean stand. dev. Hepta-P (Tween + Span) 8.3 1.8 Hepta-C (Cithrol) 11.9 1.2 Saline ≤4.6 0.0
[0280] 2.3.1.3. Aspects of Safety
[0281] Certain vaccination side-effects, typical for the use of oil-emulsion vaccines in salmon, were scored at 9 weeks post vacination: intra-abdominal adhesions and melanisation. Both were well within acceptable levels, and no significant differences were found between the two groups receiving the test vaccines, for either of these side-effects.
[0282] 2.4. Conclusions
[0283] The efficacy profile of the Cithrol-based heptavalent vaccine formulation is at least as good as that of the similar vaccine emulsified with Tween 80 and Span 80, because the Hepta-C vaccinates showed significantly better immune-response against A. salmonicida and against M. viscosa; both with higher antibody titres and with smaller spread. Aspects of vaccine-safety were also not changed.
3. Example 3: Efficacy Against SPDV
[0284] 3.1. Introduction
[0285] In this experiment the inventors expanded on the efficacy results as described in Example 2 above. Using the exact same vaccine formulations the protective capacity was tested against a challenge infection with salmon pancreas disease virus (SPDV). The side-by-side comparison was made between a prior art formulation of a heptavalent vaccine with Tween 80+Span 80 emulsifiers, against a heptavalent vaccine based on the novel emulsion with Cithrol DPHS as emulsifier. Vaccinations used only a half dose per animal, conform the registered potency test for release of efficacious batches of SPDV vaccines.
[0286] 3.2. Materials and Methods
[0287] 3.2.1. Experimental Design
[0288] In short: acclimatized Atlantic salmon parr was i.p. vaccinated with half of a full dose of each vaccine. Sterile saline was used as mock vaccine.
[0289] The treatment groups were reared in freshwater at 12° C. for 6 weeks whereby intramuscular challenge infection was performed with SPDV. Potency against SPDV was measured as relative percentage protection (RPP) of the vaccinated group compared to the control group, by means of detection of infection with SPDV via PCR of serum.
[0290] 3.2.2. Test Vaccines
[0291] Vaccines used were the same as described in Example 1: Hepta-P and Hepta-C. The control group received sterile Saline (0.9% NaCl). The vaccine given was a half dose: 0.05 ml, delivered intra-peritoneally by injection.
[0292] 3.2.3. Test Animals:
[0293] Atlantic salmon, 38 per group, strain: Salmobreed, of mixed sex, and mean weight at vaccination was 28 grams (n=20). The different groups were kept in the same tank, separated by different markings.
[0294] 3.2.4. Challenge Infection
[0295] SPDV challenge was performed at 6 weeks after vaccination. Prior to challenge, all fish were transferred to a challenge facility and kept in one tank for the remaining 10 days of the experiment.
[0296] Challenge material was SPDV SAV3 strain PD03-13p2, at 4.75 Log 10 TCID50/ml. Challenge was administered to individual fish by intramuscular injection of 0.05 ml, at the lateral line anterior to the dorsal fin.
[0297] 3.2.5. Post Challenge Blood Sampling
[0298] Ten days post SPDV challenge, individual blood samples for PCR testing were collected from the caudal vein of anesthetized fish. After o/n clotting serum was collected and kept frozen until use.
[0299] 3.2.6. SPDV Real Time PCR
[0300] RNA was extracted from individual sera, that had been spiked with inactivated Equine Influenza Virus, EIV. 35 samples per group were analysed with the SPDV gene nsP1, in a real-time PCR assay, to detect SPDV viremia prevalence as measure of protection against challenge. A real time PCR assay specific for EIV HA gene was also performed to detect the EIV spike added to the serum, as a positive control on the quality of the RNA extraction.
