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1.  Conjugation of Y. pestis F1-antigen to gold nanoparticles improves immunogenicity 
Vaccine  2012;30(48):6777-6782.
The efficacy of 15 nm gold nanoparticles (AuNP) coated with Yersinia pestis F1-antigen, as an immunogen in mice, has been assessed. The nanoparticles were decorated with F1-antigen using N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide coupling chemistry. Mice given AuNP-F1 in alhydrogel generated the greatest IgG antibody response to F1-antigen when compared with mice given AuNP-F1 in PBS or given unconjugated F1-antigen in PBS or alhydrogel. Compared with unconjugated F1-antigen, the IgG2a response was enhanced in mice dosed with AuNP-F1 in PBS (p < 0.05) but not in mice immunised with AuNP-F1 in alhydogel. All treatment groups developed a memory response to F1-antigen, the polarity of which was inflenced by formulation in alhydrogel. The sera raised against F1-antigen coupled to AuNPs was able to competitively bind to rF1-antigen, displacing protective macaque sera.
PMCID: PMC3488157  PMID: 23000121
Plague; Y. pestis; gold nanoparticle; vaccine; carbodiimide
2.  The Role of Immune Correlates and Surrogate Markers in the Development of Vaccines and Immunotherapies for Plague 
One of the difficulties in developing countermeasures to biothreat agents is the challenge inherent in demonstrating their efficacy in man. Since the first publication of the Animal Rule by the FDA, there has been increased discussion of potential correlates of protection in animal models and their use to establish surrogate markers of efficacy in man. The latter need to be relatively easy to measure in assays that are at least qualified, if not validated, in order to derive a quantitative assessment of the clinical benefit conferred. The demonstration of safety and clinical benefit is essential to achieve regulatory approval for countermeasures for which clinical efficacy cannot be tested directly, as is the case for example, for biodefence vaccines. Plague is an ancient, serious infectious disease which is still endemic in regions of the modern world and is a potential biothreat agent. This paper discusses potential immune correlates of protection for plague, from which it may be possible to derive surrogate markers of efficacy, in order to predict the clinical efficacy of candidate prophylaxes and therapies.
PMCID: PMC3182760  PMID: 21991451
3.  Immunogenicity of Recombinant Protective Antigen and Efficacy against Aerosol Challenge with Anthrax  
Infection and Immunity  2005;73(9):5978-5987.
Immunization with a recombinant form of the protective antigen (rPA) from Bacillus anthracis has been carried out with rhesus macaques. Rhesus macaques immunized with 25 μg or more of B. subtilis-expressed rPA bound to alhydrogel had a significantly increased immunoglobulin G (IgG) response to rPA compared with macaques receiving the existing licensed vaccine from the United Kingdom (anthrax vaccine precipitated [AVP]), although the isotype profile was unchanged, with bias towards the IgG1 and IgG2 subclasses. Immune macaque sera from all immunized groups contained toxin-neutralizing antibody and recognized all the domains of PA. While the recognition of the N terminus of PA (domains 1 to 3) was predominant in macaques immunized with the existing vaccines (AVP and the U.S. vaccine anthrax vaccine adsorbed), macaques immunized with rPA recognized the N- and C-terminal domains of PA. Antiserum derived from immunized macaques protected macrophages in vitro against the cytotoxic effects of lethal toxin. Passive transfer of IgG purified from immune macaque serum into naive A/J mice conferred protection against challenge with B. anthracis in a dose-related manner. The protection conferred by passive transfer of 500 μg macaque IgG correlated significantly (P = 0.003; r = 0.4) with the titers of neutralizing antibody in donor macaques. Subsequently, a separate group of rhesus macaques immunized with 50 μg of Escherichia coli-derived rPA adsorbed to alhydrogel was fully protected against a target dose of 200 50% lethal doses of aerosolized B. anthracis. These data provide some preliminary evidence for the existence of immune correlates of protection against anthrax infection in rhesus macaques immunized with rPA.
PMCID: PMC1231098  PMID: 16113318
4.  Human Immune Response to a Plague Vaccine Comprising Recombinant F1 and V Antigens  
Infection and Immunity  2005;73(6):3598-3608.
