Stability of closed and needle-punctured vials of Porvac ® subunit vaccine against classical swine fever subjected to thermal stress

doi:10.21203/rs.3.rs-4003547/v1

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Research Article

Stability of closed and needle-punctured vials of Porvac ® subunit vaccine against classical swine fever subjected to thermal stress

https://doi.org/10.21203/rs.3.rs-4003547/v1

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Background

Classical Swine Fever (CSF) is still one of the most economically important viral diseases of pigs. In endemic countries, the disease is controlled mostly through vaccination, hence, the availability of safe and effective vaccines is of utmost importance. Vaccines intended for application in developing countries must also be thermally stable, since the infrastructure needed to maintain a cold chain in those countries is usually lacking. Porvac^® is a second-generation subunit marker vaccine against CSF that has demonstrates to be safe and protective. Previous studies have also shown that the vaccine is stable for 1 week at 37 ^oC and have a shelf life of at least 36 months at 2–8 ^oC. The aim of this work was to further explore the accelerated stability of Porvac^® by assessing the physicochemical properties of the emulsion, and the safety and efficacy of the vaccine subjected to more drastic conditions of thermal stress: (1) 25 ^oC for 12 months; (2) 30^oC and 37 ^oC for one month and (3) 15 days at 37°C after the cap of the vials had been needle-punctured.

Results

The vaccine subjected to all these conditions did not show significant changes in the physicochemical properties of the emulsion; did not produce local or systemic adverse reactions in pigs, and the chromatographic profile of the recovered antigen was preserved. All vaccinated swine developed protective neutralizing antibody titers ≥ 1:1000 at 28 days post vaccination.

Conclusions

Porvac^® is stable in all the experimental conditions tested, even after cap puncture, and retains the capacity to induce protective neutralizing antibodies. These results reinforce the robustness of the vaccine, and support its use as a very attractive alternative to modified live vaccines in developing countries endemic for CSF.

Thermal stress

vaccination efficacy

vaccine stability

Montanide

NPLA

classical swine fever

virus.

Classical swine fever (CSF) is a highly contagious disease that affects domestic pigs and wild boars and represents a threat for the pig industry from sanitary and economic points of view, and its notification is mandatory to the World Organization for Animal Health [1].

CSF has been eliminated in several countries such as Canada, Australia, USA, and various countries of Western Europe [2]. In contrast, it is still epidemic in Asia, Central and South America, and East Europe [3].

There are two main strategies for CSF control: CSF free countries does not vaccinate and apply a “stamping out” policy for exposed animals during outbreaks [4]. On the other hand, in CSF endemic regions the main strategy for controlling CSF is still prophylactic vaccination with modified live attenuated vaccines (MLV) [5, 6].

MLV have been extensively use in endemic areas, but in recent decades, novel subunit vaccines based on E2 glycoprotein has also become a viable alternative by demonstrating protection against horizontal and vertical transmission and the potential to differentiate vaccinated from infected animals [4, 7].

The use of second-generation marker vaccines against CSFV could be important in the next future to control the disease [8]. In Cuba, where the virus remains endemic, vaccination is recommended to contain the virus spread and prevent massive losses and [9]

Porvac^® is a CSF subunit vaccine recently registered for its commercialization in Cuba and Viet Nam. The active ingredient of this vaccine is the chimeric protein E2-CD154, formed by the fusion of the extracellular region of E2 glycoprotein of CSFV Margarita strain and the extracellular segment of the pig CD154 molecule. E2-CD154 was formulated in Montanide^™ ISA50 V2 (SEPPIC, Paris, France) using a 60/40 proportion of aqueous/oil phase [10]. This vaccine is currently used as an important element of the national program for the control and eradication of CSF.

Unlike the first generation subunit vaccines, Porvac^® is capable of inducing a very early onset of protection [11] and interfering with the vertical transmission of the virus [12].

