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

Stability of closed and needle-punctured vials of Porvac ® subunit vaccine against classical swine fever subjected to thermal stress | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Stability of closed and needle-punctured vials of Porvac ® subunit vaccine against classical swine fever subjected to thermal stress Talía Sardina-González, Milagros Vargas-Hernández, Yusmel Sordo-Puga, and 12 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4003547/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 15 Nov, 2024 Read the published version in BMC Veterinary Research → Version 1 posted 4 You are reading this latest preprint version Abstract 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 o C and have a shelf life of at least 36 months at 2–8 o C. 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 o C for 12 months; (2) 30 o C and 37 o C 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. Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction 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 A nimal 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 o C and has a shelf life of at least 36 months at 2–8 o C. 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 o C for 15 days. Results 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 o C for twelve months; (2) Experiment 2: 37 o C and 30 o C for one month; (3) Experiment 3: 37 o C 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 o C) 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 o C, 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. 4 A). 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. 4 B). 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. 4 C). 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. Discussion 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 o C) was at least 3 years. It was also shown to remain safe and immunogenic after one week incubation at 37 o C [ 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 o C for at least one year. The average temperature in Cuba in the hottest month of the year is 27.5 o C with maximal values of 32.2 o C in the afternoons, higher than the one evaluated in this study. However, if the vaccine is stable for one year at 25 o C 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 o C and 37 o C. 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 o C and 37 o C, the average retention time of the second peak, where E2-CD154 elutes, was similar to that of the control stored at 4 o C. 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 o C 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 o C, 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 o C or 48 o C 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 o C 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 o C 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 o C for 10 days [ 36 ]. Pachauri [ 37 ] reported the stability of live attenuated CSF vaccine in liquid form at 4 o C, 25 o C and 37 o C up to 24 hours. The liquid vaccine was stable at 4 o C up to 24 h, whereas, a drop of one log 10 titer was observed at 25 o C and 37 o C 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. 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 for vaccination campaigns in developing countries endemic for CSF where the cold chain is often compromised. Materials and Methods 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 o C and 30 o C for one month One batch of Porvac ® (P-81061-1) was incubated at 30 o C 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 10 3 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 o C 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 o C. 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 0 C.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% H 2 O 2 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 > 40.3 0 C 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. Abbreviations Ho height of the emulsion Hu height of the emulsion after a time period OD optical density CSF Classical swine fever MLV modified live attenuated vaccines NPLA Neutralizing peroxidase linked-assay SEC Size Exclusion Chromatography Declarations 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. References Manual of Diagnostic Tests and Vaccines for Terrestrial, Animals. Chap 3.9.3. Classical swine fever (infection with classical swine fever virus) [ https://www.woah.org/fileadmin/Home/eng/Health_standards/tahm/3.09.03_CSF.pdf ]. Postel A, Austermann-Busch S, Petrov A, Moennig V, Becher P. Epidemiology, diagnosis and control of classical swine fever: Recent developments and future challenges. Transbound Emerg Dis. 2018;65:248–61. Zhang H, Leng C, Tian Z, Liu C, Chen J, Bai Y, Li Z, Xiang L, Zhai H, Wang Q. Complete genomic characteristics and pathogenic analysis of the newly emerged classical swine fever virus in China. BMC Vet Res. 2018;14(1):1–11. Coronado L, Perera CL, Rios L, Frías MT, Pérez LJ. A Critical Review about Different Vaccines against Classical Swine Fever Virus and Their Repercussions in Endemic Regions. Vaccines. 2021;9(2):154. Huang Y-L, Deng M-C, Wang F-I, Huang C-C, Chang C-Y. The challenges of classical swine fever control: modified live and E2 subunit vaccines. Virus Res. 2014;179:1–11. Gong W, Li J, Wang Z, Sun J, Mi S, Xu J, Cao J, Hou Y, Wang D, Huo X. Commercial E2 subunit vaccine provides full protection to pigs against lethal challenge with 4 strains of classical swine fever virus genotype 2. Vet Microbiol. 2019;237:108403. Zhou P, Huang J, Li Y, Chen H, Wu Y, Fu X, Hao X, Li Q, Zeng R, Zhang G. Efficiency Comparison of a Novel E2 Subunit Vaccine and a Classic C-Strain Vaccine against Classical Swine Fever. Veterinary Sci. 2021;8(8):148. Ganges L, Crooke HR, Bohórquez JA, Postel A, Sakoda Y, Becher P, Ruggli N. Classical swine fever virus: The past, present and future. Virus Res. 2020;289:198151. Fonseca-Rodriguez O, Coronado L, Amarán L, Perera CL, Centelles Y, Montano DN, Alfonso P, Fernández O, Santoro KR, Frías-Lepoureau MT. Descriptive epidemiology of endemic classical swine fever in cuba. Span J Agricultural Res 2018, 16(2). Suárez M, Sordo Y, Prieto Y, Rodríguez MP, Méndez L, Rodríguez EM, Rodríguez-Mallon A, Lorenzo E, Santana E, González N. A single dose of the novel chimeric subunit vaccine E2-CD154 confers early full protection against classical swine fever virus. Vaccine 2017. Sordo-Puga Y, Suárez-Pedroso M, Naranjo-Valdéz P, Pérez-Pérez D, Santana-Rodríguez E, Sardinas-Gonzalez T, Mendez-Orta MK, Duarte-Cano CA, Estrada-Garcia MP. Rodríguez-Moltó MP: Porvac® Subunit Vaccine E2-CD154 Induces Remarkable Rapid Protection against Classical Swine Fever Virus. Vaccines. 2021;9(2):167. Muñoz-González S, Sordo Y, Pérez-Simó M, Suarez M, Canturri A, Rodriguez MP, Frías-Lepoureau MT, Domingo M, Estrada MP, Ganges L. Efficacy of E2 glycoprotein fused to porcine CD154 as a novel chimeric subunit vaccine to prevent classical swine fever virus vertical transmission in pregnant sows. Vet Microbiol. 2017;205:110–6. WHO. Guidelines on stability evaluation of vaccines. Biologicals: J Int Association Biol Stand. 2009;37(6):424–34. WHO. Temperature sensitivity of vaccines. In.: World Health Organization; 2006. Fonseca O. Caracterización espaciotemporal y factores de riesgo del comportamiento endémico de la peste porcina clásica en Cuba. PhD. UNAH Fructuoso Rodríguez Pérez 2016. Fonseca-Rodríguez O, Centelles Garcia Y, Alfonso Zamora P, Ferrer-Miranda E, Montano DN, Blanco M, Gutiérrez Y, Calistri P, Santoro KR, Percedo MI. Classical Swine Fever in a Cuban Zone Intended for Eradication: Spatiotemporal Clustering and Risk Factors. Front veterinary Sci. 2020;7:38. Díaz de Arce H, Ganges L, Barrera M, Naranjo D, Sobrino F, Frias MT, Nunez JI. Origin and evolution of viruses causing classical swine fever in Cuba. Virus Res. 2005;112(1–2):123–31. Soleimani S, Rashid S. Correlation Study of the Most Important Environmental Influencing Factors on the Razi MMR Vaccine. Arch Razi Inst. 2021;76(5):1203. Oliva-Cárdenas A, Fernández-Zamora F, Santana-Rodríguez E, Sordo-Puga Y, Vargas-Hernández MdlC, Rodríguez-Moltó MP, Pérez-Pérez D, Sardina-González T, Duarte CA, León-Goñi A. Safety and immunogenicity in piglets of two immunization schedules initiated at two or three weeks of age with Porvac®, a classical swine fever subunit marker vaccine. Bionatura 2021, 6(3). Pérez-Pérez D, Sordo-Puga Y, Rodríguez-Moltó MP, Sardina T, Santana E, Montero C, Ancizar J, Cabrera Y, Tuero Á, Naranjo P. E2-CD154 vaccine candidate is safe and immunogenic in pregnant sows, and the maternal derived neutralizing antibodies protect piglets from classical swine fever virus challenge. Vet Microbiol 2021:109153. Suárez-Pedroso M, Sordo-Puga Y, Pérez-Pérez D, Rodríguez-Moltó M, Sosa-Testé I, Oliva-Cárdenas A, Duarte C, Santana-Rodríguez E, Naranjo-Valdéz P, González-Fernández N et al. Seguridad y eficacia a largo plazo de Porvac®, una vacuna de subunidad contra la peste porcina clásica. Anales de la Acad de Ciencias de Cuba 2022, 12(3). Sordo-Puga Y, Pérez-Pérez D, Montero-Espinosa C, Oliva-Cárdenas A, Sosa-Teste I, Duarte CA, Rodríguez-Moltó MP, Sardina-González T, Santana-Rodríguez E, Vargas-Hernández M. Immunogenicity of E2CD154 Subunit Vaccine Candidate against Classical Swine Fever in Piglets with Different Levels of Maternally Derived Antibodies. Vaccines. 