[0301] Relative (test vs control) prevalence of SPDV infection (by PCR detection of SPDV in sera) were used to calculate the potency of the tested vaccines, by the prevalence of fish positive for SPDV after challenge. The level of protection was expressed as absolute (+ or −) and as the relative difference in infection prevalence between vaccinated groups and the control group, as a: relative percentage protection (RPP), which is calculated as follows: RPP=[1−(% PCR positive fish in vaccinated group/% PCR positive fish in control group)]×100.
[0302] Statistical analysis of the proportion of infected fish in the groups with PCR detected SPDV in serum was performed by the Fisher's exact test, comparing the vaccinated groups to the saline group. In addition the prevalence of positive fish in groups receiving the same dose was also compared pairwise to each other using the Fisher exact test. The level of significance (a) was set to 0.05, and the test was two-sided. Statistical calculations were executed using the SAS program.
[0303] 3.3. Results and Discussion
[0304] All samples were PCR positive for the EIV spike gene, serving as internal control for RNA purification. Therefore all samples were valid for analysis.
[0305] An overview of the SPDV PCR results on sera sampled at 10 d post challenge is presented in Table 3, indicating the prevalence and calculated RPP for each group, versus prevalence in the saline group.
TABLE-US-00003 TABLE 3 Prevalence and RPP values based on PCR detection of SPDV in serum at 10 d. p.c. Group % positive (Ct ≤ 35) RPP % Hepta-P (Tween + Span) 11 88 Hepta-C (Cithrol) 29 71 Saline 97 0
[0306] The PCR results for the saline group showed that the SPDV i.m. challenge was successful since the number of positive fish in this group was 97% (34 out of 35).
[0307] The prevalence of SPDV positive fish in all vaccinated groups was significantly lower than that in the control group, as demonstrated by a p value in Fisher's exact test of 0.0001.
[0308] Importantly, even though all vaccinated animals only received a half dose of vaccine, the two groups (Hepta-P and Hepta-C) were not significantly different from each other in the prevalence of SPDV infection after challenge; the p value in the two sided Fisher test was 0.133.
[0309] 3.4. Conclusion
[0310] The results show that Cithrol based vaccine formulations protect fish effectively against a challenge infection with SPDV, and to a level of protection (from a half dose) that was not significantly less than that of current commercial vaccines.
4. Example 4: Optimisation of Vaccine Composition
[0311] 4.1. Introduction
[0312] After the new vaccine formulation using a polymeric emulsifier according to the invention was demonstrated to be useful as a safe and efficacious vaccine for fish, other aspects could be optimised. Specifically the viscosity of the new formulation, such as tested in the Hepta-C vaccine described in the above examples, was rather low. Although this clearly did not affect safety or efficacy, it was observed that the new vaccine showed so-called ‘sedimentation’ upon storage. This means that upon storage, the dispersed aqueous phase tended to move downwards under gravity. This is not the same as breaking of the emulsion, i.e. losing dispersion and showing phase separation. Also, other than breaking, sedimentation is fully reversible, and the phases can be rapidly redistributed by simple shaking by hand prior to administration.
[0313] Some level of sedimentation is common for water-in-oil emulsion vaccines, and most product leaflets will recommend a brief shaking of the emulsions before administration. Nevertheless such sedimentation could make a commercial product less attractive visually. Therefore the inventors developed some variants of the formulations tested, to optimise also this aspect of the new emulsion and vaccine.
[0314] 4.2. Variations Tested
[0315] The formulation of the Hepta-C vaccine as tested had a water to oil ratio of 45:55% w/w, and comprised 0.5% w/w Cithrol DPHS; both percentages are expressed by weight of the vaccine. This resulted in a formulation with a viscosity of about 70 mPa.Math.s. The viscosity was measured as described herein.
[0316] To prevent, or at least to considerably reduce sedimentation of the aqueous phase, both the water content and the Cithrol content were varied to increase viscosity. Variations tested were: water:oil ratios of 50:50, 60:40, and 70:30% w/w. Also, Cithrol content was increased to 1.0% w/w for some of the samples. The composition of the vaccine-variants tested was essentially the same as that of Hepta-C, apart from the test variables.