The demonstration of vaccine stability is mandatory because it is a critical factor affecting the quality, potency, and distribution of vaccines. All immunization campaigns have the challenge of validating and maintaining the cold chain during distribution, delivery, and storage of the vaccines. This aspect is especially sensitive for developing countries, frequently lacking the necessary infrastructure to guarantee the correct functioning of the cold chain during vaccination campaigns.

The World Health Organization (WHO) and other regulatory agencies have issued guidelines to regulate how stability studies should be conducted. Both, real-time and accelerated stability studies are recommended. The application of physicochemical and biological assays such as chromatographic and electrophoretic procedures, potency tests, and immunogenicity assays among others are generally encouraged [13, 14].

Previous studies have shown that Porvac^® is stable for 1 week at 37^oC and has a shelf life of at least 36 months at 2–8 ^oC. The aim of this investigation was to further explore Porvac^® stability when exposed to thermal stress. The stability of the emulsion, the safety and immunogenicity of the vaccine were studied in three independent experiments: (1) vaccine stored up to 12 months at 25°C; (2) vaccine stored up to one month at 30°C and 37°C and (3) vaccine needle-punctured vials stored at 37 ^oC for 15 days.

Stability of the emulsion

The emulsion parameters analyzed in the vaccines exposed to different temperatures are summarized in Table 1. All were seen as viscous, white bright, homogeneous emulsions throughout the study. Every specification established for the vaccine: droplet size, rheology, sterility test, mechanical and thermal stability of the emulsion remained within the acceptance criteria in all cases.

Table 1

**Effect of heat stress treatments on quality parameters of the Porvac**®
		Rheological properties		Drop size distribution						Sterility	Stability
	Groups	VI	n	A	B	C	D	E	F		TS	MS
1	I	649	0.769	11.30	60.25	24.11	4.34	0	0	No Growth	0.98	0.99
	II	682	0.763	7.58	43.73	46.14	2.55	0	0	No Growth	0.97	0.99
	III	656	0.765	8.98	51.16	37.88	1.98	0	0	No Growth	0.98	0.98
2	I	650	0.768	34.04	53.39	11.54	1.03	0	0	No Growth	0.98	0.98
	II	632	0.763	31.79	51.60	13.30	3.17	0	0	No Growth	0.99	0.99
	III	788	0.716	31.64	47.33	16.94	4.59	0	0	No Growth	0.97	0.98
3	I	644	0.773	2.50	45.17	45.8	2.50	0	0	No Growth	0.98	0.98
	II	624	0.780	4.17	70.83	24.17	0.83	0	0	No Growth	0.98	0.98
	III	643	0.756	3.53	60.30	34.04	2.13	0	0	No Growth	0.98	0.98
	IV	772	0.744	3.54	61.95	32.74	1.77	0	0	No Growth	0.99	0.99
	V	715	0.755	4.13	58.68	35.64	1.65	0	0	No Growth	0.99	0.99
(1) Experiment 1: 25 ^oC for twelve months; (2) Experiment 2: 37 ^oC and 30 ^oC for one month; (3) Experiment 3: 37 ^oC with needle-punctured caps. VI, viscosity index; N, flow index. Drop size distributions: numbers represent the percent of drops within the different ranges defines by the letters: A: ø ≤ 1, B: 1< ø ≤ 2, C: 2 <ø ≤ 3, D: 3< ø ≤ 4, E: 4< ø ≤ 5, F: ≥ 5. TS, thermal stability of the emulsion; MS, mechanical stability of the emulsion.

