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1.
Vaccine ; 39(39): 5548-5556, 2021 09 15.
Article in English | MEDLINE | ID: mdl-34419306

ABSTRACT

INTRODUCTION: Enterotoxigenic Escherichia coli (ETEC) is a common cause of infectious diarrhoea and a leading cause of morbidity and mortality in children living in resource-limited settings. It is also the leading cause of travellers' diarrhoea among civilian and military travellers. Its dual importance in global public health and travel medicine highlights the need for an effective vaccine. ETEC express colonization factors (CFs) that mediate adherence to the small intestine. An epidemiologically prevalent CF is coli surface antigen 6 (CS6). We assessed the safety and immunogenicity of a CS6-targeted candidate vaccine, CssBA, co-administered intramuscularly with the double-mutant heat-labile enterotoxin, dmLT [LT(R192G/L211A)]. METHODS: This was an open-label trial. Fifty subjects received three intramuscular injections (Days 1, 22 and 43) of CssBA alone (5 µg), dmLT alone (0.1 µg) or CssBA (5, 15, 45 µg) + dmLT (0.1 and 0.5 µg). Subjects were actively monitored for adverse events for 28 days following the third vaccination. Antibody responses (IgG and IgA) were characterized in the serum and from lymphocyte supernatants (ALS) to CS6 and the native ETEC heat labile enterotoxin, LT. RESULTS: Across all dose cohorts, the vaccine was safe and well-tolerated with no vaccine-related severe or serious adverse events. Among vaccine-related adverse events, a majority (98%) were mild with 79% being short-lived vaccine site reactions. Robust antibody responses were induced in a dose-dependent manner with a clear dmLT adjuvant effect. Response rates in subjects receiving 45 µg CssBA and 0.5 µg dmLT ranged from 50 to 100% across assays. CONCLUSION: This is the first study to demonstrate the safety and immunogenicity of CssBA and/or dmLT administered intramuscularly. Co-administration of the two components induced robust immune responses to CS6 and LT, paving the way for future studies to evaluate the efficacy of this vaccine target and development of a multivalent, subunit ETEC vaccine.


Subject(s)
Bacterial Toxins , Enterotoxigenic Escherichia coli , Escherichia coli Infections , Escherichia coli Proteins , Escherichia coli Vaccines , Antibodies, Bacterial , Child , Enterotoxins , Escherichia coli Infections/prevention & control , Escherichia coli Proteins/genetics , Escherichia coli Vaccines/adverse effects , Hot Temperature , Humans , Vaccines, Subunit
2.
Infect Immun ; 89(7): e0010621, 2021 06 16.
Article in English | MEDLINE | ID: mdl-33875477

ABSTRACT

There are no vaccines licensed for enterotoxigenic Escherichia coli (ETEC), a leading cause of diarrhea for children in developing countries and international travelers. Virulence heterogeneity among strains and difficulties identifying safe antigens for protective antibodies against STa, a potent but poorly immunogenic heat-stable toxin which plays a key role in ETEC diarrhea, are challenges in ETEC vaccine development. To overcome these challenges, we applied a toxoid fusion strategy and a novel epitope- and structure-based multiepitope fusion antigen (MEFA) vaccinology platform to construct two chimeric multivalent proteins, toxoid fusion 3xSTaN12S-mnLTR192G/L211A and adhesin CFA/I/II/IV MEFA, and demonstrated that the proteins induced protective antibodies against STa and heat-labile toxin (LT) produced by all ETEC strains or the seven most important ETEC adhesins (CFA/I and CS1 to CS6) expressed by the ETEC strains causing 60 to 70% of diarrheal cases and moderate to severe cases. Combining two proteins, we prepared a protein-based multivalent ETEC vaccine, MecVax. MecVax was broadly immunogenic; mice and pigs intramuscularly immunized with MecVax developed no apparent adverse effects but had robust antibody responses to the target toxins and adhesins. Importantly, MecVax-induced antibodies were broadly protective, demonstrated by significant adherence inhibition against E. coli bacteria producing any of the seven adhesins and neutralization of STa and cholera toxin (CT) enterotoxicity. Moreover, MecVax protected against watery diarrhea and provided over 70% and 90% protection against any diarrhea from an STa-positive or an LT-positive ETEC strain in a pig challenge model. These results indicated that MecVax induces broadly protective antibodies and prevents diarrhea preclinically, signifying that MecVax is potentially an effective injectable vaccine for ETEC. IMPORTANCE Enterotoxigenic Escherichia coli (ETEC) bacteria are a top cause of children's diarrhea and travelers' diarrhea and are responsible for over 220 million diarrheal cases and more than 100,000 deaths annually. A safe and effective ETEC vaccine can significantly improve public health, particularly in developing countries. Data from this preclinical study showed that MecVax induces broadly protective antiadhesin and antitoxin antibodies, becoming the first ETEC vaccine candidate to induce protective antibodies inhibiting adherence of the seven most important ETEC adhesins and neutralizing the enterotoxicity of not only LT but also STa toxin. More importantly, MecVax is shown to protect against clinical diarrhea from STa-positive or LT-positive ETEC infection in a pig challenge model, recording protection from antibodies induced by the protein-based, injectable, subunit vaccine MecVax against ETEC diarrhea and perhaps the possibility of intramuscularly administered protein vaccines for protection against intestinal mucosal infection.


Subject(s)
Diarrhea/microbiology , Diarrhea/prevention & control , Enterotoxigenic Escherichia coli/immunology , Escherichia coli Infections/prevention & control , Escherichia coli Vaccines/immunology , Animals , Antibodies, Bacterial/immunology , Antibodies, Neutralizing/immunology , Antigens, Bacterial/immunology , Bacterial Toxins/immunology , Diarrhea/immunology , Disease Models, Animal , Epitopes/immunology , Escherichia coli Proteins/immunology , Escherichia coli Vaccines/administration & dosage , Escherichia coli Vaccines/adverse effects , Mice , Recombinant Fusion Proteins/immunology , Swine , Vaccines, Combined/genetics , Vaccines, Combined/immunology
3.
Biotechnol Lett ; 42(11): 2223-2230, 2020 Nov.
Article in English | MEDLINE | ID: mdl-32500473

ABSTRACT

OBJECTIVES: Earlier studies have demonstrated the use of inactivated recombinant E. coli (bacterins), to protect against Clostridium spp. in vaccinated animals. These bacterins have a simpler, safer, and faster production process. However, these bacterins carry expression plasmids, containing antibiotic resistance gene, which could be assimilate accidentally by environmental microorganisms. Considering this, we aimed to impair this plasmids using formaldehyde at different concentrations. RESULTS: This compound inactivated the highest density of cells in 24 h. KanR cassette amplification was found to be impaired with 0.8% for 24 h or 0.4% for 72 h. Upon electroporation, E. coli DH5α ultracompetent cells were unable to acquire the plasmids extracted from the bacterins after inactivation procedure. Formaldehyde-treated bacterins were incubated with other viable strains of E. coli, leading to no detectable gene transfer. CONCLUSIONS: We found that this compound is effective as an inactivation agent. Here we demonstrate the biosafety involving antibiotic resistance gene of recombinant E. coli vaccines allowing to industrial production and animal application.


