TY - JOUR
AU - Lee, John Hwa
AB - ABSTRACT We compared the effects of two antacid formulations based on sodium bicarbonate and magnesium hydroxide on a Salmonella-delivered oral Brucella live attenuated vaccine. We conducted a series of in vitro and in vivo experiments to investigate the pH buffering capacity, buffering longevity and the effects of these formulations on the survival of Salmonella under neutralized pH conditions and its impact on immune responses. Magnesium hydroxide had a greater, stable and prolonged buffering capacity than sodium bicarbonate and was safer when administered orally. Oral administration of sodium bicarbonate resulted in discomfort as reflected by mouse behavior and mild muscle tremors, whereas mice treated with magnesium hydroxide and PBS were completely normal. Gastric survival studies using BALB/c mice revealed that a higher number of Salmonella reached the intestine when the magnesium hydroxide-based antacid buffer was administrated. Co-administration with attenuated Salmonella secreting Brucella antigens, SodC and Omp19 along with individual antacid formulations, significantly enhanced the antigen-specific protective immune responses against virulent Brucella challenge. Together, our results indicated that the pre vaccinated oral administration of bicarbonate-citric acid or magnesium hydroxide-based neutralizing buffers significantly counteract stomach acidity by maintaining the viability of an oral enteric vaccine formulation. Salmonella, antacid formulation, gastric survival, immune response, sodium bicarbonate, magnesium hydroxide INTRODUCTION Orally administered vaccines are simpler and more acceptable than hypodermic needle-administered vaccines (Lal and Jarrahian 2017). Being a non-invasive approach, oral administration is more suitable for infants and vaccinations trials in underdeveloped regions of the world where adequate sanitation and health care facilities are scarce (Levine 2010; Zhu and Berzofsky 2013). Vaccine antigens experience higher levels of destruction due to the harsh environment present in the stomach and the rest of the gastrointestinal tract. Therefore, antigens preparing for oral administration essentially require special formulations to protect target antigens from the harsh gastric environment (Sack et al. 1997). One common option is the utilization of acid-neutralization agents known as antacid buffers that neutralize gastric pH and protects therapeutic antigens making them available for immune responses. The importance of antacid buffers is further asserted for live attenuated vaccines, where the maximum number of attenuated bacteria must enter the mucosal surfaces of the intestines for a successful immune elicitation (Pasetti et al. 2011). The tolerance towards environmental stresses are less for attenuated bacteria than the wild type strains, thus, antacid mediated protection can be a critical factor for live attenuated vaccines. Among several pathogenic bacteria species such as Escherichia coli, Listeria monocytogenes, Shigella flexneri, researchers have found the suitability of Salmonella strains for vaccine delivery due to several advantages. Salmonella’s well-curated genome, maneuverable virulence and the ability to target the host immune system for elicitation of strong mucosal and cell-mediated immune responses are some of them (Cárdenas and Clements 1992; Schödel and Curtiss 1995). Upon oral administration, Salmonella reaches the ileum, where it enters into the systemic circulation via Peyer's patches (Jensen, Harty and Jones 1998; Jepson and Clark 2001). Over the past few years, our lab developed Salmonella-vectored Brucella vaccines that have the potential to be developed into safe and effective vaccines against brucellosis (Lalsiamthara and Lee 2017a; Senevirathne, Hewawaduge and Lee 2019). However, as oral vaccines, these Salmonella mediated Brucella vaccines require further optimization by incorporating a suitable antacid formulation to enhance the efficacy. The addition of antacid could enhance the survival of live attenuated Salmonella, maximizing the numbers reaching the small intestine for an efficient immune elicitation. Herein we formulated two frequently used antacid formulations, one containing sodium bicarbonate (NaHCO3) and the other containing magnesium hydroxide [Mg (OH)2] as these are common constituents of commercial vaccine formulations. Using the mice model, we investigated the Salmonella gastric survival, intestinal arrival, immune responses, protective efficacy against sub-lethal Brucella infection and post-vaccinated animal behavior to assess the safety concerns. The use of antacids for various health reasons is not a novel concept. Antacids are the oldest effective medications for heartburn (Robinson et al. 2002). Chewing calcium carbonate is a traditional medical treatment for heartburn and remains a popular remedy among some populations (Collings et al. 2002). Most commercially available antacid formulations are a combination of aluminum and magnesium hydroxide [Mg(OH)2] (Martin et al. 2008). Certain effervescent antacid formulations contain sodium bicarbonate (NaHCO3;Tacket et al. 1997; Hindle et al. 2002). Sodium bicarbonate has a lower acid neutralization capacity than Mg(OH)2 (Dhawal and Barve 2019). These two compounds may also contain unwanted side effects such as fluid retention, diarrhea, alkalosis for NaHCO3 and magnesium toxicity [Mg(OH)2] (Joo Suk 2008; Shirasawa et al. 2008). Therefore, the effect of NaHCO3 and Mg(OH)2 as vaccine constituents must be carefully investigated. Besides, acid-neutralizing compounds, oral formulations contain palatability enhancers. For example, the commercial Ty21a vaccine formulation (Vivotiff®) contains sucrose, ascorbic acid, amino acid mixture, lactose and magnesium stearate as major components (Ferreccio et al. 1989). Sucrose acts as a stabilizer, ascorbic acid and lactose add flavor, while magnesium stearate functions as a flow agent that facilitates better distribution (Li and Wu 2014; Pelliccia et al. 2016; Olayan et al. 2019). Considering these factors, we created two antacid formulations: one containing sodium bicarbonate (NaHCO3) and the other containing magnesium hydroxide (Mg(OH)2), each supplemented with palatability enhancers citric acid, sucrose and lactose. For the live Salmonella vaccine, we used attenuated Salmonella that secretes Brucella antigens, Cu–Zn superoxide dismutase (SodC) and outer membrane protein 19 (Omp19) which are already known protective antigens brucellosis. The SodC antigen elicits a mixed Th1 and Th2 immune response (Muñoz-Montesino et al. 2004), whereas omp19 induces IL-17 cytokine production, which is essential in mucosal immunity (Pasquevich et al. 2009). Previous studies conducted by Lalsiamthara and Lee (2017b) demonstrated the efficacy of these antigens against brucellosis in a mouse model. Brucellosis, being one of the top zoonotic diseases in humans and animals and a potential bioterror agent according to the World Health Organization (Hull and Schumaker 2018). Until to date, no fully safe or effective vaccine available for this highly debilitating disease (Li et al. 2017). Currently available live attenuated Brucella vaccine strains occasionally cause disease in vaccinated animals and cannot be used on human subjects due to virulence (Corbel 2006). Therefore, the development of appreciable safe vaccines is a timely requirement. In the present investigation, we formulated the live attenuated Salmonella vaccine as a co-mix of Salmonella secreting SodC or Omp19 antigens in a 1:1 ratio for mice oral inoculation along with pretreatment with antacid buffers. We then investigated the post-vaccinated behavioral changes, immune responses and level of protective immunity after administration. Finally, mice were challenged with Brucella and overall protective efficacy was evaluated by enumerating Brucella load in spleen and liver tissues to reveal the effect of antacid buffers. We found that treatment of mice with the antacid-containing formulations significantly enhanced antigen-specific immune responses and subsequent protection. A comparison of each antacid formulation suggests that the Mg(OH)2 buffer and NaHCO3 buffer can be comparable concerning protective efficacy. However, Mg(OH)2 was significantly safe to mice and outperformed concerning immune responses in mice compared to the NaHCO3 antacid buffer formulation. MATERIALS AND METHODS Bacterial strains, primers and plasmids Bacterial strains, plasmids and primers used in this study are described in Table 1. Attenuated Salmonella strains and E. coli strains were grown in Luria–Bertani (LB) broth (BD, Sparks, MD) with agitation at 37°C using appropriate antibiotics whenever applicable. For bacterial enumerations, Brilliant Green Agar plates (BD) were used. For the challenge, Brucella abortus 544 strain was grown in Brucella medium (BD) with agitation at 37°C in a 5% CO2 atmosphere. Table 1. Bacterial strains and plasmid used in the study. Strain/plasmid . Description . Reference . S. typhimurium JOL401 S. typhimurium wild type, challenge strain Lab stock JOL1800 ∆lon, ∆cpxR, ∆asd and ∆wpaB mutant of S. typhimurium Lab stock JOL1878 JOL1800 containing pJHL65:: SodC and expressing SodC Lab stock JOL1879 JOL1800 containing pJHL65:: Omp19 and expressing Omp19 Lab stock JOL2400 JOL1800 containing pMMP65:: GFPuv plasmid Lab stock E. coli BL21(DE3)pLysS F–, ompT, hsdSB (rB–, mB–), dcm, gal, λ (DE3), pLysS, Cmr Progma, USA JOL1921 BL21(DE3)pLysS harboring pET28a+:: SodC Lab stock JOL1922 BL21(DE3)pLyS harboring pET28a+:: Omp19 Lab stock B.abortus 544 Wild type- Challenge strain Lab stock Plasmid pET28a(+) IPTG-inducible expression vector; Kanamycin resistant Novagen, USA pET28a+:: SodC pET28a + derivative containing SodC Lab stock pET28a+::Omp19 pET28a(+) derivative containing Omp19 Lab stock pJHL65 asd+ vector, pBR ori, β-lactamase signal sequence-based periplasmic secretion plasmid, 6xHis, high copy number Lab stock pJHL65:: SodC pJHL65 harboring SodC of B. abortus Lab stock pJHL65:: Omp19 pJHL65 harboring Omp19 of B. abortus Lab stock Primer SodC EcoRI F 5′-ccgcGAATTCaagtccttatttattgcatcg-3′ SodC HindIII R 5′-ccgcAAGCTTttattcgacacgccgcagg-3′ Omp19 EcoRI F 5′-GAATTCggaatttcaaaagcaagtctgctc-3′ Omp19 HindIII R 5′-AAGCTTTcagcgcgacagcgtcggc-3′ Strain/plasmid . Description . Reference . S. typhimurium JOL401 S. typhimurium wild type, challenge strain Lab stock JOL1800 ∆lon, ∆cpxR, ∆asd and ∆wpaB mutant of S. typhimurium Lab stock JOL1878 JOL1800 containing pJHL65:: SodC and expressing SodC Lab stock JOL1879 JOL1800 containing pJHL65:: Omp19 and expressing Omp19 Lab stock JOL2400 JOL1800 containing pMMP65:: GFPuv plasmid Lab stock E. coli BL21(DE3)pLysS F–, ompT, hsdSB (rB–, mB–), dcm, gal, λ (DE3), pLysS, Cmr Progma, USA JOL1921 BL21(DE3)pLysS harboring pET28a+:: SodC Lab stock JOL1922 BL21(DE3)pLyS harboring pET28a+:: Omp19 Lab stock B.abortus 544 Wild type- Challenge strain Lab stock Plasmid pET28a(+) IPTG-inducible expression vector; Kanamycin resistant Novagen, USA pET28a+:: SodC pET28a + derivative containing SodC Lab stock pET28a+::Omp19 pET28a(+) derivative containing Omp19 Lab stock pJHL65 asd+ vector, pBR ori, β-lactamase signal sequence-based periplasmic secretion plasmid, 6xHis, high copy number Lab stock pJHL65:: SodC pJHL65 harboring SodC of B. abortus Lab stock pJHL65:: Omp19 pJHL65 harboring Omp19 of B. abortus Lab stock Primer SodC EcoRI F 5′-ccgcGAATTCaagtccttatttattgcatcg-3′ SodC HindIII R 5′-ccgcAAGCTTttattcgacacgccgcagg-3′ Omp19 EcoRI F 5′-GAATTCggaatttcaaaagcaagtctgctc-3′ Omp19 HindIII R 5′-AAGCTTTcagcgcgacagcgtcggc-3′ Open in new tab Table 1. Bacterial strains and plasmid used in the study. Strain/plasmid . Description . Reference . S. typhimurium JOL401 S. typhimurium wild type, challenge strain Lab stock JOL1800 ∆lon, ∆cpxR, ∆asd and ∆wpaB mutant of S. typhimurium Lab stock JOL1878 JOL1800 containing pJHL65:: SodC and expressing SodC Lab stock JOL1879 JOL1800 containing pJHL65:: Omp19 and expressing Omp19 Lab stock JOL2400 JOL1800 containing pMMP65:: GFPuv plasmid Lab stock E. coli BL21(DE3)pLysS F–, ompT, hsdSB (rB–, mB–), dcm, gal, λ (DE3), pLysS, Cmr Progma, USA JOL1921 BL21(DE3)pLysS harboring pET28a+:: SodC Lab stock JOL1922 BL21(DE3)pLyS harboring pET28a+:: Omp19 Lab stock B.abortus 544 Wild type- Challenge strain Lab stock Plasmid pET28a(+) IPTG-inducible expression vector; Kanamycin resistant Novagen, USA pET28a+:: SodC pET28a + derivative containing SodC Lab stock pET28a+::Omp19 pET28a(+) derivative containing Omp19 Lab stock pJHL65 asd+ vector, pBR ori, β-lactamase signal sequence-based periplasmic secretion plasmid, 6xHis, high copy number Lab stock pJHL65:: SodC pJHL65 harboring SodC of B. abortus Lab stock pJHL65:: Omp19 pJHL65 harboring Omp19 of B. abortus Lab stock Primer SodC EcoRI F 5′-ccgcGAATTCaagtccttatttattgcatcg-3′ SodC HindIII R 5′-ccgcAAGCTTttattcgacacgccgcagg-3′ Omp19 EcoRI F 