2021;9(1):7. Chan CP. Forced degradation studies: current trends and future perspectives for protein-based therapeutics. Expert Rev Proteomics. 2016;13(7):651–8. Sordo-Puga Y, Sardina-González T, Sosa-Testé I, Naranjo-Valdéz P, Rodríguez-Moltó M, Santana-Rodríguez E, Vargas-Hernández M, Méndez-Orta M, Pérez-Pérez D, Oliva-Cárdenas A, et al. Shelf Life and Accelerated Stability Studies of Porvac®, a Marker Subunit Vaccine Against Classical Swine Fever. Int J Sci Res Biol Sci. 2022;9(3):16–24. Gerdts V. Adjuvants for veterinary vaccines–types and modes of action. Berl Munch Tierarztl Wochenschr. 2015;128(11–12):456–63. Lin G-J, Liu T-Y, Tseng Y-Y, Chen Z-W, You C-C, Hsuan S-L, Chien M-S, Huang C. Yeast-expressed classical swine fever virus glycoprotein E2 induces a protective immune response. Vet Microbiol. 2009;139(3–4):369–74. Hua RH, Huo H, Li YN, Xue Y, Wang XL, Guo LP, Zhou B, Song Y, Bu ZG. Generation and efficacy evaluation of recombinant classical swine fever virus E2 glycoprotein expressed in stable transgenic mammalian cell line. PLoS ONE. 2014;9(9):e106891. Madera R, Gong W, Wang L, Burakova Y, Lleellish K, Galliher-Beckley A, Nietfeld J, Henningson J, Jia K, Li P. Pigs immunized with a novel E2 subunit vaccine are protected from subgenotype heterologous classical swine fever virus challenge. BMC Vet Res. 2016;12(1):197. Suárez-Pedroso M, Sordo-Puga Y, Rodríguez-Moltó MP, Naranjo-Valdés P, Pérez-Pérez D, Sosa-Testé I, Montero-Espinosa C, Fuentes-Rodríguez Y, Sardina-González T, Santana-Rodríguez E et al. Neutralizing antibodies as a correlate of protection against classical swine fever in Porvac® vaccinated pigs. Bionatura 2023, 8(1). Biront P, Leunen J, Vandeputte J. Inhibition of virus replication in the tonsils of pigs previously vaccinated with a Chinese strain vaccine and challenged oronasally with a virulent strain of classical swine fever virus. Vet Microbiol. 1987;14(2):105–13. Terpstra C, Wensvoort G. The protective value of vaccine-induced neutralising antibody titres in swine fever. Vet Microbiol. 1988;16(2):123–8. Fernández E, Toledo JR, Méndez L, González N, Parra F, Martín-Alonso JM, Limonta M, Sánchez K, Cabrales A, Estrada MP. Conformational and thermal stability improvements for the large-scale production of yeast-derived rabbit hemorrhagic disease virus-like particles as multipurpose vaccine. PLoS ONE. 2013;8(2):e56417. Vargas-Hernández M, Santana-Rodríguez E, Montero-Espinosa C, Sordo-Puga Y, Acosta-Hernández A, Fuentes-Rodríguez Y, Pérez-Pérez D, Oliva-Cárdenas A, González-Ramos E, Duarte CA. Stability, safety and protective immunity of Gavac® vaccine subjected to heat stress. Biotecnología Aplicada. 2018;35(1):1501–3. Acharya M, Adhikari S, Awasthi H, Jha A, Singh U. Field Verification Trial of ND I-2 Vaccine in Nepal. Nepal Veterinary J. 2019;36:15–22. Barrera M, Sanchez O, Prieto Y, Castell S, Naranjo P, Rodriguez MP, Farnós O, Aguilera A, Frias M, Fonseca O. Thermal stress treatment does not affect the stability and protective capacity of goat milk derived E2-marker vaccine formulation against CSFV. Vet Immunol Immunopathol. 2009;127(3–4):325–31. Zuo X-x, Zhao Y-h, Zhou M-x, Deng B-h, Lv HL-g, Lu F, Hou Y. -b: Live vaccine preserved at room temperature: Preparation and characterization of a freeze-dried classical swine fever virus vaccine. Vaccine. 2020;38(52):8371–8. Pachauri R, Manu M, Vishnoi P, Preethi BO, Tiwari AK, Dhar P. Stability of live attenuated classical swine fever cell culture vaccine virus in liquid form for developing an oral vaccine. Biologicals. 2020;68:108–11. Pimentel-Pérez R, Zamora-Sánchez J, López-Sáez Y, Segura-Silva RM, Olivera-Romero V, Pérez-Sánchez A, UN ADYUVANTE DE ORIGEN VEGETAL PARA EL PROCESO DE FORMULACIÓN DE LA VACUNA GAVAC®. EVALUACIÓN DE. Revista Cientifica de la Facultade de Veterinaria 2018, 28(6). Enríquez A, Canales M, Ramos E, Dandie H, Boué O, Soto A, Cabrera D. Production of a recombinant vaccine against Boophilus microplus. In.: Elfos Scientiae Havana, Cuba; 1995: 79–102. VICH. Harmonization of criteria to waive target animal batch safety testing (TABST) for inactivated vaccines for veterinary use, GL50 2014. Santana-Rodríguez E, Méndez-Orta M, Sardina-González T, Rodríguez-Moltó M, Castell-Brizuela S, Sordo-Puga Y, Pérez-Pérez D, Fundora-Llera A, Oliva-Cárdenas A, Vargas-Hernández M, et al. Consistency of the Neutralizing Peroxidase Linked Assay for Classical Swine Fever and Homologation with an OIE Reference Laboratory. Int J Sci Res Biol Sci. 2022;9(2):30–4. VICH. Target Animal Safety for Veterinary live and inactivated Vaccines, GL44. In.; 2010. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 15 Nov, 2024 Read the published version in BMC Veterinary Research → Version 1 posted Editorial decision: Revision requested 18 Apr, 2024 Submission checks completed at journal 16 Apr, 2024 Editor assigned by journal 16 Apr, 2024 First submitted to journal 01 Mar, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4003547","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":292850270,"identity":"d1e712f1-12dd-44ae-a667-2a6206908d10","order_by":0,"name":"Talía Sardina-González","email":"","orcid":"","institution":"Centro de Ingeniería Genética y Biotecnología","correspondingAuthor":false,"prefix":"","firstName":"Talía","middleName":"","lastName":"Sardina-González","suffix":""},{"id":292850271,"identity":"4d7c208a-a682-4118-a009-eb7a1f00691c","order_by":1,"name":"Milagros Vargas-Hernández","email":"data:image/png;base64,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","orcid":"","institution":"Centro de Ingeniería Genética y Biotecnología","correspondingAuthor":true,"prefix":"","firstName":"Milagros","middleName":"","lastName":"Vargas-Hernández","suffix":""},{"id":292850272,"identity":"fa114fe4-81e8-4bda-be19-3279fc440055","order_by":2,"name":"Yusmel Sordo-Puga","email":"","orcid":"","institution":"Centro de Ingeniería Genética y Biotecnología","correspondingAuthor":false,"prefix":"","firstName":"Yusmel","middleName":"","lastName":"Sordo-Puga","suffix":""},{"id":292850273,"identity":"e7710b5f-06c0-4214-a88d-c2e15e5907d3","order_by":3,"name":"Paula Naranjo-Valdéz","email":"","orcid":"","institution":"Unidad Central de Laboratorios de Salud Animal","correspondingAuthor":false,"prefix":"","firstName":"Paula","middleName":"","lastName":"Naranjo-Valdéz","suffix":""},{"id":292850274,"identity":"b5d57ed2-85fd-4695-8356-fb2c0e2196c7","order_by":4,"name":"María Pilar Rodríguez-Moltó","email":"","orcid":"","institution":"Centro de Ingeniería Genética y Biotecnología","correspondingAuthor":false,"prefix":"","firstName":"María","middleName":"Pilar","lastName":"Rodríguez-Moltó","suffix":""},{"id":292850275,"identity":"ab1c8857-84b4-4a71-a0e3-4e778f01664f","order_by":5,"name":"Mary Karla Méndez-Orta","email":"","orcid":"","institution":"Centro de Ingeniería Genética y Biotecnología","correspondingAuthor":false,"prefix":"","firstName":"Mary","middleName":"Karla","lastName":"Méndez-Orta","suffix":""},{"id":292850276,"identity":"59d8f56a-adf7-4a38-8a22-0c51b42edfa3","order_by":6,"name":"Mara Laura Hernández-García","email":"","orcid":"","institution":"Centro de Ingeniería Genética y Biotecnología","correspondingAuthor":false,"prefix":"","firstName":"Mara","middleName":"Laura","lastName":"Hernández-García","suffix":""},{"id":292850277,"identity":"22a99855-2d0e-4ee4-98c1-11749bc5d313","order_by":7,"name":"Elaine Santana-Rodríguez","email":"","orcid":"","institution":"Centro de Ingeniería Genética y Biotecnología","correspondingAuthor":false,"prefix":"","firstName":"Elaine","middleName":"","lastName":"Santana-Rodríguez","suffix":""},{"id":292850278,"identity":"19c1530d-85ea-47ff-af6f-4c51bee49f21","order_by":8,"name":"William Pena-Guimaraes","email":"","orcid":"","institution":"Centro de Ingeniería Genética y Biotecnología de Camaguey","correspondingAuthor":false,"prefix":"","firstName":"William","middleName":"","lastName":"Pena-Guimaraes","suffix":""},{"id":292850279,"identity":"a9fa641a-2d67-462b-91ee-a8851aeb4a07","order_by":9,"name":"Alain