Subject(s)
Escherichia coli/genetics , Formaldehyde/pharmacology , Kanamycin Resistance/drug effects , Plasmids/drug effects , Escherichia coli/drug effects , Escherichia coli Vaccines/adverse effects , Escherichia coli Vaccines/genetics , Gene Transfer, Horizontal/drug effects , Plasmids/genetics , Vaccines, Inactivated , Vaccines, Synthetic
4.
Vaccine ; 38(21): 3773-3779, 2020 05 06.
Article in English | MEDLINE | ID: mdl-32253098

ABSTRACT

BACKGROUND: Trials assessing the safety of novel vaccine candidates are essential in the evaluation and development of candidate vaccines. Immunogenicity and dose-sparing features of vaccination approaches which target skin and associated tissues have garnered increased interest; for enteric vaccines, cutaneous vaccination has been of particular interest. Cutaneous vaccine site reactions are among the most common and visible vaccine related adverse events (AEs) when skin routes are used. Regulatory guidelines governing classification of severity focus on functional impact but are insufficient to characterize a spectrum of skin reaction and allow for comparisons of routes, doses and products with similar local cutaneous AEs. OBJECTIVES: Our group developed a grading scale to evaluate and compare cutaneous vaccine site reactions ahead of early-phase clinical trials of intradermal (ID) and transcutaneous immunization (TCI) with enterotoxigenic E.coli (ETEC) vaccine candidates (adhesin-based vaccine co-administered with LTR192G). We reviewed existing methods for characterizing the appearance and severity of local vaccine site reactions following TCI and ID vaccination and devised a standardized vaccine site appearance grading scale (VSAGS) for use in the clinical development of novel ETEC vaccine candidates which focused on pathophysiologic manifestation of skin findings. RESULTS: Available data from published reports revealed erythematous papules and pruritus were the most common local AEs associated with TCI. Frequency of reactions varied notably across studies as did TCI vaccination methodologies and products. ID vaccination commonly results in erythema and induration at the vaccine site as well as pigmentation changes. There was no published methodology to characterize the spectrum of dermatologic findings. CONCLUSION: ID and TCI vaccination are associated with a largely predictable range of cutaneous AEs. A grading scale focused on the appearance of cutaneous changes was useful in comparing cutaneous AEs. A standardized grading scale will facilitate documentation and comparison of cutaneous AEs.


Subject(s)
Drug-Related Side Effects and Adverse Reactions/classification , Escherichia coli Vaccines/adverse effects , Skin/pathology , Vaccination/adverse effects , Administration, Cutaneous , Clinical Trials as Topic , Enterotoxigenic Escherichia coli , Humans , Immunization , Injections, Intradermal/adverse effects
5.
Vet Microbiol ; 240: 108537, 2020 Jan.
Article in English | MEDLINE | ID: mdl-31902490

ABSTRACT

The safety of the live Escherichia coli vaccine Poulvac® E. coli was tested with a flock (10,000) of layer parents aged 30 weeks. Three and 7 days after vaccination, 60 whole unbroken eggs, the egg white and yolk of 60 eggs and 60 cloacal swabs were enriched in MacConkey broth. At both sampling times, 6 out of 60 whole eggs were found positive for coliform bacteria. None of the enriched samples of yolk + egg white were positive for coliform bacteria. Three and seven days after vaccination 44 and 37, respectively out of 60 swabs were positive for coliform bacteria in MacConkey broth. All coliform isolates collected from whole eggs and cloacal swabs were tested in parallel for growth on minimal agar and blood agar to identify the vaccine strain. Some isolates showed reduced growth on minimal agar compared to blood agar and they were tested further with a PCR for the aroA gene mutation and all were found with the wild type version of the gene. Only two isolates did not grow on minimal agar but grew on blood agar and they were tested both with PCR and PFGE. They also showed the wild type version of the aroA gene and their PFGE profile was different from the vaccine strain of Poulvac® E. coli. In conclusion, the Poulvac® E. coli vaccine strain of E. coli was not identified at the detection limit of one CFU on one egg or in the content of one egg or from a cloacal swab of one hen with at least 95 % probability on flock level. The use of the vaccine is safe for hens in lay with lack of survival of the vaccine strain and lack of negative effects on the hens including egg production.


Subject(s)
Escherichia coli Vaccines/analysis , Escherichia coli/isolation & purification , Vaccination/veterinary , Animals , Chickens/microbiology , Cloaca/microbiology , Culture Media/chemistry , Egg Shell/microbiology , Egg Yolk/microbiology , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Escherichia coli Vaccines/adverse effects , Female , Food Microbiology , Limit of Detection , Ovum/microbiology , Vaccines, Live, Unattenuated/adverse effects , Vaccines, Live, Unattenuated/analysis , Whole Genome Sequencing
6.
Sci China Life Sci ; 63(4): 582-591, 2020 Apr.
Article in English | MEDLINE | ID: mdl-31231780

ABSTRACT

A new HPV-16/18 bivalent vaccine expressed by the Escherichia coli has been proven to be efficacious in adult women. A randomized, immunogenicity noninferiority study of this candidate vaccine was conducted in December 2015 in China. Girls aged 9-14 years were randomized to receive 2 doses at months 0 and 6 (n=301) or 3 doses at months 0, 1 and 6 (n=304). Girls aged 15-17 years (n=149) and women aged 18-26 years (n=225) received 3 doses. The objectives included noninferiority analysis of the IgG geometric mean concentration (GMC) ratio (95% CI, lower bound>0.5) to HPV-16 and HPV-18 at month 7 in girls compared with women. In the per-protocol set, the GMC ratio of IgG was noninferior for girls aged 9-17 years receiving 3 doses compared with women (1.76 (95% CI, 1.56, 1.99) for HPV-16 and 1.93 (95% CI, 1.69, 2.21) for HPV-18) and noninferior for girls aged 9-14 years receiving 2 doses compared with women (1.45 (95% CI, 1.25, 1.62) for HPV-16 and 1.17 (95% CI, 1.02, 1.33) for HPV-18). Noninferiority was also demonstrated for neutralizing antibodies. The immunogenicity of the HPV vaccine in girls receiving 3 or 2 doses was noninferior compared with that in young adult women.