5′-GAATTCggaatttcaaaagcaagtctgctc-3′ Omp19 HindIII R 5′-AAGCTTTcagcgcgacagcgtcggc-3′ Strain/plasmid . Description . Reference . S. typhimurium JOL401 S. typhimurium wild type, challenge strain Lab stock JOL1800 ∆lon, ∆cpxR, ∆asd and ∆wpaB mutant of S. typhimurium Lab stock JOL1878 JOL1800 containing pJHL65:: SodC and expressing SodC Lab stock JOL1879 JOL1800 containing pJHL65:: Omp19 and expressing Omp19 Lab stock JOL2400 JOL1800 containing pMMP65:: GFPuv plasmid Lab stock E. coli BL21(DE3)pLysS F–, ompT, hsdSB (rB–, mB–), dcm, gal, λ (DE3), pLysS, Cmr Progma, USA JOL1921 BL21(DE3)pLysS harboring pET28a+:: SodC Lab stock JOL1922 BL21(DE3)pLyS harboring pET28a+:: Omp19 Lab stock B.abortus 544 Wild type- Challenge strain Lab stock Plasmid pET28a(+) IPTG-inducible expression vector; Kanamycin resistant Novagen, USA pET28a+:: SodC pET28a + derivative containing SodC Lab stock pET28a+::Omp19 pET28a(+) derivative containing Omp19 Lab stock pJHL65 asd+ vector, pBR ori, β-lactamase signal sequence-based periplasmic secretion plasmid, 6xHis, high copy number Lab stock pJHL65:: SodC pJHL65 harboring SodC of B. abortus Lab stock pJHL65:: Omp19 pJHL65 harboring Omp19 of B. abortus Lab stock Primer SodC EcoRI F 5′-ccgcGAATTCaagtccttatttattgcatcg-3′ SodC HindIII R 5′-ccgcAAGCTTttattcgacacgccgcagg-3′ Omp19 EcoRI F 5′-GAATTCggaatttcaaaagcaagtctgctc-3′ Omp19 HindIII R 5′-AAGCTTTcagcgcgacagcgtcggc-3′ Open in new tab Construction of Salmonella expressing Brucella antigens Construction of attenuated Salmonella delivering Brucella antigens SodC and Omp19 was described previously (Lalsiamthara and Lee 2017b). Briefly, the sodC and omp19 genes of B. abortus were PCR amplified and cloned in-frame directly downstream of a beta-lactamase signal sequence of pJHL65 which is an asd complemented constitutive expression vector. To construct a balance host-vector system, plasmids pJHL65-sodC and pJHL-omp19 were then electroporated into an O antigen deficient asd auxotrophic S. typhimurium strain, JOL1800 and the resulting strains were designated JOL1878 and JOL1879, respectively. Expression levels of each antigen secreted by S. typhimurium were analyzed by western blot using polyclonal anti-SodC, anti-Omp19 specific hyperimmune sera raised in rabbits according to a previously described procedure (Hur and Lee 2011). Formulation of antacid buffers The antacid formulations were prepared by dissolving NaHCO3, 130 mg, 1.3% (w/v) and 365 mg, 3.6% of Mg(OH)2 each in 10 mL of phosphate-buffered saline (PBS). To enhance oral palatability, citric acid 1.6 g, 16%, 3.3 g, 33% of sucrose and 180 mg, 18% of lactose were added into each formulation. In vitro evaluation of antacid buffers The buffering capacities of antacid formulations NaHCO3 and Mg(OH)2 were assessed under in vitro conditions by mimicking the average pH of mice stomach which is pH 3 (McConnell, Basit and Murdan 2008). The oral dose was determined to be 200 µL by considering the average stomach volume of mice to be 400 µL in volume. To achieve measurable volumes for pH measurements, we used 10 mL of buffer volumes, keeping the ratio of the inoculation volume to the total stomach volume 1:2. Herein, 10 mL of PBS was adjusted to pH 3 and two antacid formulations were added to each volume of PBS using the aforementioned ratio. The immediate effect of antacid addition was assessed by measuring the initial pH, the pH immediately after antacid addition and 30 min after addition (which is the normal gastric retention time for mice), using an electronic pH meter (Thermo Scientific, Waltham, MA). Evaluation of the in vitro buffering longevity and in vivo buffering capacity In vitro antacid buffering activity was assayed by dissolving each antacid formulation separately in 150 mL of PBS at 37°C. The pH levels were pre-adjusted to pH 3 using hydrochloric acid with a pH of 1.2. The antacid formulation was prepared in a round-bottomed flask and swirled three times for even distribution and then stirred at 60 rpm in a rotary shaker to mimic the churning process in the stomach following Fordtran's model (Wu, Chen and Chen 2010). The pH was constantly measured at 20 min intervals for 100 min duration using an electronic pH meter (Orion Star A211; Waltham, MA). A graph was plotted between the pH and time in minutes. A low pH mouse model was generated by fasting to evaluate the buffering capacity of antacids and their effect on the survival of Salmonella in the intestine (Brenneman et al. 2014). The survival efficacy of attenuated Salmonella under each buffered condition was evaluated by inoculating at 2 × 108 CFU/100 µL in antacid treated mice (n = 5). First, mice were orally administered with 200 µL of each antacid formulations and rested for 30 min, and subjected to bacterial inoculation. After 1 h rest period, mice were sacrificed and the entire intestines were harvested to enumerate the number of Salmonella survived during the gastric pass. The total intestines were homogenized using a mechanical homogenizer (T10 basic; Ika, Staufen, Germany) in 3 mL of PBS and plated in decimal dilutions for bacterial enumeration. Density and distribution of Salmonella in the intestine The gastric protective efficacy of two antacid formulations was compared in mice models using GFP-expressing Salmonella strain JOL1800. After oral inoculation in mice, the fluorescence intensity in whole intestines was imaged using FOBI in vivo fluorescent imaging system (NeoScience, Daejeon, Korea). Briefly, a green fluorescent protein (GFP)-expressing Salmonella strain JOL1800 was prepared by transforming the pMMP65:: GFPuv plasmid. Group A received 100 µL of PBS, group B received 2 × 108 CFU/100 µL of GFP-Salmonella, group C received 2 × 108 CFU/100 µL of GFP-Salmonella in a NaHCO3-based formulation and group D received 2 × 108 CFU/100 µL of GFP-Salmonella in a Mg(OH)2-based formulation, and group E was considered as naïve control. A total of 1 h after oral administration, mice were sacrificed and the entire gastrointestinal tract was harvested for fluorescent examination. The relative fluorescence expression, corresponding to bacterial density and distribution, was compared among groups. Effect of antacids on Salmonella colonization and invasion of internal organs The effect of the antacid formulations on Salmonella colonization was evaluated in a separate mouse group (n = 10) inoculated according to the scheme presented in Table S1 (Supporting Information). The number of systemic bacteria in each group