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1.
Sci Rep ; 13(1): 393, 2023 01 09.
Article in English | MEDLINE | ID: mdl-36624251

ABSTRACT

Salmonella enterica serovar Typhi is the causative agent of typhoid fever restricted to humans and does not replicate in commonly used inbred mice. Genetic variation in humans is far greater and more complex than that in a single inbred strain of mice. The Collaborative Cross (CC) is a large panel of recombinant inbred strains which has a wider range of genetic diversity than laboratory inbred mouse strains. We found that the CC003/Unc and CC053/Unc strains are permissive to intraperitoneal but not oral route of S. Typhi infection and show histopathological changes characteristic of human typhoid. These CC strains are immunocompetent, and immunization induces antigen-specific responses that can kill S. Typhi in vitro and control S. Typhi in vivo. Our results indicate that CC003/Unc and CC053/Unc strains can help identify the genetic basis for typhoid susceptibility, S. Typhi virulence mechanism(s) in vivo, and serve as a preclinical mammalian model system to identify effective vaccines and therapeutics strategies.


Subject(s)
Typhoid Fever , Typhoid-Paratyphoid Vaccines , Animals , Humans , Mice , Salmonella typhi , Collaborative Cross Mice , Mammals
2.
J Pharm Biomed Anal ; 181: 113100, 2020 Mar 20.
Article in English | MEDLINE | ID: mdl-31991319

ABSTRACT

Glycoconjugate vaccines consisting of the Salmonella enterica subsp. enterica serovar Typhi (S. Typhi) Vi capsular polysaccharide (PS) covalently attached to a suitable carrier protein have become available to support mass paediatric vaccination campaigns against typhoid. One developmental vaccine from the International Vaccine Institute (IVI) uses diphtheria toxoid (DTx) as the carrier protein. Several investigational conjugates with different PS:protein ratios were prepared, as previously reported by the IVI group, for physicochemical and immunochemical characterisation. We describe here the further spectroscopic characterisation of this series of glycoconjugate immunogen bulks using NMR spectroscopy, circular dichroism and absorption spectroscopy. We have used several mathematical approaches to extract information from the spectroscopic data not previously applied to glycoconjugates. These complementary approaches provide information on (i) the integrity of the carrier protein, (ii) consistency between batches of vaccine components, (iii) the polysaccharide: protein ratio (iv) the O-acetylation of the Vi in the conjugate (v) the stability of the O-acetylation of the Vi, and (vi) the presence of residual process reagents in the bulk. The utility of the data analysis approaches is discussed. Together, these analytical methods provide important characterisation of Vi-DTx conjugates to support development and quality control of commercial products.


Subject(s)
Diphtheria Toxoid/analysis , Glycoconjugates/analysis , Polysaccharides, Bacterial/analysis , Salmonella typhi/chemistry , Circular Dichroism/methods , Nuclear Magnetic Resonance, Biomolecular/methods , Vaccines, Conjugate/chemistry , X-Ray Absorption Spectroscopy
3.
Vaccine ; 36(26): 3794-3801, 2018 06 18.
Article in English | MEDLINE | ID: mdl-29776750

ABSTRACT

BACKGROUND: Typhoid fever remains a major public health problem in low- and middle-income countries where children aged 2-14 years bear the greatest burden. Vi polysaccharide is poorly immunogenic in children <2 years of age, and protection in adults is modest. The limitations of Vi polysaccharide vaccines can be overcome by conjugation of the Vi to a carrier protein. A typhoid conjugate vaccine composed of Vi polysaccharide conjugated to diphtheria toxoid (Vi-DT) has been developed. The Phase I study results are presented here. METHODS: This was a randomized, observer-blinded Phase I study to assess the safety and immunogenicity of Vi-DT compared to Vi polysaccharide vaccine, conducted in Manila, Philippines. Participants enrolled in an age de-escalation manner (18-45, 6-17 and 2-5 years) were randomized between Test (Vi-DT, 25 µg) administered at 0 and 4 weeks and Comparator (Vi polysaccharide, Typhim Vi® and Vaxigrip®, Sanofi Pasteur) vaccines. RESULTS: A total of 144 participants were enrolled (48 by age strata, 24 in Test and Comparator groups each). No serious adverse event was reported in either group. Solicited and unsolicited adverse events were mild or moderate in both groups with the exception of a 4-year old girl in Test group with grade 3 fever which resolved without sequelae. All participants in Test group seroconverted after first and second doses of Vi-DT while the proportions in the Comparator group were 97.1% and 97.2%, after first dose of Typhim Vi® and second dose of Vaxigrip®, respectively. Vi-DT showed 4-fold higher Geometric Mean Titers (GMT) compared to Typhim Vi® (adjusted for age strata, p < 0.001). No further increase of GMT was detected after the second dose of Vi-DT. Anti-DT IgG seroresponse rates were 81.2% and 84.5% post first and second Vi-DT doses, respectively. CONCLUSIONS: Vi-DT vaccine was safe, well-tolerated and immunogenic in participants aged 2-45 years. ClinicalTrials.gov registration number: NCT02645032.