Subject(s)
Escherichia coli Vaccines/administration & dosage , Human papillomavirus 16/immunology , Human papillomavirus 18/immunology , Papillomavirus Infections/prevention & control , Papillomavirus Vaccines/administration & dosage , Adolescent , Adult , Antibodies, Neutralizing/metabolism , Child , China , Dose-Response Relationship, Radiation , Escherichia coli/metabolism , Escherichia coli Vaccines/adverse effects , Female , Humans , Immunogenicity, Vaccine , Papillomavirus Vaccines/adverse effects , Treatment Outcome
7.
Lancet Infect Dis ; 20(2): 208-219, 2020 02.
Article in English | MEDLINE | ID: mdl-31757774

ABSTRACT

BACKGROUND: Enterotoxigenic Escherichia coli causes diarrhoea, leading to substantial mortality and morbidity in children, but no specific vaccine exists. This trial tested an oral, inactivated, enterotoxigenic E coli vaccine (ETVAX), which has been previously shown to be safe and highly immuongenic in Swedish and Bangladeshi adults. We tested the safety and immunogenicity of ETVAX, consisting of four E coli strains overexpressing the most prevalent colonisation factors (CFA/I, CS3, CS5, and CS6) and a toxoid (LCTBA) administered with or without a double-mutant heat-labile enterotoxin (dmLT) as an adjuvant, in Bangladeshi children. METHODS: We did a randomised, double-blind, placebo-controlled, dose-escalation, age-descending, phase 1/2 trial in Dhaka, Bangladesh. Healthy children in one of three age groups (24-59 months, 12-23 months, and 6-11 months) were eligible. Children were randomly assigned with block randomisation to receive either ETVAX, with or without dmLT, or placebo. ETVAX (half [5·5 × 1010 cells], quarter [2·5 × 1010 cells], or eighth [1·25 × 1010 cells] adult dose), with or without dmLT adjuvant (2·5 µg, 5·0 µg, or 10·0 µg), or placebo were administered orally in two doses 2 weeks apart. Investigators and participants were masked to treatment allocation. The primary endpoint was safety and tolerability, assessed in all children who received at least one dose of vaccine. Antibody responses to vaccine antigens, defined as at least a two-times increase in antibody levels between baseline and post-immunisation, were assessed as secondary endpoints. This trial is registered with ClinicalTrials.gov, NCT02531802. FINDINGS: Between Dec 7, 2015, and Jan 10, 2017, we screened 1500 children across the three age groups, of whom 430 were enrolled and randomly assigned to the different treatment groups (130 aged 24-59 months, 100 aged 12-23 months, and 200 aged 6-11 months). All participants received at least one dose of vaccine. No solicited adverse events occurred that were greater than moderate in severity, and most were mild. The most common solicited event was vomiting (ten [8%] of 130 patients aged 24-59 months, 13 [13%] of 100 aged 12-23 months, and 29 [15%] of 200 aged 6-11 months; mostly of mild severity), which appeared related to dose and age. The addition of dmLT did not modify the safety profile. Three serious adverse events occurred but they were not considered related to the study drug. Mucosal IgA antibody responses in lymphocyte secretions were detected against all primary vaccine antigens (CFA/I, CS3, CS5, CS6, and the LCTBA toxoid) in most participants in the two older age groups, whereas such responses to four of the five antigens were less frequent and of lower magnitude in infants aged 6-11 months than in older children. Faecal secretory IgA immune responses were recorded against all vaccine antigens in infants aged 6-11 months. 78 (56%) of 139 infants aged 6-11 months who were vaccinated developed mucosal responses against at least three of the vaccine antigens versus 14 (29%) of 49 of the infants given placebo. Addition of the adjuvant dmLT enhanced the magnitude, breadth, and kinetics (based on number of responders after the first dose of vaccine) of immune responses in infants. INTERPRETATION: The encouraging safety and immunogenicity of ETVAX and benefit of dmLT adjuvant in young children support its further assessment for protective efficacy in children in enterotoxigenic E coli-endemic areas. FUNDING: PATH (Bill & Melinda Gates Foundation and the UK's Department for International Development), the Swedish Research Council, and The Swedish Foundation for Strategic Research.


Subject(s)
Antibody Formation/immunology , Enterotoxigenic Escherichia coli/immunology , Escherichia coli Vaccines/adverse effects , Escherichia coli Vaccines/immunology , Vaccines, Inactivated/adverse effects , Vaccines, Inactivated/immunology , Adjuvants, Immunologic/administration & dosage , Administration, Oral , Antibodies, Bacterial/immunology , Bangladesh , Child , Child, Preschool , Diarrhea/immunology , Double-Blind Method , Enterotoxins/immunology , Escherichia coli Proteins/immunology , Female , Humans , Immunization/methods , Immunoglobulin A/immunology , Immunoglobulin G/immunology , Infant , Male
8.
Vaccine ; 37(37): 5645-5656, 2019 09 03.
Article in English | MEDLINE | ID: mdl-30473185