was determined by enumerating the number of bacteria present in Peyer's patches, spleen and liver tissues after 3 days of oral inoculation (Yin et al. 2015). For immunohistochemical detection of internalized Salmonella, intestinal tissues were collected from mice in each group on the 3rd day of immunization and processed by antibody labeling (Lalsiamthara and Lee 2017c). Briefly, tissues were formalin-fixed, paraffin-embedded and sectioned into 5 μm slices. Antigen unmasking was performed by heat treatment in citrate buffer for 30 min. Endogenous peroxidase activity was blocked by adding 0.3% hydrogen peroxide. Blocking was conducted with 5% bovine serum albumin (BioShop, Burlington, ON) for 1 h at room temperature. Sections were incubated with Salmonella specific polyclonal antibody at 1:200 dilution for 1 h at 37°C. The secondary goat anti-rabbit HRP conjugated antibody (Cat No. 4030–05, SouthernBiotech, Birmingham, AL) was used at 1:250 dilution. The final color development was achieved by adding 3, 3′diaminobenzidine (DAB; Sigma, St. Louise, MO). Sections were counterstained with methyl green and mounted with MM 24 mounting media (Leica Biosystems, Wetzlar, Germany) for microscopic examination (Zeiss, Germany). Evaluation of the effect of the antacid buffer on mouse behavior Three mice per group received 200 µL PBS, Mg(OH)2, or NaHCO3 antacid formulation via the oral route. Mice were observed for signs of discomfort, morbidity, or any other adverse reactions. Movements of mice within their cages and their activity were video-recorded for comparison. Immunization and challenge studies All experiments with animals were approved by the Chonbuk National University Animal Ethics Committee (CBNU-2018–00264) under the guidelines of the Korean Council on animal care and the Korean Animal Protection Law, 2001; Article 13. A total of 50, 6-week-old specific pathogen-free (SPF) female BALB/c mice were purchased (KOTEC, Busan, Korea) and equally divided into five groups, each containing 10 mice per group. All mice were orally immunized according to the scheme presented in Table 2. Each mouse in groups C and D ingested 200 µL of Mg(OH)2-based antacid formulation or NaHCO3-based formulation, 30 min before vaccination. Booster immunization was conducted with the Salmonella co-mix for mice in groups B, C and D, 3 weeks post-primary immunization. A total of 2 weeks following the booster immunization, five mice from each group were euthanized and splenocytes were aseptically collected for cytokine measurement by quantitative real-time-PCR and to assess T-cell response by flow cytometry. All mice in groups A, B, C and D were challenged at the 5th-week post-primary immunization with a challenge dose of 2 × 105 CFU/mice using virulent B. abortus strain 544 via the intraperitoneal route. Mice in group E served as naïve controls. A total of 2 weeks after the challenge, five mice from groups A, B, C, D and E were euthanized, and Brucella load in organs was enumerated as described previously using whole spleen and liver (Bosseray and Plommet 1990). Brucella cell counts of each group were mathematically transformed as follows: y = log (x/log x) as described previously (Lalsiamthara and Lee 2017a). Table 2. Summary of immunization in BALB/c mice. Group (n = 50)* . Strains/immunogen . Route . Formulation and dose (CFU/100 µL) . Antacid buffer . Wild-type challenge . Brucella recovery . A Non-immunized control Oral PBS - 21 days post-final immunization (2 × 105CFU, i.p) 14 days post-challenge B JOL1878 + JOL1879 Oral 2 × 108 (each strain) - C JOL1878 + JOL1879 Oral 2 × 108 (each strain) Magnesium formulated D JOL1878 + JOL1879 Oral 2 × 108 (each strain) Bicarbonate formulated E Naïve control Oral PBS - No challenged control Group (n = 50)* . Strains/immunogen . Route . Formulation and dose (CFU/100 µL) . Antacid buffer . Wild-type challenge . Brucella recovery . A Non-immunized control Oral PBS - 21 days post-final immunization (2 × 105CFU, i.p) 14 days post-challenge B JOL1878 + JOL1879 Oral 2 × 108 (each strain) - C JOL1878 + JOL1879 Oral 2 × 108 (each strain) Magnesium formulated D JOL1878 + JOL1879 Oral 2 × 108 (each strain) Bicarbonate formulated E Naïve control Oral PBS - No challenged control i.p, intraperitoneal; PBS, phosphate-buffered saline; CFU, colony-forming unit. * n = 10 mice per group; JOL1878 rough S. typhimurium delivering pJHL65-SodC; JOL1879 rough S. typhimurium delivering pJHL65- Omp19. Open in new tab Table 2. Summary of immunization in BALB/c mice. Group (n = 50)* . Strains/immunogen . Route . Formulation and dose (CFU/100 µL) . Antacid buffer . Wild-type challenge . Brucella recovery . A Non-immunized control Oral PBS - 21 days post-final immunization (2 × 105CFU, i.p) 14 days post-challenge B JOL1878 + JOL1879 Oral 2 × 108 (each strain) - C JOL1878 + JOL1879 Oral 2 × 108 (each strain) Magnesium formulated D JOL1878 + JOL1879 Oral 2 × 108 (each strain) Bicarbonate formulated E Naïve control Oral PBS - No challenged control Group (n = 50)* . Strains/immunogen . Route . Formulation and dose (CFU/100 µL) . Antacid buffer . Wild-type challenge . Brucella recovery . A Non-immunized control Oral PBS - 21 days post-final immunization (2 × 105CFU, i.p) 14 days post-challenge B JOL1878 + JOL1879 Oral 2 × 108 (each strain) - C JOL1878 + JOL1879 Oral 2 × 108 (each strain) Magnesium formulated D JOL1878 + JOL1879 Oral 2 × 108 (each strain) Bicarbonate formulated E Naïve control Oral PBS - No challenged control i.p, intraperitoneal; PBS, phosphate-buffered saline; CFU, colony-forming unit. * n = 10 mice per group; JOL1878 rough S. typhimurium delivering pJHL65-SodC; JOL1879 rough S. typhimurium delivering pJHL65- Omp19. Open in new tab Assessment of humoral and cell-mediated immune responses The effect of the antacid formulations on protective immune responses was evaluated in mice. Anti-Brucella IgG and intestinal secretory IgA (sIgA) levels were measured using an indirect enzyme-linked immunosorbent assay (ELISA) method from serum samples collected on the 2nd, 3rd and 5th weeks and intestinal wash samples that were collected on the 5th-week post-primary vaccination. Cytokine mRNA levels were measured after the 5th week of the primary vaccination. Splenocytes were seeded at 1 × 106 cells/well in 96-well plates in Roswell Park Memorial Institute 1640 medium (Lonza, Basel, Switzerland) supplemented with 10% fetal bovine serum (FBS;Gibco, Carlsbad, CA). Cells were stimulated with 400 ng/well of