[0317] To assess the effect of the different compositions on sedimentation, the different vaccine compositions were filled into 500 ml bottles, all to the same volume, and these were stored static for 24 hrs at 4° C. After this period the vertical height of a sedimentation line (if visible) was measured in millimetres, and this was divided by the vertical height of the total volume. Any result of this height ratio below 1 indicated that some level of sedimentation had occurred.
[0318] In a smal experiment, similar to the setup in Example 1, the variants of the emulsion vaccines were also tested for their capacity to induce protective levels of antibodies against A. salmonicida, and M. viscosa. Table 5 shows the results of the ELISA titrations; protective Ab titres are levels above 10.7 or 5.8 Log 2 respectively.
[0319] 4.3. Results
[0320] Combined results of viscosity and sedimentation are presented in Table 4. Serology results are presented in Table 5.
TABLE-US-00004 TABLE 4 Effect of variations in composition, on viscosity of water-in-oil formulations with Cithrol as emulsifier water:oil ratio % w/w viscosity Sedimentation (% w/w) Cithrol (mPa .Math. s) height ratio 45:55 0.5 57.5 0.67 50:50 72.0 0.79 60:40 135 0.89 70:30 265 1 50:50 1.0 84.0 0.83 60:40 154 0.98 70:30 341 1
TABLE-US-00005 TABLE 5 Log2 ELISA titres induced by vaccination with the variants of the emulsion vaccines Vaccine composition A. salmonicida M. viscosa Saline ≤6.6 ≤4.6 Hepta-P 14.0 9.1 Hepta-C, 0.5%, 45:55 16.0 12.8 Hepta-C, 0.5%, 60:40 14.7 11.5 Hepta-C, 0.5%, 70:30 16.1 12.5 Hepta-C, 1%, 50:50 15.4 12.2 Hepta-C, 1%, 60:40 15.9 12.3 Hepta-C, 1%, 70:30 15.8 11.4
[0321] 4.4. Conclusions
[0322] Several observations could be made: [0323] the increase in water content in the emulsion had more effect on viscosity than the increase of Cithrol content [0324] (almost) complete prevention of sedimentation (after 24 hrs at 4° C.) could be achieved by increasing the water content in the emulsion to a 70:30 water:oil ratio, and/or by increasing the Cithrol content to 1% w/w. [0325] all vaccine compositions induced protective levels of antibodies against A. salmonicida and M. viscosa.
5. Example 5: Stability Assays of Vaccine Samples
[0326] The vaccines Hepta-P and Hepta-C as tested in Example 2 above were subjected to stability assays: an ‘in use’ stability assay by incubation as 25° C.; where this incubation continued after 8 hours it represents an enhanced stability assay.
[0327] At 25° C., Hepta-P vaccine emulsions broke after 5 days, while Hepta-C emulsions remained intact up to the end of the stability experiment at 8 days.
LEGEND TO THE FIGURES
[0328]
[0329] Overlay chromatographs comparing the peak patterns of the fatty acids from specific samples:
[0330] Panel A: standard sample of oleic acid (2 mg/ml) [solid line], and sample of the prior art emulsifiers [dotted line].
[0331] Panel B: samples of test emulsion vaccine 1 (complete heptavalent vaccine) [solid line], and test emulsion vaccine 4 (vaccine without antigens of M. viscosa and of A. salmonicida) [dotted line]. Horizontal axis: time (minutes); vertical axis: refractive index (nRI).
[0332]
[0333] Results of the detection by size exclusion chromatography, of the generation of free fatty acids resulting from the degradation of emulsifiers in prior art emulsion vaccines.
[0334] The horizontal axis indicates time in months, of storage at 4° C.; the vertical axis indicates the amount of free fatty acid (FA) measured.