Moreira-Rubio","email":"","orcid":"","institution":"Centro de Ingeniería Genética y Biotecnología de Camaguey","correspondingAuthor":false,"prefix":"","firstName":"Alain","middleName":"","lastName":"Moreira-Rubio","suffix":""},{"id":292850280,"identity":"d30ac8fe-406c-40a7-ac61-82387894b412","order_by":10,"name":"Rosaili Mateu-Hernández","email":"","orcid":"","institution":"Centro de Ingeniería Genética y Biotecnología","correspondingAuthor":false,"prefix":"","firstName":"Rosaili","middleName":"","lastName":"Mateu-Hernández","suffix":""},{"id":292850281,"identity":"d030fb20-3122-496f-957e-1b54afad5545","order_by":11,"name":"Ania Cabrales-Rico","email":"","orcid":"","institution":"Centro de Ingeniería Genética y Biotecnología","correspondingAuthor":false,"prefix":"","firstName":"Ania","middleName":"","lastName":"Cabrales-Rico","suffix":""},{"id":292850282,"identity":"82b3b7cf-f7a8-4e31-b5b9-ef799cc77929","order_by":12,"name":"Carlos A. Duarte","email":"","orcid":"","institution":"Centro de Ingeniería Genética y Biotecnología","correspondingAuthor":false,"prefix":"","firstName":"Carlos","middleName":"A.","lastName":"Duarte","suffix":""},{"id":292850283,"identity":"a39b53e3-4193-49c1-99ee-299006428e75","order_by":13,"name":"Danny Pérez-Pérez","email":"","orcid":"","institution":"Centro de Ingeniería Genética y Biotecnología","correspondingAuthor":false,"prefix":"","firstName":"Danny","middleName":"","lastName":"Pérez-Pérez","suffix":""},{"id":292850284,"identity":"7cfb978d-600a-4696-8610-c85ef1223fa7","order_by":14,"name":"Marisela Suárez-Pedroso","email":"","orcid":"","institution":"Centro de Ingeniería Genética y Biotecnología","correspondingAuthor":false,"prefix":"","firstName":"Marisela","middleName":"","lastName":"Suárez-Pedroso","suffix":""}],"badges":[],"createdAt":"2024-03-01 14:48:46","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4003547/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4003547/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12917-024-04354-8","type":"published","date":"2024-11-15T15:57:09+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":55088115,"identity":"9d7628e6-54fe-4a91-84d8-44790c8d41cc","added_by":"auto","created_at":"2024-04-22 11:49:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":108506,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of E2-CD154 integrity in samples of Porvac\u003csup\u003e®\u003c/sup\u003e incubated at 4 \u003csup\u003eo\u003c/sup\u003eC, 30 \u003csup\u003eo\u003c/sup\u003eC or 37\u003csup\u003eo\u003c/sup\u003eC. The emulsion aqueous phase containing E2-CD154 was separated after two freeze thawing cycles and centrifugation and loaded in a Superose6 chromatographic column. The first peak (1) correspond to chromosomal DNA, the second peak (2) contains E2-CD154 and the third peak includes other lower molecular weight contaminants. E2-CD154 was detected and quantified in the chromatographic fractions by and in house sandwich ELISA using monoclonal antibodies. 37-7-P: incubated 7 days at 37°C in punctured vials; 37-15-P: incubated 15 days at 37°C in punctured vials; 37-15-S: incubated 15 days at 37°C in sealed vials; 4-15-S: incubated 15 days at 4°C in sealed vials; P01002, P11002 and P11007, Porvac\u003csup\u003e®\u003c/sup\u003e lots incubated during 30 days at 25 °C in sealed vials.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4003547/v1/c86e7767f1ab4f2ebe9d341d.png"},{"id":55088112,"identity":"0264dd2e-b6fa-412f-af0f-d52809de5fd6","added_by":"auto","created_at":"2024-04-22 11:49:32","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":293014,"visible":true,"origin":"","legend":"\u003cp\u003eTime course of the body temperature of the animals after the 1st and 2nd immunizations with Porvac\u003csup\u003e®\u003c/sup\u003e subjected to real time stability study. The temperature was measured one hour before and one hour after immunization, and once daily during the next four days.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4003547/v1/d7656a4a14956b1e1e98afeb.png"},{"id":55089304,"identity":"0eee6fad-d26e-42fe-a87a-0d911e5e0de7","added_by":"auto","created_at":"2024-04-22 12:05:32","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":92937,"visible":true,"origin":"","legend":"\u003cp\u003eTime course of the body temperature of the animals after the 1st and 2nd immunizations with Porvac\u003csup\u003e®\u003c/sup\u003e subjected to (A): accelerated stability; (B): in use stability. The temperature was measured one hour before and one hour after immunization, and once daily during the next four days.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4003547/v1/69429160e5792306505c1a31.png"},{"id":55088698,"identity":"e9784e70-75fd-47a4-b934-910e1f37b466","added_by":"auto","created_at":"2024-04-22 11:57:32","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":152385,"visible":true,"origin":"","legend":"\u003cp\u003eE2-CD154 antibody response. A: experiment 1 (stability at 25°C); times 0, 3, 6, 9, and 12 refer to the time of incubation of the vaccine. B: experiment 2, (stability at 30 °C and 37 °C); C: Experiment 3; (in use stability); S: sealed vials; P: needle-punctured vials. Dpv: days post vaccination.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4003547/v1/fd8825faadc56dcf99e8d4e5.png"},{"id":69285494,"identity":"4b3ae933-0c67-40bb-8f60-cb22425384bc","added_by":"auto","created_at":"2024-11-18 19:26:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1304852,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4003547/v1/294c0092-376d-4d22-bbaa-67b7edae5cc3.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Stability of closed and needle-punctured vials of Porvac ® subunit vaccine against classical swine fever subjected to thermal stress","fulltext":[{"header":"Introduction","content":"\u003cp\u003eClassical 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 \u003cb\u003eA\u003c/b\u003enimal Health [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCSF has been eliminated in several countries such as Canada, Australia, USA, and various countries of Western Europe [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In contrast, it is still epidemic in Asia, Central and South America, and East Europe [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThere are two main strategies for CSF control: CSF free countries does not vaccinate and apply a \u0026ldquo;stamping out\u0026rdquo; policy for exposed animals during outbreaks [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. On the other hand, in CSF endemic regions the main strategy for controlling CSF is still prophylactic vaccination with modified live attenuated vaccines (MLV) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMLV 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 [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe use of second-generation marker vaccines against CSFV could be important in the next future to control the disease [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In Cuba, where the virus remains endemic, vaccination is recommended to contain the virus spread and prevent massive losses and [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/p\u003e \u003cp\u003ePorvac\u003csup\u003e\u0026reg;\u003c/sup\u003e 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\u003csup\u003e\u0026trade;\u003c/sup\u003e ISA50 V2 (SEPPIC, Paris, France) using a 60/40 proportion of aqueous/oil phase [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. This vaccine is currently used as an important element of the national program for the control and eradication of CSF.\u003c/p\u003e \u003cp\u003eUnlike the first generation subunit vaccines, Porvac\u003csup\u003e\u0026reg;\u003c/sup\u003e is capable of inducing a very early onset of protection [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] and interfering with the vertical transmission of the virus [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe 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.\u003c/p\u003e \u003cp\u003eThe 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 [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePrevious studies have shown that Porvac\u003csup\u003e\u0026reg;\u003c/sup\u003e is stable for 1 week at 37\u003csup\u003eo\u003c/sup\u003eC and has a shelf life of at least 36 months at 2\u0026ndash;8 \u003csup\u003eo\u003c/sup\u003eC. The aim of this investigation was to further explore Porvac\u003csup\u003e\u0026reg;\u003c/sup\u003e 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\u0026deg;C; (2) vaccine stored up to one month at 30\u0026deg;C and 37\u0026deg;C and (3) vaccine needle-punctured vials stored at 37 \u003csup\u003eo\u003c/sup\u003eC for 15 days.