Chromatographic profiles of the proteins extracted from vaccines exposed to thermal stress (for 7, 15 and 30 days at 37 ^oC) were also verified and compared with the control samples stored at 2–8°C (Fig. 1). For 25°C stability, the profile analyzed was at 12 months. The typical chromatographic profile in all samples was characterized by the presence of a first peak, usually higher and narrow, which corresponds to fragmented chromosomal DNA as has been documented by agarose gel electrophoresis (manuscript in preparation). The second, broader peak includes mainly corresponds to E2-CD154, as indicated by the results of the E2 sandwich competition ELISA; and the last peak contain other lower molecular weight protein contaminants. The retention time of the second peak in the samples subjected to heat stress was between 40 min and 45min, similar to the control sample stored at 4 ^oC, therefore thermal stress did not affect this pattern.

Safety

To evaluate the safety in pigs, local and systemic adverse reactions were monitored in all animals after vaccination. During 21 days after the administration of the immunogens exposed to different temperatures (real time, accelerated, or in use stability) all animals were healthy; no systemic adverse effects of diagnostic importance for the welfare of the animals were reported. No signs of inflammation in the injection site were detected after palpation at 2 hours post immunization. No signs of arthritis, uveitis, anorexia or lethargy were documented. The vaccine was well tolerated in all cases, regardless of the thermal treatment it underwent.

The body temperature is one of the more typical side effects associated with the response to vaccination. The rectal temperature of all pigs in the study remained within the physiological values before and after the immunization (Figs. 2 and 3).

Immunogenicity

CSFV specific NAb were detected in the serum all vaccinated animals after the first immunization with titers that ranged from 1:100 to 1:800 (Fig. 3). The booster inoculation in the third week induced a marked increase in the CSFV specific NAb response, with titers higher than 1:1500 for all vaccinated animals in the study.

In the 25°C stability study, significant differences were found at 28 dpv, in the NAb titers developed by the animals immunized with batch P01002 with respect to the other two batches (Fig. 4A).

In the second stability experiment, significant differences (p = 0.0020) were found after the first immunization between the groups 1 and 2 vaccinated with Porvac^® stored at 30°C and 37°C, respectively, with respect to group 3, (animals vaccinated with the vaccine stored at 4°C). However, after the booster, all animals developed similar NAb titers (Kruskal-Wallis, Dunn test, p > 0.05) (Fig. 4B).

In the third experiment, pigs immunized with the vaccine from needle-punctured vials conserved at 37°C also developed high NAb titers and did not show differences with the control vials stored at 4°C (Fig. 4C).

In spite of the particular differences observed, NAb titers remained very high in all animals, well above the theoretical protection threshold, regardless of the thermal treatment received.

Vaccines are the primary tools for the prevention and control of viral diseases in animals in endemic areas. Despite significant efforts to control and eliminate CSF with mandatory vaccination policies, using MLV, the disease is still endemic in Cuba and outbreaks continue to occur [4, 15, 16]. Previous reports have suggested that the virus has evolved into low pathogenicity strains, driven by the implementation of inefficient vaccination programs [17].

Vaccines can be very susceptible to environmental conditions. In particular, temperature changes significantly affect the integrity of this type of product during storage, transportation and handling. The distribution of biologicals for the treatment of domestic animals in Cuba is carried out in polystyrene boxes with refrigerant bags, through commercial transport, which guarantees their quality. However, technical problems during cold-chain could occur and expose the products to the environmental conditions. MLV vaccines are very susceptible to these problems. Temperature changes during vaccine storage, transportation, delivery and handling affect significantly the virus replication capacity. Failures in the cold-chain and manipulation issues regarding these types of vaccines might lead to a diminished efficacy [18].

The structural instability of vaccine antigens is one of the biggest challenges affecting the quality of vaccines. Therefore, a more stable subunit vaccine against CSFV could perform better in developing countries endemic for this disease.

The safety and effectiveness of Porvac® in pigs from different categories has been widely documented [19–22]. This vaccine has also been capable of providing a very rapid onset of protection[11] and to protect against vertical transmission [12]. Due to these benefits, Porvac® has been proposed for the control of CSF in endemic regions, and in consequence, the stability of the vaccine is of utmost importance.