ABSTRACT

The safety and immunogenicity of the second generation oral enterotoxigenic Escherichia coli (ETEC) vaccine ETVAX, consisting of inactivated recombinant E. coli strains over-expressing the colonization factors (CFs) CFA/I, CS3, CS5 and CS6 and the heat labile toxoid LCTBA, were evaluated in Bangladeshi volunteers. To enable analysis of antibody responses against multiple vaccine antigens for subsequent use in small sample volumes from children, a sensitive electrochemiluminescence (ECL) assay for analysis of intestine-derived antibody-secreting cell responses using the antibodies in lymphocyte secretions (ALS) assay was established using Meso Scale Discovery technology. Three groups of Bangladeshi adults (n = 15 per group) received two oral doses of ETVAX with or without double mutant LT (dmLT) adjuvant or placebo in the initial part of a randomized, double-blind, placebo-controlled, age-descending, dose-escalation trial. CF- and LTB-specific ALS and plasma IgA responses were analyzed by ECL and/or ELISA. ETVAX was safe and well tolerated in the adults. Magnitudes of IgA ALS responses determined by ECL and ELISA correlated well (r = 0.85 to 0.98 for the five primary antigens, P < 0.001) and ECL was selected as the ALS readout method. ALS IgA responses against each of the primary antigens were detected in 87-100% of vaccinees after the first and in 100% after the second vaccine dose. Plasma IgA responses against different CFs and LTB were observed in 62-93% and 100% of vaccinees, respectively. No statistically significant adjuvant effect of dmLT on antibody responses to any antigen was detected, but the overall antigenic breadth of the plasma IgA response tended to favor the adjuvanted vaccine when responses to 4 or more or 5 vaccine antigens were considered. Responses in placebo recipients were infrequent and mainly detected against single antigens. The promising results in adults supported testing ETVAX in descending age groups of children. ClinicalTrials.gov Identifier: NCT02531802.


Subject(s)
Enterotoxigenic Escherichia coli/immunology , Escherichia coli Infections/prevention & control , Escherichia coli Vaccines/immunology , Immunogenicity, Vaccine , Adolescent , Adult , Antibodies, Bacterial/blood , Antibodies, Bacterial/immunology , Bangladesh/epidemiology , Electrochemical Techniques , Enzyme-Linked Immunosorbent Assay , Escherichia coli Vaccines/administration & dosage , Escherichia coli Vaccines/adverse effects , Female , Humans , Immunoglobulin A/immunology , Immunoglobulin G/immunology , Luminescent Measurements , Male , Middle Aged , Young Adult
9.
Hum Vaccin Immunother ; 14(9): 2150-2157, 2018.
Article in English | MEDLINE | ID: mdl-29771596

ABSTRACT

This Phase 1, randomized, double-blind, placebo-controlled study was conducted to evaluate the safety, tolerability and immunogenicity of different doses of ExPEC4V conjugate vaccine (4-16µg Polysaccharide [PS]/serotype) in healthy Japanese participants, stratified into younger (≥20 to <50 years) or older age groups (≥50 years). Within each age group, participants were randomized to a single vaccination with 1 of 3 dose levels of ExPEC4V (4, 8 and 16 µg PS/serotype) or placebo. Safety and tolerability were the primary objectives; immunogenicity was secondary. Of the 48 participants, 47 (98%) completed; one (2%) in the placebo group discontinued. A total of 48% participants had ≥1 AE (younger group: n = 13 [54%]; older group: n = 10 [41.7%]). Solicited and unsolicited AEs were reported in 44% and 8% participants, respectively in the combined ExPEC4V groups. Pain/tenderness (n = 11 [31%]) and redness (n = 9 [25%]) were the most frequently reported solicited local AEs, whereas fatigue (n = 4 [11%]), headache (n = 4 [11%]), muscle pain (n = 2 [6%]), and malaise (n = 5 [14%]) were the most common solicited systemic AEs in the combined ExPEC4V group. No serious AEs, deaths, or discontinuation due to AEs were reported. All doses were immunogenic with an increase in IgG (ELISA) geometric mean titers of at least 5-fold from baseline to Days 15 and 30 for all serotypes. Of participants vaccinated with ExPEC4V, 75% - 100% demonstrated an ELISA titer increase of ≥2-fold. Strong correlation observed between ELISA and OPK. ExPEC4V was well tolerated and elicited an immunogenic response at all dose levels (up to 16 µg PS/serotype) in healthy Japanese participants.


Subject(s)
Bacteremia/prevention & control , Escherichia coli Infections/prevention & control , Escherichia coli Vaccines/adverse effects , Escherichia coli Vaccines/immunology , Extraintestinal Pathogenic Escherichia coli/immunology , Adult , Aged , Aged, 80 and over , Antibodies, Bacterial/blood , Asian People , Bacteremia/microbiology , Double-Blind Method , Drug-Related Side Effects and Adverse Reactions/epidemiology , Drug-Related Side Effects and Adverse Reactions/pathology , Enzyme-Linked Immunosorbent Assay , Escherichia coli Infections/microbiology , Escherichia coli Vaccines/administration & dosage , Female , Healthy Volunteers , Humans , Immunization Schedule , Immunoglobulin G/blood , Male , Middle Aged , Placebos/administration & dosage , Vaccines, Conjugate/administration & dosage , Vaccines, Conjugate/adverse effects , Vaccines, Conjugate/immunology , Young Adult
10.
Vaccine ; 35(49 Pt A): 6790-6792, 2017 12 14.
Article in English | MEDLINE | ID: mdl-28705515

ABSTRACT

New vaccine candidates entering the current routine immunization schedule can best be accommodated as combination vaccines. A combined Shigella and enterotoxigenic E. coli (ETEC) vaccine could greatly benefit children in disease-endemic areas. New candidates are getting closer to being able to meet these needs, but they raise numerous strategic questions related to presentation, formulation, and regulatory approach. The "Combination Vaccine Strategies to Prevent Enteric Infections" workshop at the 2016 Vaccines Against Shigella and ETEC (VASE) Conference examined some of the considerations for developing such vaccines against enteric pathogens.


Subject(s)
Congresses as Topic , Dysentery, Bacillary/prevention & control , Escherichia coli Infections/prevention & control , Vaccination/methods , Vaccines, Combined/administration & dosage , Child , Clinical Trials as Topic , Diarrhea/prevention & control , Dysentery, Bacillary/microbiology , Enterotoxigenic Escherichia coli/immunology , Escherichia coli Infections/microbiology , Escherichia coli Vaccines/administration & dosage , Escherichia coli Vaccines/adverse effects , Escherichia coli Vaccines/immunology , Humans , Immunization Schedule , Shigella/immunology , Shigella Vaccines/administration & dosage , Shigella Vaccines/adverse effects , Shigella Vaccines/immunology , Vaccination/legislation & jurisprudence , Vaccines, Combined/adverse effects , Vaccines, Combined/immunology
11.
Vet Res ; 46: 38, 2015 Apr 10.
Article in English | MEDLINE | ID: mdl-25889651