Subject(s)
Polysaccharides, Bacterial/immunology , Typhoid Fever/prevention & control , Typhoid-Paratyphoid Vaccines/adverse effects , Typhoid-Paratyphoid Vaccines/immunology , Adolescent , Adult , Antibodies, Bacterial/blood , Child , Child, Preschool , Diphtheria Toxin/immunology , Drug-Related Side Effects and Adverse Reactions/epidemiology , Drug-Related Side Effects and Adverse Reactions/pathology , Female , Healthy Volunteers , Humans , Male , Middle Aged , Philippines , Single-Blind Method , Typhoid-Paratyphoid Vaccines/administration & dosage , Vaccines, Conjugate/administration & dosage , Vaccines, Conjugate/adverse effects , Vaccines, Conjugate/immunology , Young Adult
4.
Vaccine ; 33(13): 1614-9, 2015 Mar 24.
Article in English | MEDLINE | ID: mdl-25659268

ABSTRACT

The majority of conjugate vaccines focus on inducing an antibody response to the polysaccharide antigen and the carrier protein is present primarily to induce a T-cell dependent response. In this study conjugates consisting of poly(ribosylribitolphosphate) (PRP) purified from Haemophilus influenzae Type b bound to Hepatitis B virus surface antigen (HBsAg) virus like particles were prepared with the aim of inducing an antibody response to not only the PRP but also the HBsAg. A conjugate consisting of PRP bound to HBsAg via an adipic acid dihydrazide (ADH) spacer induced strong IgG antibodies to both the PRP and HBsAg. When conjugation was performed without the ADH spacer the induction of an anti-PRP response was equivalent to that seen by conjugate with the ADH spacer, however, a negligible anti-HBsAg response was induced. For comparison, PRP was conjugated to diphtheria toxoid (DT) and Vi polysaccharide purified from Salmonella Typhi conjugated to HBsAg both using an ADH spacer. The PRPAH-DT conjugate induced strong anti-PRP and anti-DT responses, the Vi-AHHBsAg conjugate induced a good anti-HBsAg response but not as strong as that induced by the PRPAH-HBsAg conjugate. This study demonstrated that in mice it was possible to induce robust antibody responses to both polysaccharide and carrier protein provided the conjugate has certain physico-chemical properties. A PRPAH-HBsAg conjugate with the capacity to induce anti-PRP and anti-HBsAg responses could be incorporated into a multivalent pediatric vaccine and simplify formulation of such a vaccine.


Subject(s)
Haemophilus Vaccines/immunology , Haemophilus influenzae type b/immunology , Hepatitis B Surface Antigens/genetics , Hepatitis B Surface Antigens/immunology , Hepatitis B Vaccines/immunology , Polysaccharides/immunology , Vaccines, Conjugate/immunology , Animals , Antibodies, Bacterial/immunology , Diphtheria Toxoid/genetics , Diphtheria Toxoid/immunology , Diphtheria-Tetanus-Pertussis Vaccine/administration & dosage , Diphtheria-Tetanus-Pertussis Vaccine/immunology , Haemophilus influenzae type b/chemistry , Haemophilus influenzae type b/genetics , Hepatitis B Surface Antigens/chemistry , Immunoglobulin G/immunology , Mice , Polysaccharides/genetics , Polysaccharides/isolation & purification , Salmonella typhi/genetics , Salmonella typhi/immunology , Vaccines, Conjugate/administration & dosage , Vaccines, Virus-Like Particle/immunology
5.
Vaccine ; 33(6): 783-8, 2015 Feb 04.
Article in English | MEDLINE | ID: mdl-25545593