The human immune response to a new recombinant plague vaccine, comprising recombinant F1 (rF1) and rV antigens, has been assessed during a phase 1 safety and immunogenicity trial in healthy volunteers. All the subjects produced specific immunoglobulin G (IgG) in serum after the priming dose, which peaked in value after the booster dose (day 21), with the exception of one individual in the lowest dose level group, who responded to rF1 only. Three subjects, found to have an anti-rV titer at screening, were excluded from the overall analysis. Human antibody functionality has been assessed by quantification of antibody competing for binding to rV in vitro and also by the transfer of protective immunity in human serum into the naïve mouse. Human and macaque IgG competed for binding to rV in vitro with a mouse monoclonal antibody, previously shown to protect mice against challenge with plague, suggesting that this protective B-cell epitope on rV is conserved between these three species. Total IgG to rV in individuals and the titer of IgG competing for binding to rV correlated significantly at days 21 (r = 0.72; P < 0.001) and 28 (r = 0.82; P < 0.001). Passive transfer of protective immunity into mice also correlated significantly with total IgG titer to rF1 plus rV at days 21 (r2 = 98.6%; P < 0.001) and 28 (r2 = 76.8%; P < 0.03). However, no significant vaccination-related change in activation of peripheral blood mononuclear cells was detected at any time. Potential serological immune correlates of protection have been investigated, but no trends specific to vaccination could be detected in cellular markers.
PMCID: PMC1111881  PMID: 15908389
5.  Regions of Yersinia pestis V antigen that contribute to protection against plague identified by passive and active immunization. 
Infection and Immunity  1997;65(11):4476-4482.
V antigen of Yersinia pestis is a multifunctional protein that has been implicated as a protective antigen, a virulence factor, and a regulatory protein. A series of V-antigen truncates expressed as glutathione S-transferase (GST) fusion proteins (GST-V truncates) have been cloned and purified to support immunogenicity and functionality studies of V antigen. Immunization studies with GST-V truncates have identified two regions of V antigen that confer protection against Y. pestis 9B (a fully virulent human pneumonic plague isolate) in a mouse model for plague. A minor protective region is located from amino acids 2 to 135 (region I), and a major protective region is found between amino acids 135 and 275 (region II). In addition, analysis of IgG titers following immunization suggested that the major antigenic region of V antigen is located between amino acids 135 and 245. A panel of monoclonal antibodies raised against recombinant V antigen was characterized by Western blotting against GST-V truncates, and epitopes of most of the monoclonal antibodies were mapped to region I or II. Monoclonal antibody 7.3, which recognizes an epitope in region II, passively protected mice against challenge with 12 median lethal doses of Y. pestis GB, indicating that region II encodes a protective epitope. This is the first report of a V-antigen-specific monoclonal antibody that will protect mice against a fully virulent strain of Y. pestis. The combined approach of passive and active immunization has therefore confirmed the importance of the central region of the protein for protection and also identified a previously unknown protective region at the N terminus of V antigen.
PMCID: PMC175643  PMID: 9353022
6.  Expression of the Yersinia pestis capsular antigen (F1 antigen) on the surface of an aroA mutant of Salmonella typhimurium induces high levels of protection against plague. 
Infection and Immunity  1997;65(5):1926-1930.
The caf operon from Yersinia pestis encoding the structural subunit (caf1), the molecular chaperone (caf1M), the outer membrane anchor (caf1A), and the regulatory protein (caf1R) was cloned into Salmonella typhimurium SL3261 aroA. The recombinant Salmonella organisms were encapsulated when cultured at 37 degrees C but not when cultured at 28 degrees C. Oral inoculation of mice with the recombinant Salmonella induced predominantly an immunoglobulin G2a response to F1 antigen, and isolated T cells showed a recall response to soluble or Salmonella-associated F1 antigen. Mice immunized with S. typhimurium SL3261 aroA expressing F1 antigen intracellularly developed lower antibody responses to F1 antigen and showed a T-cell recall response only to Salmonella-associated F1 antigen. Mice immunized orally with two doses of the recombinant Salmonella which expressed F1 antigen on the surface were protected against 10(7) 50% lethal doses (LD50) of virulent Y. pestis given by the subcutaneous route of challenge, whereas mice immunized with the recombinant Salmonella expressing F1 antigen intracellularly were only partially protected against 10(5) LD50 of Y. pestis.
PMCID: PMC175242  PMID: 9125581
7.  Recombinant V antigen protects mice against pneumonic and bubonic plague caused by F1-capsule-positive and -negative strains of Yersinia pestis. 
Infection and Immunity  1996;64(11):4580-4585.