SodC and Omp19 purified protein for 48 h, and the total RNA was isolated using an RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Purified RNA was converted to cDNA using a ReverTra Ace® qPCR RT kit (FSQ-101, TOYOBO, Japan) and approximately 1 µg of RNA was used for cytokine assessment. Cytokine mRNA levels were measured using ABI Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, California, United States). Changes in mRNA levels in immunized mice were calculated by the 2−ΔΔCT method. The expression levels were normalized against the housekeeping gene Glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Changes in T cell populations were investigated by fluorescence-activated cell sorting (FACS) analysis (Shi et al. 2014). Briefly, 1 × 105 of viable splenocytes were seeded into the wells of a 96-well plate and stimulated with 400 ng of SodC and Omp19 antigens per well or RPMI media alone for 72 h and then harvested and stained with PE-labeled anti-CD3e, PerCPVio700-labeled anti-CD4 and FITC-labeled anti-CD8a monoclonal antibodies (Miltenyi Biotec, Germany). The T-cell populations CD3+CD4+ and CD3+CD8+ were gated from the CD3+ population and analyzed using the MacsQuant analysis system (Miltenyi Biotec, Germany). Calcium influx assay Mice macrophage RAW 264.7 cells were seeded in 96 well plates at a rate of 1 × 104 cells/well. Cells were washed with serum-free medium twice and replenished with Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS. Each antacid buffer 5 mM NaHCO3 or 1.4 mM Mg(OH)2 was added to the medium. Cells were allowed to incubate 2 h at 37°C in 5% CO2 incubator. Cells were incubated with a serum-free medium containing 2 µM Fluo-3 AM (Invitrogen, Carlsbad, CA) for 30 min at 37°C in the darkness in 5% CO2 incubator. To remove any dye, cells were washed with serum-free medium twice and further incubated for 30 min to allow de-esterification. Then cells were directly assessed in a flow cytometer (Miltenyi Biotec, Germany). Cells were gated for a red fluorescence and compared against the buffer non-treated cells. Statistical analysis Data analysis was performed using GraphPad Prism 6.02 (GraphPad Software, Inc., San Diego, CA). Statistical significance was analyzed by one-way analysis of variance (ANOVA) and Student's t-test. A P value of 0.05 or less was considered statistically significant. RESULTS Construction and validation of Salmonella delivering Brucella antigens The construction of attenuated Salmonella mutant strains expressing and secreting Brucella antigens was described previously (Lalsiamthara and Lee 2017a). The Electro-transformation of the plasmid into host strains was confirmed by colony PCR using specific primers. Expression and secretion of each Brucella antigen were validated by western blot analysis using antigen-specific polyclonal antibodies. Both SodC and Omp19 proteins with approximate sizes at 21 kDa and 20.68 kDa, respectively, were detected (Figure S1, Supporting Information) on the western blot membrane. Impact of antacid agents on acidic pH The pH buffering capacity of the two antacid formulations was evaluated under in vitro conditions. The two buffer formulations were reacted with simulated stomach acid at pH 3 and then changes in pH were recorded continuously over 100 min. The results indicate that the overall pH buffering capacity of Mg(OH)2 was better than that of NaHCO3 (Fig. 1A). As soon as the Mg(OH)2 antacid buffer was added to the low pH buffer, the overall pH value became neutral, whereas, for the NaHCO3 buffer, the pH of the low pH buffer remained mildly acidic. To assess whether the antacid treatment protected Salmonella during the gastric pass, we conducted an in vivo study by orally administering either Mg(OH)2 or NaHCO3 antacid formulations in mice. Salmonella was orally administered to each mouse group 30 min after antacid treatment to allow the entry into the intestines. We previously confirmed, orally administered Salmonella enters into the intestine approximately after 30 min (data not shown). Therefore, in the present investigation, observations in the intestines were conducted 1 h after Salmonella administration, to allow entry and distribution in the intestines. Then, the whole intestines were harvested and the total number of Salmonella present in the intestine was enumerated. We observed a significantly higher level of Salmonella protection in Mg(OH)2 antacid buffer than the NaHCO3 was used as the antacid buffer (Fig. 1B). Here we observed a 1.2-fold increase in survival of Salmonella with the Mg(OH)2 buffer than the NaHCO3 buffer formulation. Figure 1. Open in new tabDownload slide In vitro assessment of pH buffering capacity of antacid formulations and in vivo gastric survival of Salmonella upon antacid oral treatment. (A) A total of 5 mL of each antacid solutions was added to 10 mL of solution at pH 3 and pH was measured at different time points over 100 min. (B) Mice were fasted for 6 h and then were administrated Mg(OH)2 or NaHCO3 antacid formulations 30 min before and vaccination. A total of 1 h later, mice were euthanized and their small intestines collected and homogenized. Salmonella survival was measured by plating on BGA plates. Results are expressed as CFU/mL. Each data point indicates the mean stimulation index ± SD of five animals. *P < 0.05. Figure 1. Open in new tabDownload slide In vitro assessment of pH buffering capacity of antacid formulations and in vivo gastric survival of Salmonella upon antacid oral treatment. (A) A total of 5 mL of each antacid solutions was added to 10 mL of solution at pH 3 and pH was measured at different time points over 100 min. (B) Mice were fasted for 6 h and then were administrated Mg(OH)2 or NaHCO3 antacid formulations 30 min before and vaccination. A total of 1 h later, mice were euthanized and their small intestines collected and homogenized. Salmonella survival was measured by plating on BGA plates. Results are expressed as CFU/mL. Each data point indicates the mean stimulation index ± SD of five animals. *P < 0.05. Antacid-mediated gastric protection of Salmonella Green fluorescence protein-expressing Salmonella was orally administered in mice 30 min after the oral antacid administration. Their distribution within mice intestines was observed using a live-in vivo imaging system (FOBI). After 1 h, a higher level of green fluorescence was observed in both groups of antacid treated mice than PBS-treated mice; however, a rapid descent of Salmonella