ABSTRACT

Previously we showed that conjugation of pneumococcal surface protein A (PspA) to Vi capsular polysaccharide from Salmonella Typhi enhanced the anti-PspA response without the need to add adjuvant. In the current study conjugates consisting of the α helical regions of PspA families 1 or 2 bound to Vi were used to vaccinate mice to test their ability to protect against a lethal intravenous challenge of a range of various strains of Streptococcus pneumoniae. Conjugate vaccine containing PspA family 1 provided good protection from PspA family 1 challenge strains but offered very little protection against PspA family 2 challenge strains. Similarly, PspA family 2 conjugates provided good protection from PspA family 2 challenge strains and poor protection against PspA family 1 challenge strains. This observation was supported by the low levels of cross-reactivity of PspA antibodies seen in ELISA plates coated with the heterologous PspA family. Cytokine profiles showed a mixed Th1/Th2 response to Vi and the Vi-PspA conjugates. IgG subclass analysis of the anti-Vi response showed a shift from predominantly IgG2a/3 to IgG1 after conjugation to PspA was consistent with other polysaccharide conjugate vaccines. The results demonstrate that conjugation of the α helical region of PspA to Vi enhances its capacity to induce a protective immune response and that a vaccine based on the α helical region of PspA should contain PspA from both families 1 and 2 to achieve broad cross-protection.


Subject(s)
Antibodies, Bacterial/blood , Bacterial Proteins/immunology , Pneumococcal Infections/prevention & control , Pneumococcal Vaccines/immunology , Polysaccharides, Bacterial/immunology , Typhoid Fever/prevention & control , Typhoid-Paratyphoid Vaccines/immunology , Animals , Antibodies, Bacterial/biosynthesis , Bacterial Proteins/chemistry , Bacterial Proteins/classification , Cross Protection , Cross Reactions , Female , Immunoconjugates/chemistry , Immunoconjugates/immunology , Immunoglobulin Class Switching , Immunoglobulin G/biosynthesis , Immunoglobulin G/blood , Mice , Mice, Inbred ICR , Pneumococcal Infections/blood , Pneumococcal Infections/immunology , Pneumococcal Infections/mortality , Pneumococcal Vaccines/administration & dosage , Pneumococcal Vaccines/chemical synthesis , Polysaccharides, Bacterial/chemistry , Salmonella typhi/drug effects , Salmonella typhi/immunology , Streptococcus pneumoniae/drug effects , Streptococcus pneumoniae/immunology , Survival Analysis , Th1-Th2 Balance , Typhoid Fever/blood , Typhoid Fever/immunology , Typhoid Fever/mortality , Typhoid-Paratyphoid Vaccines/administration & dosage , Typhoid-Paratyphoid Vaccines/chemical synthesis , Vaccines, Conjugate
6.
Vaccine ; 32(43): 5755-60, 2014 Sep 29.
Article in English | MEDLINE | ID: mdl-25171842

ABSTRACT

In the current study pneumococcal surface protein A (PspA) was conjugated to Vi capsular polysaccharide from Salmonella Typhi to make available a vaccine against typhoid fever that has the potential to also provide broad protection from Streptococcus pneumoniae. High yielding production processes were developed for the purification of PspAs from families 1 and 2. The purified PspAs were conjugated to Vi with high recovery of both Vi and PspA. The processes developed especially for PspA family 2 could readily be adapted for large scale production under cGMP conditions. Previously we have shown that conjugation of diphtheria toxoid (DT) to Vi polysaccharide improves the immune response to Vi but can also enhance the response to DT. In this study it was shown that conjugation of PspA to Vi enhanced the anti-PspA response and that PspA was a suitable carrier protein as demonstrated by the characteristics of a T-cell dependent response to the Vi. We propose that a bivalent vaccine consisting of PspA from families 1 and 2 bound to Vi polysaccharide would protect against typhoid fever and has the potential to also protect against pneumococcal disease and should be considered for use in developing countries.