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStability of the emulsion\u003c/h2\u003e \u003cp\u003eThe emulsion parameters analyzed in the vaccines exposed to different temperatures are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. 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.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eEffect of heat stress treatments on quality parameters of the Porvac\u003c/b\u003e\u0026reg;\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"13\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eRheological properties\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"6\" nameend=\"c10\" namest=\"c5\"\u003e \u003cp\u003eDrop size distribution\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003eSterility\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c13\" namest=\"c12\"\u003e \u003cp\u003eStability\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGroups\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003en\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eE\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c12\"\u003e \u003cp\u003eTS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c13\"\u003e \u003cp\u003eMS\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e649\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.769\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e11.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e60.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e24.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e4.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNo Growth\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e0.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.99\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e682\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.763\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e43.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e46.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e2.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNo Growth\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e0.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.99\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e656\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.765\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e51.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e37.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNo Growth\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e0.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.98\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e650\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.768\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e34.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e53.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e11.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNo Growth\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e0.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.98\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e632\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.763\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e31.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e51.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e13.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e3.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNo Growth\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e0.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.99\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e788\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.716\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e31.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e47.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e16.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e4.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNo Growth\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e0.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.98\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e644\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.773\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e45.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e45.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e2.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNo Growth\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e0.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.98\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e624\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.780\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e70.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e24.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNo Growth\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e0.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.98\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e643\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.756\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e60.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e34.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e2.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNo Growth\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e0.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.98\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e772\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.744\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e61.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e32.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNo Growth\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e0.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.99\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e715\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.755\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e58.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e35.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNo Growth\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e0.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.99\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"13\"\u003e(1) Experiment 1: 25 \u003csup\u003eo\u003c/sup\u003eC for twelve months; (2) Experiment 2: 37 \u003csup\u003eo\u003c/sup\u003eC and 30 \u003csup\u003eo\u003c/sup\u003eC for one month; (3) Experiment 3: 37 \u003csup\u003eo\u003c/sup\u003eC 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: \u0026oslash; \u0026le; 1, B: 1\u0026lt; \u0026oslash; \u0026le; 2, C: 2 \u0026lt;\u0026oslash; \u0026le; 3, D: 3\u0026lt; \u0026oslash; \u0026le; 4, E: 4\u0026lt; \u0026oslash; \u0026le; 5, F: \u0026ge; 5. TS, thermal stability of the emulsion; MS, mechanical stability of the emulsion.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eChromatographic profiles of the proteins extracted from vaccines exposed to thermal stress (for 7, 15 and 30 days at 37 \u003csup\u003eo\u003c/sup\u003eC) were also verified and compared with the control samples stored at 2\u0026ndash;8\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). For 25\u0026deg;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 \u003csup\u003eo\u003c/sup\u003eC, therefore thermal stress did not affect this pattern.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSafety\u003c/h3\u003e\n\u003cp\u003e 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.\u003c/p\u003e \u003cp\u003eThe 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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eImmunogenicity\u003c/h2\u003e \u003cp\u003eCSFV 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.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). 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.\u003c/p\u003e \u003cp\u003eIn the 25\u0026deg;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.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the second stability experiment, significant differences (p\u0026thinsp;=\u0026thinsp;0.0020) were found after the first immunization between the groups 1 and 2 vaccinated with Porvac\u003csup\u003e\u0026reg;\u003c/sup\u003e stored at 30\u0026deg;C and 37\u0026deg;C, respectively, with respect to group 3, (animals vaccinated with the vaccine stored at 4\u0026deg;C). However, after the booster, all animals developed similar NAb titers (Kruskal-Wallis, Dunn test, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eIn the third experiment, pigs immunized with the vaccine from needle-punctured vials conserved at 37\u0026deg;C also developed high NAb titers and did not show differences with the control vials stored at 4\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eIn 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.