ABSTRACT

Cattle are the most important reservoir for enterohemorrhagic Escherichia coli (EHEC), a subset of shigatoxigenic E. coli (STEC) capable of causing life-threatening infectious diseases in humans. In cattle, Shiga toxins (Stx) suppress the immune system thereby promoting long-term STEC shedding. First infections of animals at calves' age coincide with the lack of Stx-specific antibodies. We hypothesize that vaccination of calves against Shiga toxins prior to STEC infection may help to prevent the establishment of a persistent type of infection. The objectives of this study were to generate recombinant Shiga toxoids (rStx1mut & rStx2mut) by site-directed mutagenesis and to assess their immunomodulatory, antigenic, and immunogenic properties. Cultures of bovine primary immune cells were used as test systems. In ileal intraepithelial lymphocytes both, recombinant wild type Stx1 (rStx1WT) and rStx2WT significantly induced transcription of IL-4 mRNA. rStx1WT and rStx2WT reduced the expression of Stx-receptor CD77 (syn. Globotriaosylceramide, Gb3) on B and T cells from peripheral blood and of CD14 on monocyte-derived macrophages. At the same concentrations, rStx1mut and rStx2mut exhibited neither of these effects. Antibodies in sera of cattle naturally infected with STEC recognized the rStxmut toxoids equally well as the recombinant wild type toxins. Immunization of calves with rStx1mut plus rStx2mut led to induction of antibodies neutralizing Stx1 and Stx2. While keeping their antigenicity and immunogenicity recombinant Shiga toxoids are devoid of the immunosuppressive properties of the corresponding wild type toxins in cattle and candidate vaccines to mitigate long-term STEC shedding by the reservoir host.


Subject(s)
Bacterial Proteins/genetics , Cattle Diseases/immunology , Escherichia coli Infections/veterinary , Escherichia coli Vaccines/immunology , Shiga-Toxigenic Escherichia coli/immunology , Toxoids/pharmacology , Animals , Bacterial Proteins/metabolism , Cattle , Cattle Diseases/microbiology , Escherichia coli Infections/immunology , Escherichia coli Infections/microbiology , Escherichia coli Vaccines/adverse effects , Male , Mutagenesis, Site-Directed/veterinary , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Vaccines, Synthetic/adverse effects , Vaccines, Synthetic/immunology
12.
Clin Vaccine Immunol ; 22(1): 129-35, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25410205

ABSTRACT

Enterotoxigenic Escherichia coli (ETEC) organisms are a leading cause of infectious diarrhea in developing countries. A live, attenuated cholera strain that expresses high levels of the nontoxic B subunit of cholera toxin, which might also serve as an ETEC protective antigen, was evaluated for safety, excretion, and immunogenicity in healthy volunteers. We enrolled four inpatient dose-escalation cohorts of 15 to 16 eligible subjects to randomly (3:1) receive a single oral dose of vaccine or placebo (buffer alone), evaluating 1 ×10(7), 1 ×10(8), 1 ×10(9), and 1 ×10(10) CFU of the vaccine. The vaccine was well tolerated, although some subjects experienced moderate diarrhea. The serum Inaba vibriocidal antibody response appeared to display a dose-response relationship with increasing dosages of vaccine, plateauing at the 10(9)-CFU dosage. The serum antitoxin (cholera toxin and heat-labile enterotoxin) antibody seroconversion rate (4-fold increase over baseline) also appeared to display a dose-response relationship. The vaccine strain was excreted in stool cultures, displaying a dose-response relationship. A single oral dose of Peru-15 pCTB at dosages up to 1 ×10(10) CFU was safe and immunogenic in this first-in-human trial. These encouraging data support the ongoing clinical development of this candidate combined cholera and ETEC vaccine. (This study has been registered at ClinicalTrials.gov under registration no. NCT00654108.).


Subject(s)
Cholera Vaccines/adverse effects , Cholera Vaccines/immunology , Enterotoxigenic Escherichia coli/immunology , Escherichia coli Vaccines/adverse effects , Escherichia coli Vaccines/immunology , Vibrio cholerae/immunology , Administration, Oral , Adolescent , Adult , Antibodies, Bacterial/blood , Antitoxins/blood , Cholera Vaccines/administration & dosage , Diarrhea/pathology , Dose-Response Relationship, Immunologic , Drug-Related Side Effects and Adverse Reactions/pathology , Escherichia coli Vaccines/administration & dosage , Female , Healthy Volunteers , Humans , Male , Middle Aged , Placebos/administration & dosage , Treatment Outcome , Vaccines, Attenuated/administration & dosage , Vaccines, Attenuated/adverse effects , Vaccines, Attenuated/immunology , Young Adult
13.
Vaccine ; 32(52): 7077-84, 2014 Dec 12.
Article in English | MEDLINE | ID: mdl-25444830

ABSTRACT

BACKGROUND: We have developed a new oral vaccine against enterotoxigenic Escherichia coli (ETEC), which is the most common cause of bacterial diarrhea in children in developing countries and in travelers. METHODS: The vaccine was tested for safety and immunogenicity alone and together with double-mutant heat-labile toxin (dmLT) adjuvant in a double-blind, placebo-controlled Phase I study in 129 Swedish adults. The vaccine consists of four inactivated recombinant E. coli strains overexpressing the major ETEC colonization factors (CFs) CFA/I, CS3, CS5, and CS6 mixed with an LT B-subunit related toxoid, LCTBA. Volunteers received two oral doses of vaccine alone, vaccine plus 10 µg or 25 µg dmLT or placebo. Secretory IgA antibody responses in fecal samples and IgA responses in secretions from circulating intestine-derived antibody secreting cells were assessed as primary measures of vaccine immunogenicity. RESULTS: The vaccine was safe and well tolerated; adverse events were few and generally mild with no significant differences between subjects receiving placebo or vaccine with or without adjuvant. As many as 74% of subjects receiving vaccine alone and 83% receiving vaccine plus 10 µg dmLT showed significant mucosal IgA responses to all five primary vaccine antigens and about 90% of all vaccinees responded to at least four of the antigens. Subjects receiving vaccine plus 10 µg dmLT responded with significantly increased intestine-derived anti-CS6 responses compared to subjects receiving vaccine alone. CONCLUSIONS: The vaccine was safe and broadly immunogenic. dmLT further enhanced mucosal immune responses to CF antigens present in low amounts in the vaccine. Based on these encouraging results, the vaccine will be tested for safety and immunogenicity in different age groups including infants in Bangladesh and for protective efficacy in travelers.