Subject(s)
Bacterial Proteins/immunology , Carrier Proteins/immunology , Pneumococcal Infections/prevention & control , Pneumococcal Vaccines/immunology , Animals , Female , Mice, Inbred ICR , Polysaccharides, Bacterial/immunology , Recombinant Proteins/immunology , Salmonella typhi , Vaccines, Conjugate/immunology
7.
Vaccine ; 32(21): 2457-62, 2014 May 01.
Article in English | MEDLINE | ID: mdl-24631090

ABSTRACT

The O specific polysaccharide (OSP) of the lipopolysaccharide (LPS) of Salmonella enterica serovar Paratyphi A is a protective antigen and the target for vaccine development. LPS is the major constituent of the outer membrane of S. Paratyphi A with the OSP exposed on the surface, in addition to the cell associated LPS a large amount of free LPS was present in the fermentation broth. A purification method was developed to take advantage of both sources of LPS and to maximize recovery of OSP. After fermentation the bacterial cells were concentrated and washed, the permeate containing the free LPS was processed separately from the cells. The free LPS was concentrated and washed on a 100kD ultrafiltration membrane to remove low molecular weight impurities. The LPS was then detoxified by separation of the lipid A from the OSP using acid hydrolysis at 100°C, the precipitated lipid A was removed by 0.2µm membrane filtration. Contaminants were then removed by acid precipitation in the presence of sodium deoxycholate. The OSP was concentrated and washed with 1M NaCl then water using a 10kD ultrafiltration membrane then sterile filtered through a 0.2µm membrane filter. The cells were treated by acid hydrolysis at 100°C, the remaining cells, cell debris and precipitate was removed by centrifugation. The filtrate was then treated in the same way as described above for the free LPS. The combined yield of purified OSP from free LPS plus the cells was greater than 880mg/L of culture broth. The method developed yields large amounts of OSP, is scalable and compatible with cGMP so would be readily transferrable to developing country vaccine manufacturers for low cost production of vaccine against S. Paratyphi A.


Subject(s)
Lipopolysaccharides/isolation & purification , Salmonella paratyphi A/chemistry , Acetylation , Bioreactors , Fermentation , Filtration , Hydrolysis , Lipid A/chemistry , Lipid A/isolation & purification , Lipopolysaccharides/chemistry , O Antigens/chemistry , Technology, Pharmaceutical/methods
8.
Vaccine ; 31(42): 4714-9, 2013 Oct 01.
Article in English | MEDLINE | ID: mdl-23994374

ABSTRACT

Vi capsular polysaccharide is the major component of Vi polysaccharide typhoid vaccines. Vi is synthesized during growth of Salmonella enterica subspecies enterica serovar Typhi and is released into the fermentation broth in large quantities. Along with the Vi considerable amounts of impurities consisting of bacterial protein, nucleic acid and lipopolysaccharide (LPS) as well as media components contaminate the fermentation broth. A purification method based on selective precipitation of Vi using the cationic detergent cetavlon was developed to separate impurities from Vi. A novel method for handling the Vi precipitate using 0.2 µm sterilizing grade filters to trap and wash the Vi and then, after re-solubilization, allow the Vi to pass through the filter was developed. Cetavlon selectively precipitates Vi and is the major purification step in the process, however, the conditions must be carefully controlled otherwise LPS will co-precipitate in large quantities. Various diafiltration steps help to remove contaminating protein, nucleic acid and fermentation media components as well as chemicals added during the process to induce precipitation of either Vi or contaminants. The final yield of purified Vi was approximately 45% and the bulk concentrate complied with the specifications defined in the WHO recommendations for Vi polysaccharide vaccine. Analysis of the Vi by size exclusion chromatography revealed a uniform peak with a narrow size distribution. The Nuclear Magnetic Resonance spectrum was similar to Vi produced by other methods. The method developed produces large quantities of Vi using low cost production methods translating into Vi based vaccines that can be produced at affordable prices for use in developing countries.