into the ileum was observed in mice that received the NaHCO3 antacid buffer formulation (Fig. 2A). To investigate how each antacid formulation could protect Salmonella during the gastric pass and how it could influence protective immune responses were evaluated in mice. Mice were treated with each oral antacid formulation and inoculated with Salmonella JOL1800 strain and the bacterial colonization in the intestine, spleen and liver was evaluated by colony counting 3 days after the oral inoculation. Compared to the Salmonella-only group, the groups that received antacid formulations showed a significant increase (P < 0.05) in viable Salmonella counts present in the intestine, spleen and liver specimens (Fig. 2B). Figure 2. Open in new tabDownload slide Assessment of the intestinal distribution of Salmonella, systemic colonization and immunohistochemical assessment of ileum tissues upon oral antacid treatment. (A) Visualization of bacterial localization by fluorescence. At 1 h post-inoculation, the gastrointestinal tract was removed and examined for fluorescence. The mean GFP signal was quantified as 1 × 105 CFU. The images shown are representative results from groups of five mice. (B) Colonization of BALB/c mice by attenuated S. typhimurium vaccine strains. Bacterial numbers shown were recovered in Peyer's patches, spleen and liver at 3 days post-inoculation. Results are expressed as CFU/mL. Each data point indicates the mean stimulation index ± SD of five animals. *P < 0.05. ns; non-significant. (C) Immunohistochemical localization of Salmonella. Black arrows show clusters of Salmonella that stained brown with DAB in the ileal mucosa, spleen and liver directly under the epithelium. Figure 2. Open in new tabDownload slide Assessment of the intestinal distribution of Salmonella, systemic colonization and immunohistochemical assessment of ileum tissues upon oral antacid treatment. (A) Visualization of bacterial localization by fluorescence. At 1 h post-inoculation, the gastrointestinal tract was removed and examined for fluorescence. The mean GFP signal was quantified as 1 × 105 CFU. The images shown are representative results from groups of five mice. (B) Colonization of BALB/c mice by attenuated S. typhimurium vaccine strains. Bacterial numbers shown were recovered in Peyer's patches, spleen and liver at 3 days post-inoculation. Results are expressed as CFU/mL. Each data point indicates the mean stimulation index ± SD of five animals. *P < 0.05. ns; non-significant. (C) Immunohistochemical localization of Salmonella. Black arrows show clusters of Salmonella that stained brown with DAB in the ileal mucosa, spleen and liver directly under the epithelium. Immunohistochemical staining of intestinal mucosal layers was conducted at 3 days post-inoculation from tissues collected from naïve, PBS alone and mice treated with oral antacid formulations along with Salmonella. A relatively higher number of invaded Salmonella could be observed in Ileum tissues of the antacid buffer treated groups than the PBS control group (Fig. 2C). Antacid impact on mouse behavior The side effects of oral antacid treatments were evaluated in mice upon oral administration. Mice were orally treated with PBS, Mg(OH)2, or NaHCO3, each in 200 µL volume and observed for 30 min. Administration of PBS or Mg(OH)2 did not cause any distinguishable variation in behavior. However, mice treated with 200 µL NaHCO3 was less active than mice in the control and Mg(OH)2 groups (multimedia video file) demonstrating signs of distress. Antacid administration enhances humoral and cell-mediated immune responses The immune responses in antacid treated and non-treated mice were investigated by measuring the vaccine-induced serum IgG and sIgA levels in intestinal washes. To characterize humoral immune responses in mice that received PBS, Salmonella alone and Salmonella with antacid formulations, serum IgG and intestinal sIgA antibody concentrations were assayed. Compared to the PBS control group, concentrations of serum IgG antibodies specific to SodC and Omp19 gradually increased in all mice primed with Salmonella. After the second round of immunization, mice that received Mg(OH)2- and NaHCO3-based antacid formulations had significantly higher antibody levels than the Salmonella-only group (P < 0.05, Fig. 3A). To study mucosal immune responses, intestinal wash samples (5th week) were assessed in all mice (Fig. 3B). Figure 3. Open in new tabDownload slide Systemic IgG and secretory IgA (sIgA) responses upon antacid treatment. Mice were vaccinated orally with attenuated Salmonella with magnesium hydroxide and sodium bicarbonate-based antacid formulations, attenuated Salmonella alone, or PBS control. (A) SodC- and Omp19-specific IgG antibodies were tested using mouse sera collected at 0, 2, 3 and 5 weeks post-immunization. (B) sIgA was quantified using mouse intestinal wash samples collected at 5 weeks post-immunization. Each data point represents the mean ± standard deviation (SD) of five mice per group. *P < 0.05. ns; non-significant. Figure 3. Open in new tabDownload slide Systemic IgG and secretory IgA (sIgA) responses upon antacid treatment. Mice were vaccinated orally with attenuated Salmonella with magnesium hydroxide and sodium bicarbonate-based antacid formulations, attenuated Salmonella alone, or PBS control. (A) SodC- and Omp19-specific IgG antibodies were tested using mouse sera collected at 0, 2, 3 and 5 weeks post-immunization. (B) sIgA was quantified using mouse intestinal wash samples collected at 5 weeks post-immunization. Each data point represents the mean ± standard deviation (SD) of five mice per group. *P < 0.05. ns; non-significant. The changes in T-cell populations and the induction of immunomodulatory cytokines were evaluated in splenic T-cells. Splenocytes isolated from immunized and non-immunized mice were stimulated with SodC and Omp19 purified proteins in vitro. Within immunized groups, mice vaccinated with attenuated Salmonella that received either of the two antacid formulations showed significantly increased mRNA levels of both IFN-γ and IL-4 (Fig. 4A). Furthermore, proportions of CD3+CD4+ and CD3+CD8+ T-cell subsets were significantly increased in mice that ingested antacid formulations as compared to mice vaccinated with Salmonella alone and the PBS control group (P < 0.001; Fig. 4B and C). Figure 4. Open in new tabDownload slide Cytokine and T-cell responses upon antacid treatment. Splenocytes were