Among the procedures more often used for measuring the stability of vaccines are real times and accelerated stability studies. In the later, vaccines are subjected to temperatures at which active pharmaceutical ingredient (API) degradation occurs at a faster pace. Then, the rate at which it occurs can be extrapolated to the lower temperatures used for vaccine storage [23].

In a previous work from our group, the shelf life determined for Porvac^® at storage conditions (2–8 ^oC) was at least 3 years. It was also shown to remain safe and immunogenic after one week incubation at 37 ^oC [24]. In this paper, those previous studies were expanded to explore different temperatures and incubation times.

First, the 25°C stability study is very important, since the outcome of this experiment can predict the behavior of the vaccine at room temperature if the cold chain is affected. The results indicate that Porvac^® is stable at 25 ^oC for at least one year. The average temperature in Cuba in the hottest month of the year is 27.5 ^oC with maximal values of 32.2 ^oC in the afternoons, higher than the one evaluated in this study. However, if the vaccine is stable for one year at 25 ^oC it is most likely that it would resists for several days out of refrigeration during a vaccination campaign.

Next, two other accelerated stability studies were conducted during four weeks at 30 ^oC and 37 ^oC. The results revealed that heat stress did not affect the main quality parameters of the vaccine: white color with a homogeneous appearance, a prevalent drop size between 1 µm and 2 µm, which classifies the emulsion as fine and preserves its stability. Small droplet sizes allow a more efficient diffusion of the antigen in the animal injected to reach quickly and systematically the lymphatic tissues and thus trigger the immune response of the animal [25]. Additionally, no separation of the aqueous and oil phases was observed after this incubation period; rheological properties were maintained, no microorganism growth or changes in the organoleptic properties of the immunogen were detected. All these results indicate that the physicochemical properties of the emulsion were preserved.

Another important parameter evaluated was the chromatographic profile of the antigen after the incubation period. After disrupting the emulsion by freeze-thawing and centrifugation, the chromatographic profile of the antigen was preserved, which indicate that the antigen did not suffer significant degradation or aggregation during the incubation time. The E2-CD154 protein forms large polydisperse aggregates with and average size of 517 kD (manuscript in preparation). After four weeks of incubation at 30 ^oC and 37 ^oC, the average retention time of the second peak, where E2-CD154 elutes, was similar to that of the control stored at 4 ^oC. These findings are in correspondence with the immunogenicity results, since a direct correlation between conformational stability of the E2 glycoprotein and the induction of a protective NAbs response in pigs has been reported by several authors [26–28].

In this study, even after one month exposure to 37°C, the pigs did not manifest adverse events; the vaccine continued to be safe and well tolerated.

Another essential element is that Porvac^® subjected to heat stress retained the ability to induce high NAb titers. In a previous study from our group [29], after the analysis of 22 challenge experiments with Porvac^®, the geometric mean of the NAb titers was 1:5153. A complete correspondence between NAb titers and protection was found (pigs remained free from CSF clinical signs and pathological lesions and were negative for viral isolation after the challenge). There had been already established from previous investigations from several authors that NAb titers > 1:32 confer an adequate protection to both the individual animal and the herd [30, 31].

The results of the second accelerated stability experiment re-define the stability at 37 ^oC of Porvac^® up to one month, instead of the one week informed in the previous publication.

Finally, another important aspect studied here for the first time was the in-use stability of Porvac^®. It became evident that the vaccine retains its physicochemical properties and even the sterility for two weeks at 37 ^oC, after the cap has been needle-punctured. The safety and immunogenicity of this in-use vaccine were also preserved.

In both human and animal health, vaccines resistant to damage by heat could have great economic and health benefit. A virus like particles vaccine against rabbit hemorrhagic disease adjuvated with Montanide 888 was stable at 37 ^oC or 48 ^oC for 7 days, since the most important physic-chemical and functional properties of the vaccine remained unaffected [32]. A recombinant vaccine against cattle tick also formulated in Montanide 88 was stable for 14 days at 37°C in needle-punctured vaccine vials [33]. Newcastle disease (ND) virus vaccine exposed up to 30 ^oC for 7 days was found effective in prevention of Newcastle Disease in village chickens [34].