Subject(s)
Culture Media/chemistry , Polysaccharides, Bacterial/isolation & purification , Salmonella typhi/metabolism , Technology, Pharmaceutical/methods , Chemical Fractionation , Filtration , Humans , Magnetic Resonance Spectroscopy
9.
Vaccine ; 30(6): 1023-8, 2012 Feb 01.
Article in English | MEDLINE | ID: mdl-22192846

ABSTRACT

The influence pre-exposure of mice to Vi capsular polysaccharide, purified from Salmonella enterica Serovar Typhi, on the subsequent immune response induced by a Vi-diphtheria toxoid (Vi-DT) conjugate was evaluated. Vi induced low anti Vi IgG titers with the dominant subclass being IgG3. The Vi-DT conjugate induced high titers of anti Vi IgG with the dominant subclass being IgG1 but with considerable quantities of IgG2a, IgG2b and IgG3. Priming of mice with Vi suppressed the response to a subsequent dose of conjugate and the suppression was overcome by a second dose of conjugate. Priming with conjugate prevented suppression of the anti Vi response and subsequent dosing with Vi raised titers back to previous levels but did not boost to new higher levels. The anti DT IgG response to one dose of conjugate was relatively strong and protracted and continued to rise for 12 weeks, compared to the response to one dose of DT which was poor and peaked at two weeks. The prolonged anti DT response was most likely due to the slow release of DT from the conjugate lattice as it degrades within the mouse resulting in a continuous stimulation of the immune response. The presence of increasing amounts of un-conjugated Vi, up to 50%, administered with the conjugate resulted in increasingly higher levels of both anti Vi and anti DT. Larger amounts of un-conjugated Vi inhibited the anti Vi response. These findings have implications for vaccine quality and a limit for un-conjugated polysaccharide should not exceed 50% and from a vaccine program perspective if the results presented here translate to humans then a Vi conjugate, once it becomes available, should replace Vi polysaccharide vaccines.


Subject(s)
Diphtheria Toxoid/immunology , Polysaccharides, Bacterial/immunology , Salmonella typhi/immunology , Typhoid-Paratyphoid Vaccines/immunology , Animals , Antibodies, Bacterial/blood , Female , Immunoglobulin G/blood , Mice , Mice, Inbred ICR , Typhoid-Paratyphoid Vaccines/administration & dosage , Vaccines, Conjugate/administration & dosage , Vaccines, Conjugate/immunology
10.
Vaccine ; 29(44): 7618-23, 2011 Oct 13.
Article in English | MEDLINE | ID: mdl-21843575

ABSTRACT

In this study it was demonstrated that the immunogenicity of Vi polysaccharide-diphtheria toxoid conjugates was related to the physical and chemical structure of the conjugate. Conjugates were prepared in two steps, firstly binding adipic acid dihydrazide (ADH) spacer molecules to diphtheria toxoid (DT) carrier protein then secondly binding varying amounts of this derivatized DT to a fixed amount of Vi capsular polysaccharide purified from Salmonella enterica Serovar Typhi. As the amount of DT bound to the Vi increased the size of the conjugate increased but also the degree of cross-linking increased. The immunogenicity of the conjugates was tested in mice and measured by ELISA for anti Vi and anti DT IgG responses, and the results revealed a trend that as the amount of DT bound to the Vi increased the anti Vi responses increased. This study establishes a correlation between physico-chemical characteristics of the conjugate and the magnitude of the anti Vi and anti DT responses.


Subject(s)
Diphtheria Toxoid/chemistry , Diphtheria Toxoid/immunology , Polysaccharides, Bacterial/chemistry , Polysaccharides, Bacterial/immunology , Typhoid-Paratyphoid Vaccines/chemistry , Typhoid-Paratyphoid Vaccines/immunology , Adipates/metabolism , Animals , Antibodies, Bacterial/blood , Antitoxins/blood , Enzyme-Linked Immunosorbent Assay , Humans , Immunoglobulin G/blood , Mice , Protein Binding , Vaccines, Combined/chemistry , Vaccines, Combined/immunology , Vaccines, Conjugate/chemistry , Vaccines, Conjugate/immunology
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