Subject(s)
Adjuvants, Immunologic/administration & dosage , Enterotoxigenic Escherichia coli/immunology , Escherichia coli Infections/prevention & control , Escherichia coli Vaccines/adverse effects , Escherichia coli Vaccines/immunology , Adjuvants, Immunologic/genetics , Administration, Oral , Adolescent , Adult , Antibodies, Bacterial/analysis , Antibody-Producing Cells/immunology , Bacterial Toxins/administration & dosage , Bacterial Toxins/genetics , Double-Blind Method , Enterotoxins/administration & dosage , Enterotoxins/genetics , Escherichia coli Proteins/administration & dosage , Escherichia coli Proteins/genetics , Escherichia coli Vaccines/administration & dosage , Feces/chemistry , Female , Humans , Immunity, Mucosal , Immunoglobulin A/analysis , Male , Mutant Proteins/administration & dosage , Mutant Proteins/genetics , Placebos/administration & dosage , Sweden , Young Adult
14.
Lancet Infect Dis ; 14(3): 197-204, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24291168

ABSTRACT

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) is a major cause of travellers' diarrhoea. We investigated the efficacy and safety of a skin-patch vaccine containing the pathogen's heat-labile toxin (LT) in a population of travellers to Mexico and Guatemala. METHODS: In this phase 3, randomised, double-blind, placebo-controlled field trial, healthy adults (aged 18-64 years) travelling from Germany or the UK to Mexico or Guatemala were assigned in a 1:1 ratio by a dynamic electronic randomisation system to receive transcutaneous immunisation with a patch containing 37.5 µg of ETEC LT or a placebo patch. Participants, site staff, and the investigators who did the analyses were masked to group assignment. Participants were vaccinated before travel, with two patches given 14 days apart. In the destination country, participants tracked stool output in a diary and provided stool samples for pathogen identification if diarrhoea occurred. The primary endpoint was the proportion of participants with at least one episode of moderate-to-severe diarrhoea (defined as four or more unformed stools in a 24 h period) in which either or both ETEC enterotoxins (LT and heat-stable toxin [ST]) were detected. The study is registered at ClinicalTrials.gov, number NCT00993681. FINDINGS: 2036 participants were recruited and randomly assigned between Oct 14, 2009, and Aug 13, 2010, with 1016 allocated to receive the LT patch and 1020 the placebo patch. 821 participants in the LT-patch group and 823 in the placebo group received both vaccinations and were analysed in the per-protocol population. 30 (3.7%, 95% CI 2.5-5.2) participants in the LT-patch group and 46 (5.6%, 4.1-7.4) in the placebo group had moderate or severe ETEC diarrhoea (vaccine efficacy 34.6%, -2.2 to 58.9; p=0.0621). 9333 local (ie, patch-site) adverse events (including erythema, rash, pruritus, hyperpigmentation, pain, hypopigmentation, and oedema) occurred in 943 (93%) of 1015 participants in the LT-patch group, compared with 1444 local adverse events in 574 (56%) of 1019 participants in the placebo group (p<0.0001). Serious adverse events occurred in 25 participants (14 in the LT-patch group and 11 in the placebo group), with all regarded as either unrelated or possibly related to treatment. Vaccine-induced hyperpigmentation persisted for at least 180 days after vaccination in 150 (18%) of the 849 participants who received both vaccinations and returned for final assessment in the LT-patch group, compared with none of the 842 participants in the placebo group. The vaccine was immunogenic, with a post-vaccination geometric mean titre of LT-specific serum immunoglobulin G of 3400.29, compared with 315.41 in the placebo group. INTERPRETATION: Although the LT antigen was delivered effectively by the skin patch, the vaccine did not protect travellers against diarrhoea caused by ETEC or other organisms. Future vaccines against travellers' diarrhoea might need to include several antigens against various diarrhoeal pathogens, and might need to be able to generate mucosal and higher systemic immunity.


Subject(s)
Bacterial Toxins/immunology , Diarrhea/prevention & control , Enterotoxins/immunology , Escherichia coli Proteins/immunology , Escherichia coli Vaccines/administration & dosage , Escherichia coli/immunology , Travel , Administration, Cutaneous , Adolescent , Adult , Developing Countries , Diarrhea/microbiology , Double-Blind Method , Drug Delivery Systems , Escherichia coli Vaccines/adverse effects , Europe , Female , Guatemala , Humans , Immunization/methods , Male , Mexico , Middle Aged , Young Adult
15.
Br J Nutr ; 111(3): 465-73, 2014 Feb.
Article in English | MEDLINE | ID: mdl-23930950

ABSTRACT

To assess the effect of Lactobacillus acidophilus (American Type Culture Collection (ATCC) 700396) on enterotoxigenic Escherichia coli (ETEC) infection, in the present study, a parallel, double-blind, placebo-controlled 4-week intervention was performed in healthy males. The subjects largely consumed their habitual diet, but had to abstain from consuming dairy foods generally high in Ca. The subjects were randomised into the L. acidophilus (dose 109 colony-forming units twice daily; n 20) or the placebo (n 19) group. After an adaptation period of 2 weeks, the subjects were orally infected with a live, but attenuated, ETEC vaccine, able to induce mild, short-lived symptoms. Before and after the challenge, the subjects recorded stool consistency, bowel habits, and frequency and severity of gastrointestinal complaints. The ETEC challenge led to a significant increase in faecal output on the 2nd day and a concomitant increase in Bristol stool scale scores. Likewise, abdominal pain, bloating, flatulence, fever, headache and nausea peaked 1 d after the oral challenge. The concentrations of faecal calprotectin and IgA peaked 2 d after and that of serum IgM peaked 9 and 15 d after the oral challenge. The concentrations of serum IgA and IgG were unaffected. The ETEC challenge led to a reduction in the number of Bacteroides-Prevotella, Bifidobacterium, Clostridium cluster XIVab and total faecal bacteria. Probiotic treatment was associated with a larger increase in Bristol stool scale scores and more fever, headache and nausea after the ETEC challenge compared with the placebo treatment. These differences were, however, small and with substantial variation within the groups. Oral application of an attenuated live ETEC vaccine provides a useful model for food-borne infections. Supplementation with L. acidophilus ATCC 700396, however, was ineffective in reducing ETEC infection symptoms in healthy men.