The purified recombinant V antigen from Yersinia pestis, expressed in Escherichia coli and adsorbed to aluminum hydroxide, an adjuvant approved for human use, was used to immunize outbred Hsd:ND4 mice subcutaneously. Immunization protected mice from lethal bubonic and pneumonic plague caused by CO92, a wild-type F1+ strain, or by the isogenic F1- strain C12. This work demonstrates that a subunit plague vaccine formulated for human use provides significant protection against bubonic plague caused by an F1- strain (C12) or against substantial aerosol challenges from either F1+ (CO92) or F1-(C12) Y. pestis.
PMCID: PMC174416  PMID: 8890210
8.  Active immunization with recombinant V antigen from Yersinia pestis protects mice against plague. 
Infection and Immunity  1995;63(8):2854-2858.
The gene encoding V antigen from Yersinia pestis was cloned into the plasmid expression vector pGEX-5X-2. When electroporated into Escherichia coli JM109, the recombinant expressed V antigen as a stable fusion protein with glutathione S-transferase. The glutathione S-transferase-V fusion protein was isolated from recombinant E. coli and cleaved with factor Xa to yield purified V antigen as a stable product. Recombinant V antigen was inoculated intraperitoneally into mice and shown to induce a protective immune response against a subcutaneous challenge with 3.74 x 10(6) CFU of virulent Y. pestis. Protection correlated with the induction of a high titer of serum antibodies and a T-cell response specific for recombinant V antigen. These results indicate that V antigen should be a major component of an improved vaccine for plague.
PMCID: PMC173387  PMID: 7622205
9.  Immunization with live recombinant Salmonella typhimurium aroA producing F1 antigen protects against plague. 
Infection and Immunity  1995;63(2):563-568.
An attenuated Salmonella typhimurium strain which expressed the F1 capsular antigen of Yersinia pestis was constructed by transformation of S. typhimurium SL3261 with plasmid pFGAL2a, a derivative of pUC18 which contained the caf1 gene without the leader sequence. The recombinant was used to vaccinate mice intragastrically and intravenously. The immunity induced was able to protect mice against challenge with a virulent strain of plague. Protection correlated with the induction of high titers of immunoglobulin G in serum samples and a specific T-cell response.
PMCID: PMC173032  PMID: 7822022
10.  Epitope mapping of the alpha-toxin of Clostridium perfringens. 
Infection and Immunity  1991;59(12):4338-4342.
A panel of monoclonal antibodies specific for the Clostridium perfringens alpha-toxin was produced by the fusion of X63.Ag8-653 cells with splenocytes from mice immunized either intrasplenically or intraperitoneally with an alpha-toxoid. The toxin-binding activity of each monoclonal antibody was evaluated. The monoclonal antibodies were also screened for their toxin-neutralizing potential in vitro, as determined by the inhibition of phospholipase C and hemolytic activities. In vivo inhibition of toxicity was assessed by the survival of mice challenged with preincubated alpha-toxin-antibody mixtures. Only one monoclonal antibody (3A4D10) was protective in vivo and neutralizing in both in vitro assays. Since 3A4D10 could inhibit both activities, the evidence suggests that these are colocated in the same area of the toxin molecule. This paper identifies a significant continuous linear binding region for 3A4D10 at positions 193 to 198 in the primary amino acid sequence of alpha-toxin.
PMCID: PMC259046  PMID: 1718874
11.  Anti-idiotypic antibody-induced protection against Clostridium perfringens type D. 
Infection and Immunity  1990;58(8):2487-2492.
A monoclonal antibody (BALB/c mouse) with specificity for a neutralizing epitope on the epsilon-toxin produced by Clostridium perfringens type D was used to raise anti-idiotypic antibodies (anti-Id) in different strains of mice and rabbits. These were purified and used in cross-immunization studies to induce anti-(anti-idiotype). All strains of mice and rabbits immunized with BALB/c-derived anti-Id showed a high-titer antibody response directed towards the active site of the toxin. This protected the animals against toxin challenge and against an oral dose of the vegetative organisms. Animals immunized with other anti-Id preparations showed no specific antibody response and were not protected. Guinea pig peritoneal macrophages have a cell surface receptor for the toxin, and incubation of these cells with BALB/c anti-Id allowed them to survive toxin challenge, indicating that occupation of the receptors by the anti-Id prevented binding by the toxin. In conclusion, we have shown that an internal-image anti-Id preparation will induce protective immunity in syngeneic and xenogeneic animals and furthermore that immunity to a single epitope on the exotoxin is sufficient to protect against the toxin and clinical sequelae evoked by the disease-causing organism itself.
PMCID: PMC258845  PMID: 1695203

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