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eVaccines 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 [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Previous reports have suggested that the virus has evolved into low pathogenicity strains, driven by the implementation of inefficient vaccination programs [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eVaccines 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 [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe 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.\u003c/p\u003e \u003cp\u003eThe safety and effectiveness of Porvac\u0026reg; in pigs from different categories has been widely documented [\u003cspan additionalcitationids=\"CR20 CR21\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. This vaccine has also been capable of providing a very rapid onset of protection[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] and to protect against vertical transmission [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Due to these benefits, Porvac\u0026reg; has been proposed for the control of CSF in endemic regions, and in consequence, the stability of the vaccine is of utmost importance.\u003c/p\u003e \u003cp\u003eAmong 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 [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn a previous work from our group, the shelf life determined for Porvac\u003csup\u003e\u0026reg;\u003c/sup\u003e at storage conditions (2\u0026ndash;8 \u003csup\u003eo\u003c/sup\u003eC) was at least 3 years. It was also shown to remain safe and immunogenic after one week incubation at 37 \u003csup\u003eo\u003c/sup\u003eC [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In this paper, those previous studies were expanded to explore different temperatures and incubation times.\u003c/p\u003e \u003cp\u003eFirst, the 25\u0026deg;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\u003csup\u003e\u0026reg;\u003c/sup\u003e is stable at 25 \u003csup\u003eo\u003c/sup\u003eC for at least one year. The average temperature in Cuba in the hottest month of the year is 27.5 \u003csup\u003eo\u003c/sup\u003eC with maximal values of 32.2 \u003csup\u003eo\u003c/sup\u003eC in the afternoons, higher than the one evaluated in this study. However, if the vaccine is stable for one year at 25 \u003csup\u003eo\u003c/sup\u003eC it is most likely that it would resists for several days out of refrigeration during a vaccination campaign.\u003c/p\u003e \u003cp\u003eNext, two other accelerated stability studies were conducted during four weeks at 30 \u003csup\u003eo\u003c/sup\u003eC and 37 \u003csup\u003eo\u003c/sup\u003eC. 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 \u0026micro;m and 2 \u0026micro;m, 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 [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. 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.\u003c/p\u003e \u003cp\u003eAnother 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 \u003csup\u003eo\u003c/sup\u003eC and 37 \u003csup\u003eo\u003c/sup\u003eC, the average retention time of the second peak, where E2-CD154 elutes, was similar to that of the control stored at 4 \u003csup\u003eo\u003c/sup\u003eC. 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 [\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, even after one month exposure to 37\u0026deg;C, the pigs did not manifest adverse events; the vaccine continued to be safe and well tolerated.\u003c/p\u003e \u003cp\u003eAnother essential element is that Porvac\u003csup\u003e\u0026reg;\u003c/sup\u003e subjected to heat stress retained the ability to induce high NAb titers. In a previous study from our group [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], after the analysis of 22 challenge experiments with Porvac\u003csup\u003e\u0026reg;\u003c/sup\u003e, 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\u0026thinsp;\u0026gt;\u0026thinsp;1:32 confer an adequate protection to both the individual animal and the herd [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe results of the second accelerated stability experiment re-define the stability at 37 \u003csup\u003eo\u003c/sup\u003eC of Porvac\u003csup\u003e\u0026reg;\u003c/sup\u003e up to one month, instead of the one week informed in the previous publication.\u003c/p\u003e \u003cp\u003eFinally, another important aspect studied here for the first time was the in-use stability of Porvac\u003csup\u003e\u0026reg;\u003c/sup\u003e. It became evident that the vaccine retains its physicochemical properties and even the sterility for two weeks at 37 \u003csup\u003eo\u003c/sup\u003eC, after the cap has been needle-punctured. The safety and immunogenicity of this in-use vaccine were also preserved.\u003c/p\u003e \u003cp\u003eIn 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 \u003csup\u003eo\u003c/sup\u003eC or 48 \u003csup\u003eo\u003c/sup\u003eC for 7 days, since the most important physic-chemical and functional properties of the vaccine remained unaffected [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. A recombinant vaccine against cattle tick also formulated in Montanide 88 was stable for 14 days at 37\u0026deg;C in needle-punctured vaccine vials [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Newcastle disease (ND) virus vaccine exposed up to 30 \u003csup\u003eo\u003c/sup\u003eC for 7 days was found effective in prevention of Newcastle Disease in village chickens [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHeat stress studies have been also reported in classical swine fever vaccines: stability for 7 days at 37 \u003csup\u003eo\u003c/sup\u003eC was confirmed for a recombinant vaccine against CSF formulated in Montanide 888 [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Live attenuated classical swine fever virus vaccine was heat stable at 37 \u003csup\u003eo\u003c/sup\u003eC for 10 days [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Pachauri [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] reported the stability of live attenuated CSF vaccine in liquid form at 4 \u003csup\u003eo\u003c/sup\u003eC, 25 \u003csup\u003eo\u003c/sup\u003eC and 37 \u003csup\u003eo\u003c/sup\u003eC up to 24 hours. The liquid vaccine was stable at 4 \u003csup\u003eo\u003c/sup\u003eC up to 24 h, whereas, a drop of one log\u003csub\u003e10\u003c/sub\u003e titer was observed at 25 \u003csup\u003eo\u003c/sup\u003eC and 37 \u003csup\u003eo\u003c/sup\u003eC during the same period.\u003c/p\u003e \u003cp\u003eTo our knowledge, no other vaccine has yet proved to be stable for so long as one month at 37\u0026deg;C. Due to this remarkable thermal stability, Porvac\u003csup\u003e\u0026reg;\u003c/sup\u003e 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\u003csup\u003e\u0026reg;\u003c/sup\u003e in comparison with the thermally sensitivity of traditional MLV.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003ePorvac\u0026reg; 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.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eEthics statement\u003c/h2\u003e \u003cp\u003e 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).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eVaccine\u003c/h2\u003e \u003cp\u003eThe active ingredient of Porvac\u003csup\u003e\u0026reg;\u003c/sup\u003e 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\u003csup\u003e\u0026trade;\u003c/sup\u003e ISA50 V2 (SEPPIC, Paris, France) using a 60/40 proportion of aqueous/oil phases. The \u0026lsquo;\u0026lsquo;water in oil\u0026rdquo; emulsion was produced with an SD-41 homogenizer (IKA, Germany). The concentration of the E2-CD154 in the final emulsion was 25 \u0026micro;g/mL. Porvac\u003csup\u003e\u0026reg;\u003c/sup\u003e is produced under good manufacturing practices (GMP) at the facilities of the Center for Genetic Engineering and Biotechnology of Camag\u0026uuml;ey.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStability studies design\u003c/h2\u003e \u003cp\u003e(1) Thermal stress at 25\u0026deg;C for twelve months\u003c/p\u003e \u003cp\u003eThree batches of Porvac\u003csup\u003e\u0026reg;\u003c/sup\u003e (P01002, P11002 and P11007) were incubated at 25\u0026deg;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.