Subject(s)
Disease Resistance , Enterotoxigenic Escherichia coli/immunology , Escherichia coli Infections/prevention & control , Foodborne Diseases/prevention & control , Gastroenteritis/prevention & control , Lactobacillus acidophilus/immunology , Probiotics/therapeutic use , Abdominal Pain/etiology , Abdominal Pain/prevention & control , Adult , Diarrhea/etiology , Diarrhea/prevention & control , Double-Blind Method , Escherichia coli Infections/immunology , Escherichia coli Infections/microbiology , Escherichia coli Infections/physiopathology , Escherichia coli Vaccines/adverse effects , Escherichia coli Vaccines/immunology , Feces/chemistry , Feces/microbiology , Foodborne Diseases/immunology , Foodborne Diseases/microbiology , Foodborne Diseases/physiopathology , Gastroenteritis/immunology , Gastroenteritis/microbiology , Gastroenteritis/physiopathology , Humans , Immunoglobulin A/analysis , Immunoglobulin M/analysis , Leukocyte L1 Antigen Complex/analysis , Male , Probiotics/adverse effects , Severity of Illness Index , Vaccines, Attenuated/adverse effects , Vaccines, Attenuated/immunology , Young Adult
17.
Microbiol Spectr ; 2(6)2014 Dec.
Article in English | MEDLINE | ID: mdl-26104443

ABSTRACT

Pediatric immunization has been the most effective measure to prevent and reduce the burden of infectious diseases in children. The recent inclusion of pneumococcal and meningococcal polysaccharide conjugates in infant immunization further reinforces their importance. Currently there is no human vaccine against enterohemorrhagic Escherichia coli (EHEC) infections. This review focuses on the human EHEC vaccine that has been studied clinically, in particular, the polysaccharide conjugate against E. coli O157. The surface polysaccharide antigen, O-specific polysaccharide, was linked to rEPA, recombinant exotoxin A of Pseudomonas aeruginosa. In adults and children 2 to 5 years old, O157-rEPA conjugates, shown to be safe, induced high levels of antilipopolysaccharide immunoglobulin G with bactericidal activities against E. coli O157, a functional bioassay that mimics the killing of inoculum in vivo. A similar construct using the B subunit of Shiga toxin (Stx) 1 as the carrier protein elicited both bactericidal and toxin-neutralizing antibodies in mice. So far there is no clinical study of Stx-based human vaccine. Passive immunization of Stx-specific antibodies with humanized, chimeric, or human monoclonal antibodies, produced in transgenic mice, showed promising data in animal models and offered high prospects. Demonstrations of their safety and effectiveness in treating hemolytic-uremic syndrome or patients with EHEC infections are under way, and results are much anticipated. For future development, other virulence factors such as the nontoxic Stx B subunit or intimin should be included, either as carrier protein in conjugates or as independent components. The additional antigens from O157 may provide broader coverage to non-O157 Stx-producing E. coli and facilitate both preventive and therapeutic treatment.


Subject(s)
Escherichia coli Infections/prevention & control , Escherichia coli O157/immunology , Escherichia coli Vaccines/immunology , ADP Ribose Transferases/genetics , Adult , Animals , Antibodies, Bacterial/blood , Bacterial Toxins/genetics , Blood Bactericidal Activity , Child, Preschool , Drug-Related Side Effects and Adverse Reactions , Escherichia coli Infections/immunology , Escherichia coli Infections/microbiology , Escherichia coli O157/genetics , Escherichia coli Vaccines/administration & dosage , Escherichia coli Vaccines/adverse effects , Escherichia coli Vaccines/genetics , Exotoxins/genetics , Humans , Immunoglobulin G/blood , Mice, Transgenic , O Antigens/immunology , Treatment Outcome , Vaccines, Conjugate/administration & dosage , Vaccines, Conjugate/adverse effects , Vaccines, Conjugate/genetics , Vaccines, Conjugate/immunology , Virulence Factors/genetics , Pseudomonas aeruginosa Exotoxin A
18.
Clin Vaccine Immunol ; 20(11): 1764-70, 2013 Nov.
Article in English | MEDLINE | ID: mdl-24049109

ABSTRACT

Enterotoxigenic Escherichia coli (ETEC) is a primary cause of traveler's diarrhea for which there is no licensed vaccine. This phase 1 trial determined the safety and immunogenicity of a recombinantly produced double mutant heat-labile enterotoxin (dmLT) of ETEC. It was administered as a single oral dose of dmLT in escalating doses of 5 µg, 25 µg, 50 µg, and 100 µg, followed by a 72-h inpatient observation, outpatient visits at 8, 14, and 28 days, and telephone calls at 2 and 6 months postvaccination. Safety was assessed by frequency of adverse events, and immune responses determined after immunization included dmLT-specific serum IgA and IgG, fecal IgA, antibody-secreting cells (ASC), and antibodies in lymphocyte supernatant (ALS) responses. All doses were well tolerated by the 36 healthy adults enrolled. Immune responses were limited in the 5- and 25-µg dose recipients. The 50-µg dose recipients trended toward stronger responses than the 100-µg dose recipients by serum IgA (67% versus 33%, P = 0.22), serum IgG (58% versus 33%, P = 0.41), and fecal IgA (58% versus 33%, P = 0.41). By day 14 postvaccination, there were significantly more positive responders (≥4-fold increase from baseline) among the 50- versus 100-µg dose recipients for serum IgA (P = 0.036) but not serum IgG (P = 0.21). In conclusion, a single oral dose of dmLT was well tolerated and immunogenic, with immune responses plateauing at the 50-µg dose. (This clinical trial is registered at www.clinicaltrials.gov, registration number NCT01147445.).