isolated from vaccinated and control mice on the 5th week post-initial immunization and re-stimulated in vitro with SodC (I) and Omp19 (II). (A) Following 48 h of stimulation, IFN-γ and IL-4 transcript levels were evaluated by quantitative RT-PCR. (B) The percentages of CD3+CD4+ and CD3+CD8+ T cells among splenocytes collected from mice vaccinated with attenuated Salmonella alone (a), attenuated Salmonella with the magnesium hydroxide antacid formulation (b) and attenuated Salmonella with the sodium bicarbonate antacid formulation (c) were analyzed using flow cytometry. The acquisition of 10 000 gated cells was confirmed by CD3-labeled cells. (C) Bar graphs showing the percentages of CD3+CD4+ and CD3+CD8+ subpopulations gated from the CD3+ population. Each data point indicates the mean stimulation index ± SD of five animals. *P < 0.05. ns; non-significant. Figure 4. Open in new tabDownload slide Cytokine and T-cell responses upon antacid treatment. Splenocytes were isolated from vaccinated and control mice on the 5th week post-initial immunization and re-stimulated in vitro with SodC (I) and Omp19 (II). (A) Following 48 h of stimulation, IFN-γ and IL-4 transcript levels were evaluated by quantitative RT-PCR. (B) The percentages of CD3+CD4+ and CD3+CD8+ T cells among splenocytes collected from mice vaccinated with attenuated Salmonella alone (a), attenuated Salmonella with the magnesium hydroxide antacid formulation (b) and attenuated Salmonella with the sodium bicarbonate antacid formulation (c) were analyzed using flow cytometry. The acquisition of 10 000 gated cells was confirmed by CD3-labeled cells. (C) Bar graphs showing the percentages of CD3+CD4+ and CD3+CD8+ subpopulations gated from the CD3+ population. Each data point indicates the mean stimulation index ± SD of five animals. *P < 0.05. ns; non-significant. Antacid buffer effect on intracellular Ca2+ The effect of intracellular Ca2+ ion reservoir upon exposure to NaHCO3 and Mg(OH)2 was investigated using RAW cells. Calcium influx measured by Fluo-3 staining revealed a profound depletion in intracellular Ca2+ concentration by Na+ ions. In contrast, Mg2+ ions did not cause significant alteration in the Ca2+ reservoir in RAW cells. The effect on intracellular Ca2+ levels can affect both the innate and adaptive immune responses (Vig and Kinet 2009; Figure S2, Supporting Information). Protective efficacy of the oral vaccine formulations Protective efficacy was evaluated in immunized mice groups after challenge with the virulent B. abortus 544 strain. Post-challenge Brucella numbers in spleen and liver tissues indicated a significant reduction in bacterial load in the spleen and liver of all vaccinated groups compared to the naïve group (P < 0.001). Mice subjected to antacid treatment displayed a significant degree of protection compared with groups that received Salmonella only. However, no significant difference in protection could be observed between two antacid buffer formulations (Fig. 5). Figure 5. Open in new tabDownload slide Protective efficacy of the Salmonella oral vaccine against antacid treatment. Groups of 10 female BALB/c mice were vaccinated orally with attenuated Salmonella with magnesium hydroxide- or sodium bicarbonate-based antacid formulations, attenuated Salmonella alone, PBS control, or left unvaccinated as naive controls. At 2 weeks of post-final vaccination, all animals were challenged with 2 × 105 CFU/mouse intraperitoneally. On the 14th day post-challenge, mice were euthanized and Brucella in the (A) spleen and (B) liver were quantified. Data are expressed as mean log10CFU/mL ± SD of five mice per group. *P < 0.05. ns; non-significant. Figure 5. Open in new tabDownload slide Protective efficacy of the Salmonella oral vaccine against antacid treatment. Groups of 10 female BALB/c mice were vaccinated orally with attenuated Salmonella with magnesium hydroxide- or sodium bicarbonate-based antacid formulations, attenuated Salmonella alone, PBS control, or left unvaccinated as naive controls. At 2 weeks of post-final vaccination, all animals were challenged with 2 × 105 CFU/mouse intraperitoneally. On the 14th day post-challenge, mice were euthanized and Brucella in the (A) spleen and (B) liver were quantified. Data are expressed as mean log10CFU/mL ± SD of five mice per group. *P < 0.05. ns; non-significant. DISCUSSION Incorporation of antacid formulations in oral live attenuated vaccines is of utmost importance to have superior immune responses. In the present study, we compared the effects of two commonly used antacid formulations based on Mg(OH)2 and NaHCO3 for Salmonella survival and its subsequent effect on protective immune responses. As a Salmonella mediated model vaccine, we employed an attenuated Salmonella mediated Brucella vaccine and the protective immune responses were evaluated in the context of antacid formulations. Each Mg(OH)2 and NaHCO3 based formulations were prepared by incorporating palatability enhancers, citric acid, sucrose and lactose. The pH neutralizing ability of each formulation was assessed by in vitro assays by mimicking the gastric pH of mice, which is pH 3. The reaction volume that was sufficient for pH measurement using the electrode of a benchtop pH meter was determined by considering the ratio of antacid inoculation volume of 200 µL and the average mouse stomach volume of 400 µL (1:2 ratio; McConnell, Basit and Murdan 2008). The pH measurements revealed that Mg(OH)2 has a faster, stable and superior pH neutralization capacity than NaHCO3. The addition of Mg(OH)2 brought the pH up to the neutral pH 7 while NaHCO3maximum reached pH 6.7 maintaining a slightly acidic pH level. Buffering duration was measured over 100 min which demonstrated maintenance of near-neutral pH by Mg(OH)2 during the initial 20 min and gradually declined to pH 5 at the end of the 100 min. The pH value derived from Mg(OH)2 was always higher than the pH obtained by NaHCO3 for each time point. In an aqueous medium, NaHCO3 breaks down to Na+ and HCO3− ions. When the medium is acidic HCO3− ions further break down to form CO2 and H2O. The formation of CO2 has an effervescent effect that is favorable in an oral formulation in certain circumstances. However, it also can cause gastric discomfort. We observed a significant change in the behavior of mice that received NaHCO3. Moreover, sodium ions can cause fluid retention and edema (Goldman and Bassett 1955) which also be a concern. To evaluate mouse behavior