Heat stress studies have been also reported in classical swine fever vaccines: stability for 7 days at 37 ^oC was confirmed for a recombinant vaccine against CSF formulated in Montanide 888 [35]. Live attenuated classical swine fever virus vaccine was heat stable at 37 ^oC for 10 days [36]. Pachauri [37] reported the stability of live attenuated CSF vaccine in liquid form at 4 ^oC, 25 ^oC and 37 ^oC up to 24 hours. The liquid vaccine was stable at 4 ^oC up to 24 h, whereas, a drop of one log₁₀ titer was observed at 25 ^oC and 37 ^oC during the same period.

To our knowledge, no other vaccine has yet proved to be stable for so long as one month at 37°C. Due to this remarkable thermal stability, Porvac^® can be a potent tool for CSFV control and eradication programs, especially in developing countries, where the harsh field conditions require a robust vaccine. This is another important advantage of Porvac^® in comparison with the thermally sensitivity of traditional MLV.

Porvac® is stable in all the experimental conditions tested, even after cap puncture, and retains the capacity to induce protective neutralizing antibodies. These results reinforce the robustness of the vaccine, and support its use as a very attractive alternative to modified live vaccines for vaccination campaigns in developing countries endemic for CSF where the cold chain is often compromised.

Ethics statement

The experiments were approved and conducted following the guidelines of the Ethics Committee on Animal Experimentation. All procedures and samplings involving animals were carried out following the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011) and approved by the Ethics Committee of the Center for Genetic Engineering and Biotechnology (CIGB) (approved protocols number VE2CD-0120/2, VE2CD-0121/2 and VE2CD-0220/2).

Vaccine

The active ingredient of Porvac^® is a chimeric protein formed by the fusion of the extracellular region of E2 glycoprotein of CSFV Margarita strain, and the extracellular segment of swine CD154 molecule. E2-CD154 protein was formulated in Montanide^™ ISA50 V2 (SEPPIC, Paris, France) using a 60/40 proportion of aqueous/oil phases. The ‘‘water in oil” emulsion was produced with an SD-41 homogenizer (IKA, Germany). The concentration of the E2-CD154 in the final emulsion was 25 µg/mL. Porvac^® is produced under good manufacturing practices (GMP) at the facilities of the Center for Genetic Engineering and Biotechnology of Camagüey.

Stability studies design

(1) Thermal stress at 25°C for twelve months

Three batches of Porvac^® (P01002, P11002 and P11007) were incubated at 25°C for twelve months and immediately used to vaccinate pigs. Five animals were immunized for each experimental group, and five unvaccinated pigs were used as negative controls. Humoral response and protection were evaluated every three months.

(2) Thermal stress at 37^oC and 30^oC for one month

One batch of Porvac^® (P-81061-1) was incubated at 30 ^oC and 37°C for 30 days and immediately used to vaccinate pigs. Five animals were immunized for each experimental group, and three unvaccinated pigs were used as negative controls. A positive control group of five animals was immunized with the vaccine conserved between 2°C and 8°C. The three groups were as followed: I (30°C), II (37°C), III (4°C) and IV (non-vaccinated controls).

(3) Thermal stress in vials with cap punctures (in use stability)

Bottles from lot P11008-1 of Porvac^® were used. Each vial contains 15 doses of 2 mL (50 µg of E2-CD154 protein each dose) for a total volume of 30 mL. The bottles were needle-punctured for a first extraction, and kept at different temperatures according to the following experimental design:

Group I. Sealed vials at 37 ºC during 15 days

Group II. Needle-punctured vials at 37 ºC during 15 days

Group III. Needle-punctured vials at 37 ºC during 7 days.

