E. Diane Williamson

Vaccination against bubonic and pneumonic plague

Yersinia pestis is the etiological agent of bubonic and pneumonic plague, diseases which have caused over 200 milllion human deaths in the past. Plague still occurs throughout the world today, though for reasons that are not fully understood pandemics of disease do not develop from these outbreaks. Antibiotic treatment of bubonic plague is usually effective, but pneumonic plague is difficult to treat and even with antibiotic therapy death often results. A killed whole cell plague vaccine has been used in the past, but recent studies in animals have shown that this vaccine offers poor protection against pneumonic disease. A live attenuated vaccine is also available. Whilst this vaccine is effective, it retains some virulence and in most countries it is not considered to be suitable for use in humans. We review here work to develop improved sub-unit and live attenuated vaccines against plague. A sub-unit vaccine based on the F1- and V-antigens is highly effective against both bubonic and pneumonic plague, when tested in animal models of disease. This vaccine has been used to explore the utility of different intranasal and oral delivery systems, based on the microencapsulation or Salmonella delivery of sub-units.

A sub-unit vaccine elicits IgG in serum, spleen cell cultures and bronchial washings and protects immunized animals against pneumonic plague

In this study, the protection afforded against aerosolized Yersinia pestis by injection of an alhydrogel-adsorbed sub-unit vaccine has been compared with that given by an existing killed whole cell vaccine licensed for human use. The sub-unit vaccine protected mice against exposure to > 10(4) colony-forming units (c.f.u.) of virulent plague organisms (100 LD50 doses), whereas the whole cell vaccine provided only 50% protection against 1.8 x 10(3) c.f.u. In sub-unit vaccinees, IgG to each of the F1 and V antigens contained in the vaccine, was detected in serum, on direct secretion by spleen cells and in broncho-alveolar washings (BAL). In killed whole cell vaccinees, physiologically significant levels of IgG to F1 only were detectable in equivalent samples. Levels of F1-specific IgG in serum, secreted from spleen cells and in BAL were significantly higher (P < 0.01) in sub-unit compared with killed whole cell vaccinees. IgA was not detected in BAL from intra-muscularly dosed sub-unit vaccinees and thus the protection achieved against inhalational challenge with Yersinia pestis is attributed to the induction of systemic immunity to both the F1 and V antigens in the sub-unit vaccine. The enhanced protective efficacy of this sub-unit vaccine over an existing vaccine has been demonstrated in an animal model of pneumonic plague.

Yersinia pestis (plague) vaccines

Live attenuated and killed whole cell vaccines against disease caused by Yersinia pestis have been available since the early part of the last century. Although these vaccines indicate the feasibility of protecting against disease, they have a number of shortcomings. The live attenuated vaccine is highly reactogenic and is not licensed for use in humans. The killed whole cell vaccine, also reactogenic, provides poor protection against pneumonic plague and immunisation requires mutiple doses of the vaccine. Against this background, a range of candidate vaccines, including rationally attenuated mutants, subunit vaccines and naked DNA vaccines have been described. Of these, an injected subunit vaccine is likely to offer the best near-term solution to the provision of a vaccine that protects against both bubonic and pneumonic plague.

A Recombinant Carboxy-Terminal Domain of the Protective Antigen of Bacillus anthracis Protects Mice against Anthrax Infection

ABSTRACT The immunogenicity and protective efficacy of overlapping regions of the protective antigen (PA) polypeptide, cloned and expressed as glutathione S-transferase fusion proteins, have been assessed. Results show that protection can be attributed to individual domains and imply that it is domain 4 which contains the dominant protective epitopes of PA.