immediately after antacid administration, here we orally administered two antacid formulations or PBS, in 200 µL volume per each. Then the behavioral changes of mice were observed over a 1 h. Mice that ingested NaHCO3 demonstrated signs of alkalosis whereas ingestion of the Mg(OH)2 was completely normal compared to the PBS control group (multimedia). The bacterial numbers descended into the intestines after 1 h of ingestion revealed a significantly high level of Salmonella protection with Mg(OH)2 than NaHCO3. This can be attributed to a higher duration of neutral pH maintained by Mg(OH)2. This observation could be linked to the present Salmonella strain JOL1800 which is a mutant of lon and cpxR genes (Takaya et al. 2003). The deletion of the cpxR gene eliminates stress responsiveness of the Salmonella strain (Raivio, Leblanc, and Price 2013), thus it can be more susceptible to acidic pH conditions. Therefore Mg(OH)2 may be a choice for highly attenuated strains such as JOL1800. In preliminary experiments, we observed most Salmonella has descended to the intestine after 30 min of inoculation. Therefore, a GFP tracking assay was conducted after 1 h of GFP-Salmonella ingestion leaving time for distribution in the intestines. Compared to PBS control, both antacid formulations significantly protected Salmonella during the gastric pass. However, we observed a higher rate of descending in NaHCO3 treated mice which can be caused by the effervescent effect generated by CO2 that could have propelled Salmonella into the terminal ileum. The evaluation of the number of bacteria entered into systemic infection were observed after 3 days of oral inoculation, that revealed significantly high level of Salmonella presence in Payer's patches, Spleen and liver tissues in mice treated with Mg(OH)2. During the entry level we could observe relative variations caused by Mg(OH)2 and NaHCO3 on invasion of Salmonella into intestinal epithelium by using immunohistochemical staining which revealed number of dark spots (representing Salmonella) was comparable for both buffers compared to the PBS control. The number of bacteria present in the systemic circulation must be correlated with the induction of antigen-specific humoral and cell-mediated immune responses. Both Mg(OH)2 and NaHCO3 oral administration resulted in elevated levels of IgG and sIgA antibody levels generated at the end of 5th-week post-primary inoculation. The humoral responses generated by Mg(OH)2 were significantly higher than the NaHCO3 at 5th-week post-primary inoculation. Besides, the induction of IFN- γ and IL-4 cytokine levels and CD3+CD4+ T-cell responses were also superior in Mg(OH)2 treated mice. Both NaHCO3 and Mg(OH)2 consist of anti-inflammatory properties that can affect vaccine-induced inflammatory responses. T-cell responses demonstrate a skew towards Th2 type immune response. This can partly be due to the choice of selected antigens such as Omp19 which is known to induce humoral immune responses in the immunized host. The anti-inflammatory propensity of NaHCO3 can be much stronger than Mg(OH)2 which may reflect by lower induction of cytokines such as IFN- γ. Anti-inflammatory responses can be favorable to alleviate vaccine-induced endotoxicty. However, concerning the present Salmonella strain which is already attenuated and low in cytotoxicity and endotoxicity, the level of suppression of inflammatory responses generated by NaHCO3 could negatively impact the overall immune response. Therefore, the outperformance we observed with Mg(OH)2 concerning immune responses may be a function of higher pH neutralization ability and anti-inflammatory/immunomodulation potential that favored the present vaccine strain. Besides, neutralized pH may contribute to an increased level of protein expression and protein survival (Olson 1993). Interestingly, we also observed, the sodium ions demonstrate a stronger inhibitory effect on cellular calcium uptake and maintenance of intracellular calcium reservoir than the effect inflicted by Mg(OH)2 on mice macrophage cell line, RAW cells. As an important secondary messenger in a myriad of immune responses and signaling pathways, the depletion in intracellular calcium reservoir can affect subsequent immune responses. Such an effect was not evident with Mg(OH)2 (Figure S2, Supporting Information). The protection efficacy against the wild type Brucella stain 544 challenge in mice revealed, both Mg(OH)2 and NaHCO3 were derived significantly lower numbers of challenged Brucella in spleen and liver tissues whereas the lowest averages resulted in Mg(OH)2 oral antacid buffer treated mice than the non-treated groups. Even though the level of protection derived by both Mg(OH)2 and NaHCO3 was statistically comparable, this study reveals the potential influence generated by the immunomodulatory properties of each buffer formulation. The level of protection is more related to the selected antigens and the species of challenged pathogen rather than the selected antacid buffer. However, it is possible to conclude, that the choice of the antacid buffer should be decided upon many factors such as the level of attenuation, endotoxicity, selected protective antigens and intended pathogen, etc. Summarizing our results, the addition of an antacid formulation can enhance the survival of Salmonella during the gastric pass and enable a higher level of inoculation in the immune elicitation area of the intestines. Even though both sodium bicarbonate and magnesium hydroxide can enhance bacterial survival, magnesium hydroxide may be a better choice due to its rapid and long term neutralization ability with minimal side effects. The effervescent effect created by sodium bicarbonate may create a certain level of discomfort, alkalosis and edema due to excessive intake of sodium ions into the body. The addition of antacid to live attenuated formulation of Salmonella vaccines can significantly enhance immune responses and subsequent protection. 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TI - Comparative study of sodium bicarbonate- and magnesium hydroxide-based gastric antacids for the effectiveness of Salmonella delivered Brucella antigens against wild type challenge in BALB/c mice
JF - Pathogens and Disease
DO - 10.1093/femspd/ftab002
DA - 2021-02-19
UR - https://www.deepdyve.com/lp/oxford-university-press/comparative-study-of-sodium-bicarbonate-and-magnesium-hydroxide-based-CuszJcJSfv
VL - 79
IS - 2
DP - DeepDyve
ER -