\u003c/p\u003e \u003cp\u003e(2) Thermal stress at 37\u003csup\u003eo\u003c/sup\u003eC and 30\u003csup\u003eo\u003c/sup\u003eC for one month\u003c/p\u003e \u003cp\u003eOne batch of Porvac\u003csup\u003e\u0026reg;\u003c/sup\u003e (P-81061-1) was incubated at 30 \u003csup\u003eo\u003c/sup\u003eC and 37\u0026deg;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\u0026deg;C and 8\u0026deg;C. The three groups were as followed: I (30\u0026deg;C), II (37\u0026deg;C), III (4\u0026deg;C) and IV (non-vaccinated controls).\u003c/p\u003e \u003cp\u003e(3) Thermal stress in vials with cap punctures (in use stability)\u003c/p\u003e \u003cp\u003eBottles from lot P11008-1 of Porvac\u003csup\u003e\u0026reg;\u003c/sup\u003e were used. Each vial contains 15 doses of 2 mL (50 \u0026micro;g 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:\u003c/p\u003e \u003cp\u003eGroup I. Sealed vials at 37 \u0026ordm;C during 15 days\u003c/p\u003e \u003cp\u003eGroup II. Needle-punctured vials at 37 \u0026ordm;C during 15 days\u003c/p\u003e \u003cp\u003eGroup III. Needle-punctured vials at 37 \u0026ordm;C during 7 days.\u003c/p\u003e \u003cp\u003eGroup IV. Sealed vials at 4 \u0026ordm;C during 15 days\u003c/p\u003e \u003cp\u003eGroup V. Needle-punctured vials at 4 \u0026ordm;C during 15 days\u003c/p\u003e \u003cp\u003eTen animals were immunized for each experimental group, and ten unvaccinated pigs were used as negative controls\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eOrganoleptic properties\u003c/h2\u003e \u003cp\u003eThe 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\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eRheological properties\u003c/h2\u003e \u003cp\u003eThe 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\u0026deg;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\u0026thinsp;\u0026le;\u0026thinsp;1500 mPa-sec and the flow index, n\u0026thinsp;\u0026lt;\u0026thinsp;1.These parameters were measured at the beginning of the study and after the thermal stress [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eThermal stability\u003c/h2\u003e \u003cp\u003eThe 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\u0026thinsp;\u0026plusmn;\u0026thinsp;2 \u0026ordm;C 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 [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eMechanical Stability\u003c/h2\u003e \u003cp\u003eTo 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eSterility test\u003c/h2\u003e \u003cp\u003eVolumes of 2 mL of Porvac\u003csup\u003e\u0026reg;\u003c/sup\u003e were extracted from a vial and incubated at either 4\u0026deg;C or 37\u0026deg;C, as described before. Afterwards, they were added to 200 mL of dispersant solution (Peptone\u0026thinsp;+\u0026thinsp;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 10\u003csup\u003e3\u003c/sup\u003e c.f.u. /mL of the respective microorganisms were used as positive controls (\u003cem\u003eStaphylococcus\u003c/em\u003e aureus, \u003cem\u003ePseudomonas aureginosa\u003c/em\u003e and \u003cem\u003eClostridium sporogenes\u003c/em\u003e for bacteria and \u003cem\u003eBacillus subtilis\u003c/em\u003e, \u003cem\u003eCandida albicans\u003c/em\u003e, \u003cem\u003eAspergillus niger\u003c/em\u003e for fungi). Flasks were then incubated for 14 days at 30 \u0026ordm;C to 35 \u0026ordm;C for bacteria and 20 \u0026ordm;C to 25 \u0026ordm;C 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eDrop size distribution\u003c/h2\u003e \u003cp\u003eSamples of 20 \u0026micro;L of the vaccines subjected to each treatment were diluted in 980 \u0026micro;L 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 \u0026micro;m; B: from 1 \u0026micro;m to 2 \u0026micro;m; C: from 2 \u0026micro;m to 3 \u0026micro;m; D: from 3 \u0026micro;m to 4 \u0026micro;m; E: from 4 \u0026micro;m to 5 \u0026micro;m and F: greater than 5 \u0026micro;m. The following formula was used to calculate the percent of drops below the acceptance limits (drop size\u0026thinsp;\u0026le;\u0026thinsp;5 \u0026micro;m). H = (A\u0026thinsp;+\u0026thinsp;B\u0026thinsp;+\u0026thinsp;C\u0026thinsp;+\u0026thinsp;D\u0026thinsp;+\u0026thinsp;E / A\u0026thinsp;+\u0026thinsp;B\u0026thinsp;+\u0026thinsp;C\u0026thinsp;+\u0026thinsp;D\u0026thinsp;+\u0026thinsp;E\u0026thinsp;+\u0026thinsp;F) * 100. Where: H: is the percent of drops\u0026thinsp;\u0026le;\u0026thinsp;5 \u0026micro;m. To comply with the test, 80% of the drops in the sample must have a diameter equal to or lower than 5 \u0026micro;m [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis by size exclusion chromatography\u003c/h2\u003e \u003cp\u003eAfter 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 \u003csup\u003eo\u003c/sup\u003eC 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 \u003csup\u003eo\u003c/sup\u003eC.\u003c/p\u003e \u003cp\u003eThe 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\u003csup\u003e\u0026trade;\u003c/sup\u003e 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eSandwich ELISA\u003c/h2\u003e \u003cp\u003eIdentity 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 Sp\u0026iacute;ritus (Sancti Sp\u0026iacute;ritus, Cuba). Nunc MaxiSorp\u003csup\u003e\u0026trade;\u003c/sup\u003e ELISA plates (Fisher Scientific) were coated with 10 \u0026micro;g/mL of 1G6 MAb in carbonate-bicarbonate buffer solution, pH 9.6, and incubated overnight at 4 \u003csup\u003e0\u003c/sup\u003eC.A volume of 100 \u0026micro;L/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)\u0026thinsp;+\u0026thinsp;Tween-20 (0.05%), and blocked with PBS\u0026thinsp;+\u0026thinsp;1% skim milk for 1 h at 37\u0026deg;C. Next, a standard curve of E2-CD154 diluted in PBS\u0026thinsp;+\u0026thinsp;1% skim milk\u0026thinsp;+\u0026thinsp;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\u0026deg;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% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and arrested with 50 \u0026micro;L/well of 2M sulfuric acid. The optical density at 492 nm was measured in a Sunrise\u003csup\u003e\u0026trade;\u003c/sup\u003e, ELISA reader (Tecan Life Sciences, M\u0026auml;nnedorf, Switzerland).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eAnimals and immunization schedule\u003c/h2\u003e \u003cp\u003eCross breeding York-Land swine weighting about 20\u0026ndash;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 [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFor the immunization scheme, two doses of 2 mL of the vaccine (containing 50 \u0026micro;g 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eSample collection\u003c/h2\u003e \u003cp\u003eBlood 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).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eNeutralizing antibodies detection\u003c/h2\u003e \u003cp\u003eThe 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 [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. 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.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eClinical observation\u003c/h2\u003e \u003cp\u003e 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 [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Body temperature\u0026thinsp;\u0026gt;\u0026thinsp;40.3\u003csup\u003e0\u003c/sup\u003eC for two consecutive days was defined as fever according to the Cuban norms.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe 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\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was indicative of statistical significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHo\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eheight of the emulsion\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHu\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eheight of the emulsion after a time period\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eOD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eoptical density\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCSF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eClassical swine fever\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMLV\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emodified live attenuated vaccines\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNPLA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNeutralizing peroxidase linked-assay\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSEC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSize Exclusion Chromatography\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgments\u003c/h2\u003e\n\u003cp\u003eThe 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONFLICTS OF INTEREST\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo external funding was received for this investigation\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available on request from the corresponding author\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMVH: 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.