Mucosal or Parenteral Administration of Microsphere-Associated Bacillus anthracis Protective Antigen Protects against Anthrax Infection in Mice

ABSTRACT Existing licensed anthrax vaccines are administered parenterally and require multiple doses to induce protective immunity. This requires trained personnel and is not the optimum route for stimulating a mucosal immune response. Microencapsulation of vaccine antigens offers a number of advantages over traditional vaccine formulations, including stability without refrigeration and the potential for utilizing less invasive routes of administration. Recombinant protective antigen (rPA), the dominant antigen for protection against anthrax infection, was encapsulated in poly-l-lactide 100-kDa microspheres. Alternatively, rPA was loosely attached to the surfaces of microspheres by lyophilization. All of the microspheric formulations were administered to A/J mice with a two-dose schedule by either the intramuscular route, the intranasal route, or a combination of these two routes, and immunogenicity and protective efficacy were assessed. An intramuscular priming immunization followed by either an intramuscular or intranasal boost gave optimum anti-rPA immunoglobulin G titers. Despite differences in rPA-specific antibody titers, all immunized mice survived an injected challenge consisting of 103 median lethal doses of Bacillus anthracis STI spores. Immunization with microencapsulated and microsphere-associated formulations of rPA also protected against aerosol challenge with 30 median lethal doses of STI spores. These results show that rPA can be encapsulated and surface bound to polymeric microspheres without impairing its immunogenicity and also that mucosal or parenteral administration of microspheric formulations of rPA efficiently protects mice against both injected and aerosol challenges with B. anthracis spores. Microspheric formulations of rPA could represent the next generation of anthrax vaccines, which could require fewer doses because they are more potent, are less reactogenic than currently available human anthrax vaccines, and could be self-administered without injection.

Intra nasal administration of poly-lactic acid microsphere co-encapsulated Yersinia pestis subunits confers protection from pneumonic plague in the mouse

Equivocal doses of soluble, or high molecular weight poly (lactic acid) microsphere co-encapsulated, F1 and V subunit antigens of Yersinia pestis were used to immunize mice intra-nasally. Animals were dosed on day 1 and 7 with 2.724 micrograms V plus 0.956 micrograms F1. Co-encapsulated antigens induced superior systemic and mucosal immunity in comparison with free F1 and V. All of the mice immunized with soluble antigens died shortly after an aerosol challenge consisting of 1 x 10(5) colony-forming units of plague bacteria. In contrast, 66% of the co-encapsulated subunit vaccinees survived this lethal challenge. Humoral immunity to plague was improved further, resulting in 80% protection from challenge, if a relatively high dose (10 micrograms) of cholera toxin B subunit was added to the microsphere suspension prior to intra-nasal delivery. Significantly, by adding 10 micrograms cholera toxin B subunit to the free antigen solution, a 100% post-challenge survival rate was attained. We conclude that in this animal model of pneumonic plague, intra-nasal administration of microgram quantities of Yersinia pestis subunits confers protective immunity, provided the vaccines are microencapsulated or admixed with a strong mucosal adjuvant, such as the cholera toxin B subunit.

Human Monoclonal Antibodies against Anthrax Lethal Factor and Protective Antigen Act Independently To Protect against Bacillus anthracis Infection and Enhance Endogenous Immunity to Anthrax

ABSTRACT The unpredictable nature of bioterrorism and the absence of real-time detection systems have highlighted the need for an efficient postexposure therapy for Bacillus anthracis infection. One approach is passive immunization through the administration of antibodies that mitigate the biological action of anthrax toxin. We isolated and characterized two protective fully human monoclonal antibodies with specificity for protective antigen (PA) and lethal factor (LF). These antibodies, designated IQNPA (anti-PA) and IQNLF (anti-LF), were developed as hybridomas from individuals immunized with licensed anthrax vaccine. The effective concentration of IQNPA that neutralized 50% of the toxin in anthrax toxin neutralization assays was 0.3 nM, while 0.1 nM IQNLF neutralized the same amount of toxin. When combined, the antibodies had additive neutralization efficacy. IQNPA binds to domain IV of PA containing the host cell receptor binding site, while IQNLF recognizes domain I containing the PA binding region in LF. A single 180-μg dose of either antibody given to A/J mice 2.5 h before challenge conferred 100% protection against a lethal intraperitoneal spore challenge with 24 50% lethal doses [LD50s] of B. anthracis Sterne and against rechallenge on day 20 with a more aggressive challenge dose of 41 LD50s. Mice treated with either antibody and infected with B. anthracis Sterne developed detectable murine anti-PA and anti-LF immunoglobulin G antibody responses by day 17 that were dependent on which antibody the mice had received. Based on these results, IQNPA and IQNLF act independently during prophylactic anthrax treatment and do not interfere with the establishment of endogenous immunity.