Group IV. Sealed vials at 4 ºC during 15 days

Group V. Needle-punctured vials at 4 ºC during 15 days

Ten animals were immunized for each experimental group, and ten unvaccinated pigs were used as negative controls

Organoleptic properties

The effects of thermal stress on the organoleptic characteristics of the vaccine bottles were evaluated by visual observation. Three vaccine vials for each temperature group were used. The bottles were opened. The content was poured into 15 mL tubes and allowed to settle in upright position for 10 min, and the contents of the tubes visually inspected for color and appearance. The emulsion must be bright white with a homogeneous appearance to past the test, according to the manufacturer’s instructions.

Rheological properties

The viscosity of the vaccine was determined using a Brookfield DV-III ultrarheometer, coupled to a cryostat to allow control of the temperature of the sample. The rheometer was calibrated using the Brookfield 100 mPas-sec, and Brookfield 1000 mPas-sec reference materials, with rotor speeds of 60 and 250 rpm, respectively. After the cryostat reached a temperature of 20°C, the emulsion was poured into a reservoir designed for this purpose. The rotor speed was initially set at 60 rpm and gradually increased by 10 rpm up to 250 rpm. Three independent replicates of the viscosity readings were made for each sample. The acceptance limits for viscosity are VI ≤ 1500 mPa-sec and the flow index, n < 1.These parameters were measured at the beginning of the study and after the thermal stress [38].

Thermal stability

The test was designed to determine the stability of the emulsion after exposure to elevated temperatures. A sample of 10 mL of the emulsion was poured into 15 mL centrifuge tubes. The height of the emulsion (Ho) in the tube was measured. The tubes were placed in the humid chamber at 37 ± 2 ºC in a vertical position for 14 days, and the height of the emulsion was measured again after that time (Hu). The Hu/Ho ratio was then calculated. This ratio must be equal to or higher than 0.9 to comply with the quality standards established for the vaccine [39].

Mechanical Stability

To measure the mechanical stability of the emulsion, ten milliliters of the emulsion were poured into a 15 mL centrifuge tube. The initial height (Ho) of the emulsion was determined with a graduated ruler. The tubes were centrifuged for 1 hour at 3000 rpm in a SCT-5B centrifuge (Hitachi, Japan).The final height of the emulsified column (Hu) was then measured. The mechanical stability was determined through the Hu/Ho quotient for each replicate of the samples. According to the quality specifications of the product, the formulation passes the test if the average Hu/Ho ratio of three replicates was equal to or higher than 0.80.

Sterility test

Volumes of 2 mL of Porvac^® were extracted from a vial and incubated at either 4°C or 37°C, as described before. Afterwards, they were added to 200 mL of dispersant solution (Peptone + Tween 80). After homogenization of the mixture, a 20mL sample was taken and added to flasks with different culture media (Tryptone Soy Broth for fungi and Thioglycolate for bacteria). Other flasks with the same media inoculated with 10³ c.f.u. /mL of the respective microorganisms were used as positive controls (Staphylococcus aureus, Pseudomonas aureginosa and Clostridium sporogenes for bacteria and Bacillus subtilis, Candida albicans, Aspergillus niger for fungi). Flasks were then incubated for 14 days at 30 ºC to 35 ºC for bacteria and 20 ºC to 25 ºC for fungi. Negative controls treated with each medium were included. Flasks were monitored for the presence of turbidity, biofilms, lumps or any other form of microbial growth on days 3, 5, 7, 9, 11 and 14.