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eManual of Diagnostic Tests and Vaccines for Terrestrial, Animals. Chap 3.9.3. Classical swine fever (infection with classical swine fever virus) [\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.woah.org/fileadmin/Home/eng/Health_standards/tahm/3.09.03_CSF.pdf\u003c/span\u003e\u003cspan address=\"https://www.woah.org/fileadmin/Home/eng/Health_standards/tahm/3.09.03_CSF.pdf\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e ].\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePostel A, Austermann-Busch S, Petrov A, Moennig V, Becher P. Epidemiology, diagnosis and control of classical swine fever: Recent developments and future challenges. Transbound Emerg Dis. 2018;65:248\u0026ndash;61.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang H, Leng C, Tian Z, Liu C, Chen J, Bai Y, Li Z, Xiang L, Zhai H, Wang Q. Complete genomic characteristics and pathogenic analysis of the newly emerged classical swine fever virus in China. BMC Vet Res. 2018;14(1):1\u0026ndash;11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCoronado L, Perera CL, Rios L, Fr\u0026iacute;as MT, P\u0026eacute;rez LJ. A Critical Review about Different Vaccines against Classical Swine Fever Virus and Their Repercussions in Endemic Regions. Vaccines. 2021;9(2):154.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang Y-L, Deng M-C, Wang F-I, Huang C-C, Chang C-Y. The challenges of classical swine fever control: modified live and E2 subunit vaccines. Virus Res. 2014;179:1\u0026ndash;11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGong W, Li J, Wang Z, Sun J, Mi S, Xu J, Cao J, Hou Y, Wang D, Huo X. Commercial E2 subunit vaccine provides full protection to pigs against lethal challenge with 4 strains of classical swine fever virus genotype 2. Vet Microbiol. 2019;237:108403.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou P, Huang J, Li Y, Chen H, Wu Y, Fu X, Hao X, Li Q, Zeng R, Zhang G. Efficiency Comparison of a Novel E2 Subunit Vaccine and a Classic C-Strain Vaccine against Classical Swine Fever. Veterinary Sci. 2021;8(8):148.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGanges L, Crooke HR, Boh\u0026oacute;rquez JA, Postel A, Sakoda Y, Becher P, Ruggli N. Classical swine fever virus: The past, present and future. Virus Res. 2020;289:198151.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFonseca-Rodriguez O, Coronado L, Amar\u0026aacute;n L, Perera CL, Centelles Y, Montano DN, Alfonso P, Fern\u0026aacute;ndez O, Santoro KR, Fr\u0026iacute;as-Lepoureau MT. Descriptive epidemiology of endemic classical swine fever in cuba. Span J Agricultural Res 2018, 16(2).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSu\u0026aacute;rez M, Sordo Y, Prieto Y, Rodr\u0026iacute;guez MP, M\u0026eacute;ndez L, Rodr\u0026iacute;guez EM, Rodr\u0026iacute;guez-Mallon A, Lorenzo E, Santana E, Gonz\u0026aacute;lez N. A single dose of the novel chimeric subunit vaccine E2-CD154 confers early full protection against classical swine fever virus. \u003cem\u003eVaccine\u003c/em\u003e 2017.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSordo-Puga Y, Su\u0026aacute;rez-Pedroso M, Naranjo-Vald\u0026eacute;z P, P\u0026eacute;rez-P\u0026eacute;rez D, Santana-Rodr\u0026iacute;guez E, Sardinas-Gonzalez T, Mendez-Orta MK, Duarte-Cano CA, Estrada-Garcia MP. Rodr\u0026iacute;guez-Molt\u0026oacute; MP: Porvac\u0026reg; Subunit Vaccine E2-CD154 Induces Remarkable Rapid Protection against Classical Swine Fever Virus. Vaccines. 2021;9(2):167.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMu\u0026ntilde;oz-Gonz\u0026aacute;lez S, Sordo Y, P\u0026eacute;rez-Sim\u0026oacute; M, Suarez M, Canturri A, Rodriguez MP, Fr\u0026iacute;as-Lepoureau MT, Domingo M, Estrada MP, Ganges L. Efficacy of E2 glycoprotein fused to porcine CD154 as a novel chimeric subunit vaccine to prevent classical swine fever virus vertical transmission in pregnant sows. Vet Microbiol. 2017;205:110\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWHO. Guidelines on stability evaluation of vaccines. Biologicals: J Int Association Biol Stand. 2009;37(6):424\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWHO. Temperature sensitivity of vaccines. In.: World Health Organization; 2006.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFonseca O. Caracterizaci\u0026oacute;n espaciotemporal y factores de riesgo del comportamiento end\u0026eacute;mico de la peste porcina cl\u0026aacute;sica en Cuba. \u003cem\u003ePhD.\u003c/em\u003e UNAH Fructuoso Rodr\u0026iacute;guez P\u0026eacute;rez 2016.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFonseca-Rodr\u0026iacute;guez O, Centelles Garcia Y, Alfonso Zamora P, Ferrer-Miranda E, Montano DN, Blanco M, Guti\u0026eacute;rrez Y, Calistri P, Santoro KR, Percedo MI. Classical Swine Fever in a Cuban Zone Intended for Eradication: Spatiotemporal Clustering and Risk Factors. Front veterinary Sci. 2020;7:38.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eD\u0026iacute;az de Arce H, Ganges L, Barrera M, Naranjo D, Sobrino F, Frias MT, Nunez JI. Origin and evolution of viruses causing classical swine fever in Cuba. Virus Res. 2005;112(1\u0026ndash;2):123\u0026ndash;31.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSoleimani S, Rashid S. Correlation Study of the Most Important Environmental Influencing Factors on the Razi MMR Vaccine. Arch Razi Inst. 2021;76(5):1203.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOliva-C\u0026aacute;rdenas A, Fern\u0026aacute;ndez-Zamora F, Santana-Rodr\u0026iacute;guez E, Sordo-Puga Y, Vargas-Hern\u0026aacute;ndez MdlC, Rodr\u0026iacute;guez-Molt\u0026oacute; MP, P\u0026eacute;rez-P\u0026eacute;rez D, Sardina-Gonz\u0026aacute;lez T, Duarte CA, Le\u0026oacute;n-Go\u0026ntilde;i A. Safety and immunogenicity in piglets of two immunization schedules initiated at two or three weeks of age with Porvac\u0026reg;, a classical swine fever subunit marker vaccine. Bionatura 2021, 6(3).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eP\u0026eacute;rez-P\u0026eacute;rez D, Sordo-Puga Y, Rodr\u0026iacute;guez-Molt\u0026oacute; MP, Sardina T, Santana E, Montero C, Ancizar J, Cabrera Y, Tuero \u0026Aacute;, Naranjo P. E2-CD154 vaccine candidate is safe and immunogenic in pregnant sows, and the maternal derived neutralizing antibodies protect piglets from classical swine fever virus challenge. Vet Microbiol 2021:109153.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSu\u0026aacute;rez-Pedroso M, Sordo-Puga Y, P\u0026eacute;rez-P\u0026eacute;rez D, Rodr\u0026iacute;guez-Molt\u0026oacute; M, Sosa-Test\u0026eacute; I, Oliva-C\u0026aacute;rdenas A, Duarte C, Santana-Rodr\u0026iacute;guez E, Naranjo-Vald\u0026eacute;z P, Gonz\u0026aacute;lez-Fern\u0026aacute;ndez N et al. Seguridad y eficacia a largo plazo de Porvac\u0026reg;, una vacuna de subunidad contra la peste porcina cl\u0026aacute;sica. Anales de la Acad de Ciencias de Cuba 2022, 12(3).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSordo-Puga Y, P\u0026eacute;rez-P\u0026eacute;rez D, Montero-Espinosa C, Oliva-C\u0026aacute;rdenas A, Sosa-Teste I, Duarte CA, Rodr\u0026iacute;guez-Molt\u0026oacute; MP, Sardina-Gonz\u0026aacute;lez T, Santana-Rodr\u0026iacute;guez E, Vargas-Hern\u0026aacute;ndez M. Immunogenicity of E2CD154 Subunit Vaccine Candidate against Classical Swine Fever in Piglets with Different Levels of Maternally Derived Antibodies. Vaccines. 2021;9(1):7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChan CP. Forced degradation studies: current trends and future perspectives for protein-based therapeutics. Expert Rev Proteomics. 2016;13(7):651\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSordo-Puga Y, Sardina-Gonz\u0026aacute;lez T, Sosa-Test\u0026eacute; I, Naranjo-Vald\u0026eacute;z P, Rodr\u0026iacute;guez-Molt\u0026oacute; M, Santana-Rodr\u0026iacute;guez E, Vargas-Hern\u0026aacute;ndez M, M\u0026eacute;ndez-Orta M, P\u0026eacute;rez-P\u0026eacute;rez D, Oliva-C\u0026aacute;rdenas A, et al. Shelf Life and Accelerated Stability Studies of Porvac\u0026reg;, a Marker Subunit Vaccine Against Classical Swine Fever. Int J Sci Res Biol Sci. 2022;9(3):16\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGerdts V. Adjuvants for veterinary vaccines\u0026ndash;types and modes of action. Berl Munch Tierarztl Wochenschr. 2015;128(11\u0026ndash;12):456\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin G-J, Liu T-Y, Tseng Y-Y, Chen Z-W, You C-C, Hsuan S-L, Chien M-S, Huang C. Yeast-expressed classical swine fever virus glycoprotein E2 induces a protective immune response. Vet Microbiol. 