A single dose sub-unit vaccine protects against pneumonic plague

In this study, the protection afforded against aerosolised Yersinia pestis by injection of a single dose of an alhydrogel-adsorbed sub-unit vaccine has been compared with that given by an existing killed whole cell vaccine licensed for human use. The sub-unit vaccine, prepared by admixing F1 antigen derived from a Y. pestis cell culture supernatant with recombinant V antigen derived from an E. coli cell lysate, fully protected an outbred strain of mouse against exposure to 10(6) CFU of virulent plague organisms (10(4) mouse lethal doses, MLD). In contrast, the whole cell vaccine provided only 16% protection against the same level of challenge. Furthermore, sub-unit vaccinees were able to clear the bacteria from their lungs post-challenge whereas bacteria were cultured from the lungs of a surviving KWC vaccinee post-challenge. In killed whole cell vaccinees, physiologically significant levels of IgG to F1 only were detectable and the levels of F1-specific IgG in serum and in broncho-alveolar washings were significantly lower (p<0.05) compared with sub-unit vaccinees. In sub-unit vaccinees, an IgG titre to the F1 and V antigens was detected in serum where it was significantly higher (p<0.05) compared with broncho-alveolar washings suggesting that, at the time of challenge, protection is attributable mainly to the combined circulating IgG titre to the F1 and V sub-units. The enhanced protective efficacy of this sub-unit vaccine administered as a single dose compared with an existing vaccine has been demonstrated in an outbred animal model of pneumonic plague.

Synergistic Protection of Mice against Plague with Monoclonal Antibodies Specific for the F1 and V Antigens of Yersinia pestis

ABSTRACT Monoclonal antibodies specific for Yersinia pestis V antigen and F1 antigen, administered singly or in combination, protected mice in models of bubonic and pneumonic plague. Antibodies showed synergy when administered prophylactically and as a therapy 48 h postinfection. Monoclonal antibodies therefore have potential as a treatment for plague.

Induction of protective immunity against lethal anthrax challenge with a patch.

BACKGROUND Transcutaneous immunization (TCI) is a needle-free technique that delivers antigens and adjuvants to potent epidermal immune cells. To address critical unmet needs in biodefense against anthrax, we have designed a novel vaccine delivery system using a dry adhesive patch that simplifies administration and improves tolerability of a subunit anthrax vaccine. METHODS Mice and rabbits were vaccinated with recombinant protective antigen of Bacillus anthracis and the heat-labile toxin of Escherichia coli. Serologic changes, levels of toxin-neutralizing antibodies (TNAs), and pulmonary and nodal responses were monitored in the mice. A lethal aerosolized B. anthracis challenge model was used in A/J mice, to demonstrate efficacy. RESULTS The level of systemic immunity and protection induced by TCI was comparable to that induced by intramuscular vaccination, and peak immunity could be achieved with only 2 doses. The addition of adjuvant in the patch induced superior TNA levels, compared with injected vaccination. CONCLUSIONS Anthrax vaccine patches stimulated robust and functional immune responses that protected against lethal challenge. Demonstration of responses in the lung suggests that a mechanism exists for protection against challenge with aerosolized anthrax spores. A formulated, pressure-sensitive, dry adhesive patch, which is stable and can be manufactured in large scale, elicited comparable immunoglobulin G and TNA responses, suggesting that an anthrax vaccine patch is feasible and should advance into clinical evaluation.