Drop size distribution

Samples of 20 µL of the vaccines subjected to each treatment were diluted in 980 µL of 10% Montanide adjuvant solution (Seppic, France). The mixture was stirred gently to preserve the original drop sizes. The emulsion was then observed microscopically and a total of 100 drops were measured. The photography area was adjusted using the Periplan 12.5 x 20 eyepieces, which have a square in the center of the circle (photography area) engraved on its lens. For phase contrasting, the image was observed with the 10/0.25 CP-A CHROMAT objectives and the 100/1.25 oil DPlan 100. A representative area was chosen to count the number of drops in the following ranges: A: less than 1 µm; B: from 1 µm to 2 µm; C: from 2 µm to 3 µm; D: from 3 µm to 4 µm; E: from 4 µm to 5 µm and F: greater than 5 µm. The following formula was used to calculate the percent of drops below the acceptance limits (drop size ≤ 5 µm). H = (A + B + C + D + E / A + B + C + D + E + F) * 100. Where: H: is the percent of drops ≤ 5 µm. To comply with the test, 80% of the drops in the sample must have a diameter equal to or lower than 5 µm [39].

Analysis by size exclusion chromatography

After the heat stress in vials with or without cap puncture, the emulsion was disrupted to recover the antigen. A volume of 2mL the emulsion was frozen at -70 ^oC during 1 h. After thawing, the sample was centrifuged at 10,000 g for 10 min. Two phases were formed: aqueous phase (translucent) and semi-oil phase (white). The aqueous phase was extracted and stored at -20 ^oC.

The E2-CD154 antigen integrity was evaluated by Size Exclusion Chromatography (SEC) at the aqueous phase (after emulsion split-up). Chromatographic separation was accomplished in a Superose^™ 6 Increase 10/300 GL column (GE, EE UU) coupled to an Akta Pure 25 M system (GE, EE UU), previously equilibrated with phosphate buffer. The flow rate and wavelength were 0.5 mL/min and 280 nm, respectively. The absorbance at 220 nm was registered. Data acquisition and processing were carried out with UNICORN v7.0 software.

Sandwich ELISA

Identity and quantification the protein E2-CD154 was performed by sandwich enzyme-linked immunosorbent assay (ELISA), using E2 specific monoclonal antibodies (MAb), purchased by the Center of Genetic Engineering and Biotechnology of Sancti Spíritus (Sancti Spíritus, Cuba). Nunc MaxiSorp^™ ELISA plates (Fisher Scientific) were coated with 10 µg/mL of 1G6 MAb in carbonate-bicarbonate buffer solution, pH 9.6, and incubated overnight at 4 ⁰C.A volume of 100 µL/well was used in this and in the rest of the steps, except for the stop solution. Plates were washed three times with Phosphate buffer saline (PBS) + Tween-20 (0.05%), and blocked with PBS + 1% skim milk for 1 h at 37°C. Next, a standard curve of E2-CD154 diluted in PBS + 1% skim milk + 0.05% Tween 20 was added. The standard curve included six points from 60 ng/ml and other to 1.5 ng/mL. Serial dilutions of the problem samples in the same dilution buffer were incubated in parallel during 1 h at 37°C. The microplate was washed again prior to the addition of MAb CBSSE2.3 conjugated to horseradish peroxidase. The reaction was revealed with 0.5 mg/mL orthophenylenediamine and 0.015% H₂O₂ and arrested with 50 µL/well of 2M sulfuric acid. The optical density at 492 nm was measured in a Sunrise^™, ELISA reader (Tecan Life Sciences, Männedorf, Switzerland).

Animals and immunization schedule

Cross breeding York-Land swine weighting about 20–25 Kg, serologically negative to CSFV and belonging to a non-vaccinated and CSF free herd were used, animals were identified with earrings, housed by experimental groups and handled according to international guidelines for experimentation with animals [40].

For the immunization scheme, two doses of 2 mL of the vaccine (containing 50 µg of E2-CD154 antigen) were applied by deep intramuscular injection in the neck. The first immunization was performed at day 0 on the right side of the neck and the second at day 21 on the left side, using 18 G x 1 inch needles and in compliance with good veterinary clinical practices. Ten animals were included in each experimental group.