2009;139(3\u0026ndash;4):369\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHua RH, Huo H, Li YN, Xue Y, Wang XL, Guo LP, Zhou B, Song Y, Bu ZG. Generation and efficacy evaluation of recombinant classical swine fever virus E2 glycoprotein expressed in stable transgenic mammalian cell line. PLoS ONE. 2014;9(9):e106891.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMadera R, Gong W, Wang L, Burakova Y, Lleellish K, Galliher-Beckley A, Nietfeld J, Henningson J, Jia K, Li P. Pigs immunized with a novel E2 subunit vaccine are protected from subgenotype heterologous classical swine fever virus challenge. BMC Vet Res. 2016;12(1):197.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSu\u0026aacute;rez-Pedroso M, Sordo-Puga Y, Rodr\u0026iacute;guez-Molt\u0026oacute; MP, Naranjo-Vald\u0026eacute;s P, P\u0026eacute;rez-P\u0026eacute;rez D, Sosa-Test\u0026eacute; I, Montero-Espinosa C, Fuentes-Rodr\u0026iacute;guez Y, Sardina-Gonz\u0026aacute;lez T, Santana-Rodr\u0026iacute;guez E et al. Neutralizing antibodies as a correlate of protection against classical swine fever in Porvac\u0026reg; vaccinated pigs. Bionatura 2023, 8(1).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBiront P, Leunen J, Vandeputte J. Inhibition of virus replication in the tonsils of pigs previously vaccinated with a Chinese strain vaccine and challenged oronasally with a virulent strain of classical swine fever virus. Vet Microbiol. 1987;14(2):105\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTerpstra C, Wensvoort G. The protective value of vaccine-induced neutralising antibody titres in swine fever. Vet Microbiol. 1988;16(2):123\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFern\u0026aacute;ndez E, Toledo JR, M\u0026eacute;ndez L, Gonz\u0026aacute;lez N, Parra F, Mart\u0026iacute;n-Alonso JM, Limonta M, S\u0026aacute;nchez K, Cabrales A, Estrada MP. Conformational and thermal stability improvements for the large-scale production of yeast-derived rabbit hemorrhagic disease virus-like particles as multipurpose vaccine. PLoS ONE. 2013;8(2):e56417.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVargas-Hern\u0026aacute;ndez M, Santana-Rodr\u0026iacute;guez E, Montero-Espinosa C, Sordo-Puga Y, Acosta-Hern\u0026aacute;ndez A, Fuentes-Rodr\u0026iacute;guez Y, P\u0026eacute;rez-P\u0026eacute;rez D, Oliva-C\u0026aacute;rdenas A, Gonz\u0026aacute;lez-Ramos E, Duarte CA. Stability, safety and protective immunity of Gavac\u0026reg; vaccine subjected to heat stress. Biotecnolog\u0026iacute;a Aplicada. 2018;35(1):1501\u0026ndash;3.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAcharya M, Adhikari S, Awasthi H, Jha A, Singh U. Field Verification Trial of ND I-2 Vaccine in Nepal. Nepal Veterinary J. 2019;36:15\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarrera M, Sanchez O, Prieto Y, Castell S, Naranjo P, Rodriguez MP, Farn\u0026oacute;s O, Aguilera A, Frias M, Fonseca O. Thermal stress treatment does not affect the stability and protective capacity of goat milk derived E2-marker vaccine formulation against CSFV. Vet Immunol Immunopathol. 2009;127(3\u0026ndash;4):325\u0026ndash;31.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZuo X-x, Zhao Y-h, Zhou M-x, Deng B-h, Lv HL-g, Lu F, Hou Y. -b: Live vaccine preserved at room temperature: Preparation and characterization of a freeze-dried classical swine fever virus vaccine. Vaccine. 2020;38(52):8371\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePachauri R, Manu M, Vishnoi P, Preethi BO, Tiwari AK, Dhar P. Stability of live attenuated classical swine fever cell culture vaccine virus in liquid form for developing an oral vaccine. Biologicals. 2020;68:108\u0026ndash;11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePimentel-P\u0026eacute;rez R, Zamora-S\u0026aacute;nchez J, L\u0026oacute;pez-S\u0026aacute;ez Y, Segura-Silva RM, Olivera-Romero V, P\u0026eacute;rez-S\u0026aacute;nchez A, UN ADYUVANTE DE ORIGEN VEGETAL PARA EL PROCESO DE FORMULACI\u0026Oacute;N DE LA VACUNA GAVAC\u0026reg;. EVALUACI\u0026Oacute;N DE. \u003cem\u003eRevista Cientifica de la Facultade de Veterinaria\u003c/em\u003e 2018, 28(6).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEnr\u0026iacute;quez A, Canales M, Ramos E, Dandie H, Bou\u0026eacute; O, Soto A, Cabrera D. Production of a recombinant vaccine against Boophilus microplus. In.: Elfos Scientiae Havana, Cuba; 1995: 79\u0026ndash;102.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVICH. Harmonization of criteria to waive target animal batch safety testing (TABST) for inactivated vaccines for veterinary use, GL50 2014.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSantana-Rodr\u0026iacute;guez E, M\u0026eacute;ndez-Orta M, Sardina-Gonz\u0026aacute;lez T, Rodr\u0026iacute;guez-Molt\u0026oacute; M, Castell-Brizuela S, Sordo-Puga Y, P\u0026eacute;rez-P\u0026eacute;rez D, Fundora-Llera A, Oliva-C\u0026aacute;rdenas A, Vargas-Hern\u0026aacute;ndez M, et al. Consistency of the Neutralizing Peroxidase Linked Assay for Classical Swine Fever and Homologation with an OIE Reference Laboratory. Int J Sci Res Biol Sci. 2022;9(2):30\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVICH. Target Animal Safety for Veterinary live and inactivated Vaccines, GL44. In.; 2010.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-veterinary-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Veterinary Research](http://bmcvetres.biomedcentral.com/)","snPcode":"12917","submissionUrl":"https://submission.nature.com/new-submission/12917/3?","title":"BMC Veterinary Research","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Thermal stress, vaccination efficacy, vaccine stability, Montanide, NPLA, classical swine fever, virus.","lastPublishedDoi":"10.21203/rs.3.rs-4003547/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4003547/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eClassical 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\u003csup\u003e\u0026reg;\u003c/sup\u003e 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 \u003csup\u003eo\u003c/sup\u003eC and have a shelf life of at least 36 months at 2\u0026ndash;8 \u003csup\u003eo\u003c/sup\u003eC. The aim of this work was to further explore the accelerated stability of Porvac\u003csup\u003e\u0026reg;\u003c/sup\u003e 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 \u003csup\u003eo\u003c/sup\u003eC for 12 months; (2) 30\u003csup\u003eo\u003c/sup\u003eC and 37 \u003csup\u003eo\u003c/sup\u003eC for one month and (3) 15 days at 37\u0026deg;C after the cap of the vials had been needle-punctured.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003e 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\u0026thinsp;\u0026ge;\u0026thinsp;1:1000 at 28 days post vaccination.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003ePorvac\u003csup\u003e\u0026reg;\u003c/sup\u003e 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.\u003c/p\u003e","manuscriptTitle":"Stability of closed and needle-punctured vials of Porvac ® subunit vaccine against classical swine fever subjected to thermal stress","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-22 11:49:28","doi":"10.21203/rs.3.rs-4003547/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-04-18T15:06:01+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-04-17T03:15:10+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-17T03:15:10+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Veterinary Research","date":"2024-03-01T14:46:16+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-veterinary-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Veterinary Research](http://bmcvetres.biomedcentral.com/)","snPcode":"12917","submissionUrl":"https://submission.nature.com/new-submission/12917/3?","title":"BMC Veterinary Research","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e41a2612-71f1-4486-a04b-1e0fcb4f9d1c","owner":[],"postedDate":"April 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-11-18T19:20:54+00:00","versionOfRecord":{"articleIdentity":"rs-4003547","link":"https://doi.org/10.1186/s12917-024-04354-8","journal":{"identity":"bmc-veterinary-research","isVorOnly":false,"title":"BMC Veterinary Research"},"publishedOn":"2024-11-15 15:57:09","publishedOnDateReadable":"November 15th, 2024"},"versionCreatedAt":"2024-04-22 11:49:28","video":"","vorDoi":"10.1186/s12917-024-04354-8","vorDoiUrl":"https://doi.org/10.1186/s12917-024-04354-8","workflowStages":[]},"version":"v1","identity":"rs-4003547","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4003547","identity":"rs-4003547","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}