Subject(s)
Bacterial Toxins/immunology , Enterotoxigenic Escherichia coli/immunology , Enterotoxins/immunology , Escherichia coli Proteins/immunology , Escherichia coli Vaccines/adverse effects , Escherichia coli Vaccines/immunology , Administration, Oral , Adult , Antibodies, Bacterial/analysis , Antibodies, Bacterial/blood , Antibody-Producing Cells/immunology , Drug-Related Side Effects and Adverse Reactions/epidemiology , Drug-Related Side Effects and Adverse Reactions/pathology , Escherichia coli Vaccines/administration & dosage , Feces/chemistry , Female , Healthy Volunteers , Humans , Immunoglobulin A/analysis , Immunoglobulin A/blood , Immunoglobulin G/analysis , Immunoglobulin G/blood , Male , Vaccines, Subunit/administration & dosage , Vaccines, Subunit/adverse effects , Vaccines, Subunit/immunology , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/adverse effects , Vaccines, Synthetic/immunology , Young Adult
19.
Vaccine ; 31(20): 2457-64, 2013 May 07.
Article in English | MEDLINE | ID: mdl-23541621

ABSTRACT

A first-generation oral inactivated whole-cell enterotoxigenic Escherichia coli (ETEC) vaccine, comprising formalin-killed ETEC bacteria expressing different colonization factor (CF) antigens combined with cholera toxin B subunit (CTB), when tested in phase III studies did not significantly reduce overall (generally mild) ETEC diarrhea in travelers or children although it reduced more severe ETEC diarrhea in travelers by almost 80%. We have now developed a novel more immunogenic ETEC vaccine based on recombinant non-toxigenic E. coli strains engineered to express increased amounts of CF antigens, including CS6 as well as an ETEC-based B subunit protein (LCTBA), and the optional combination with a nontoxic double-mutant heat-labile toxin (LT) molecule (dmLT) as an adjuvant. Two test vaccines were prepared under GMP: (1) A prototype E. coli CFA/I-only formalin-killed whole-cell+LCTBA vaccine, and (2) A "complete" inactivated multivalent ETEC-CF (CFA/I, CS3, CS5 and CS6 antigens) whole-cell+LCTBA vaccine. These vaccines, when given intragastrically alone or together with dmLT in mice, were well tolerated and induced strong intestinal-mucosal IgA antibody responses as well as serum IgG and IgA responses to each of the vaccine CF antigens as well as to LT B subunit (LTB). Both mucosal and serum responses were further enhanced (adjuvanted) when the vaccines were co-administered with dmLT. We conclude that the new multivalent oral ETEC vaccine, both alone and especially in combination with the dmLT adjuvant, shows great promise for further testing in humans.


Subject(s)
Adjuvants, Immunologic , Bacterial Toxins/immunology , Cholera Toxin/immunology , Enterotoxigenic Escherichia coli/immunology , Enterotoxins/immunology , Escherichia coli Proteins/immunology , Escherichia coli Vaccines/immunology , Fimbriae Proteins/immunology , Vaccines, Inactivated/genetics , Vaccines, Inactivated/immunology , Adjuvants, Immunologic/administration & dosage , Administration, Oral , Animals , Antibody Formation/immunology , Antigens, Bacterial/genetics , Antigens, Bacterial/immunology , Bacterial Toxins/administration & dosage , Bacterial Toxins/genetics , Cholera Toxin/administration & dosage , Cholera Toxin/genetics , Enterotoxins/administration & dosage , Enterotoxins/genetics , Escherichia coli Infections/immunology , Escherichia coli Infections/microbiology , Escherichia coli Infections/prevention & control , Escherichia coli Proteins/administration & dosage , Escherichia coli Proteins/genetics , Escherichia coli Vaccines/administration & dosage , Escherichia coli Vaccines/adverse effects , Escherichia coli Vaccines/genetics , Female , Fimbriae Proteins/genetics , Immunity, Mucosal/immunology , Immunoglobulin A/analysis , Immunoglobulin A/blood , Immunoglobulin A/immunology , Immunoglobulin G/analysis , Immunoglobulin G/blood , Immunoglobulin G/immunology , Intestines/immunology , Mice , Mice, Inbred BALB C , Mutant Proteins/administration & dosage , Mutant Proteins/immunology , Vaccines, Inactivated/administration & dosage , Vaccines, Inactivated/adverse effects
20.
Vaccine ; 31(11): 1458-63, 2013 Mar 01.
Article in English | MEDLINE | ID: mdl-23375979

ABSTRACT

ETEC strains expressing K88 (F4) or F18 fimbriae and enterotoxins are the predominant cause of porcine post-weaning diarrhea (PWD). PWD continues causing significant economic losses to swine producers worldwide. Vaccines effectively protecting against PWD are needed. Our recent study revealed that a tripartite adhesin-toxin monomer (FaeG-FedF-LT(A2-B)) elicited protective antibodies. In this study, we constructed a new adhesin-toxoid fusion, expressed it as a 1A:5B holotoxin-structured antigen (1FaeG-FedF-LT(192A2):5LT(B)) in an avirulent Escherichia coli strain, and evaluated its vaccine potential in pig challenge studies. Piglets orally inoculated with this live strain showed no adverse effects but developed systemic and mucosal antibodies that neutralized cholera toxin and inhibited adherence of K88 and F18 fimbriae in vitro. Moreover, the immunized piglets, when were challenged with ETEC strain 3030-2 (K88ac/LT/STb), had significant fewer bacteria colonized at small intestines and did not develop diarrhea; whereas the control piglets developed severe diarrhea and died. These results indicated the 1FaeG-FedF-LT(192A2):5LT(B) fusion antigen induced protective antiadhesin and antitoxin immunity in pigs, and suggested a live attenuated vaccine can be potentially developed against porcine ETEC diarrhea. Additionally, presenting antigens in a holotoxin structure to target host local mucosal immunity can be used in vaccine development against other enteric diseases.


Subject(s)
Enterotoxigenic Escherichia coli/immunology , Escherichia coli Infections/veterinary , Escherichia coli Vaccines/administration & dosage , Escherichia coli Vaccines/immunology , Swine Diseases/prevention & control , Adhesins, Bacterial/metabolism , Administration, Oral , Animals , Antibodies, Bacterial/analysis , Antibodies, Bacterial/blood , Bacterial Load , Bacterial Toxins/antagonists & inhibitors , Diarrhea/prevention & control , Enterotoxigenic Escherichia coli/genetics , Escherichia coli Infections/prevention & control , Escherichia coli Vaccines/adverse effects , Escherichia coli Vaccines/genetics , Intestinal Mucosa/immunology , Intestine, Small/microbiology , Neutralization Tests , Serum/immunology , Swine , Vaccines, Attenuated/administration & dosage , Vaccines, Attenuated/adverse effects , Vaccines, Attenuated/genetics , Vaccines, Attenuated/immunology , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/adverse effects , Vaccines, Synthetic/genetics , Vaccines, Synthetic/immunology
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