Sample collection

Blood samples were collected before the first inoculation, and at the end of the experiment, 7 days after the second immunization. The animals were bled by ophthalmic venous sinus punctures using sterile tubes without anticoagulant at 0, 21, 28 days post-vaccination (dpv).

Neutralizing antibodies detection

The sera were screened for the ability to neutralize the cell culture adapted Margarita CSFV strain (National Center for Animal and Plant Health, Mayabeque Cuba) using neutralizing peroxidase linked-assay (NPLA), as described elsewhere [1, 41]. The assay was revealed with the anti E2 Mab CBSSE2.3 (CIGB-SS, Cuba) conjugated to horseradish peroxidase, followed by 3-amino-9-ethyl carbazole (AEC), and hydrogen peroxide substrate. The viral replication was determined by visual inspection at the optical microscope. The last serum dilution without any signal of virus replication was considered as the neutralizing antibody (NAb) titer. The results are expressed as the geometric mean (GM) of the inverse of the NAb titers plus the confidence intervals.

Clinical observation

Vaccinated animals were carefully evaluated daily for clinical signs, anorexia and prostration, inflammatory reactions at the inoculation site, appreciable changes in respiratory rate, and other alterations that may or may not have been related to the vaccination. The rectal temperature of the animals was recorded 1 hour before and 1 hour after each immunization, and once a day during the four subsequent days [42]. Body temperature > 40.3⁰C for two consecutive days was defined as fever according to the Cuban norms.

Statistical analysis

The normality of the data was evaluated with D'Agostino- Pearson tests. Kruskal-Wallis test was applied for the general comparison of the antibody titers among groups, followed by Dunn's multiple comparisons test to evaluate differences between individual groups. For the comparison of the body temperature among groups a simple factorial Analysis of Variance (ANOVA) was performed. The GraphPad Prism 6 software was used for all the analysis (Prism 6 for Windows, Version 6.01, GraphPad Software, Inc., La Jolla, USA). A p < 0.05 was indicative of statistical significance.

height of the emulsion

height of the emulsion after a time period

optical density

CSF

Classical swine fever

MLV

modified live attenuated vaccines

NPLA

Neutralizing peroxidase linked-assay

SEC

Size Exclusion Chromatography

Acknowledgments

The authors acknowledge all researchers and technicians from the Veterinary State Control Laboratory (DSA), for their unconditional support in the handling and care of the animals.

CONFLICTS OF INTEREST

The authors declare no conflict of interest.

FUNDING

No external funding was received for this investigation

DATA AVAILABILITY

The data that support the findings of this study are available on request from the corresponding author

AUTHOR CONTRIBUTIONS

MVH: design of the experiments, acquisition, analysis and interpretation of data and drafted the manuscript. TSG, YSP, design of the experiments, acquisition and interpretation of data. PNV, MPRM: acquisition and interpretation of data. MLHG, ESR, AMR, WPG: acquisition of data. MKMO, ACR and RMH: acquisition, analysis and interpretation of data. CAD: analysis and interpretation of data and drafted the manuscript. DPP and MSP: conception and design of the experiments, analysis and interpretation of data and manuscript review. All authors read and approved the final manuscript.

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Stability of closed and needle-punctured vials of Porvac ® subunit vaccine against classical swine fever subjected to thermal stress

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Abstract

Background

Results

Conclusions

Figures

Introduction

Results

Stability of the emulsion

Safety

Immunogenicity

Discussion

Conclusions

Materials and Methods

Ethics statement

Vaccine

Stability studies design

Organoleptic properties

Rheological properties

Thermal stability

Mechanical Stability

Sterility test

Drop size distribution

Analysis by size exclusion chromatography

Sandwich ELISA

Animals and immunization schedule

Sample collection

Neutralizing antibodies detection

Clinical observation

Statistical analysis

Abbreviations

Declarations

Acknowledgments

References

Additional Declarations

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Version 1