S. M. Eley

A comparison of Plague vaccine, USP and EV76 vaccine induced protection against Yersinia pestis in a murine model

The median lethal dose (MLD) of a pathogenic strain of Yersinia pestis was established by three routes of administration in three strains of mouse. There was no significant difference between the MLDs in the different strains of mouse. The MLD by the subcutaneous route in Balb/C and an outbred line was approximately 1 c.f.u.; the MLD following intraperitoneal administration was tenfold higher. There were significant differences in the mean times to death after administration of the challenge by different routes. The relative efficacy of a live attenuated vaccine strain of Y. pestis (EV76) was compared with that of the formaldehyde-killed vaccine (Plague vaccine, USP). EV76 protected against high challenge doses (up to 5.75 x 10(6) MLD), though immunized animals showed side effects of varying severity. The killed vaccine was less effective in terms of dose-protection (deaths occurred after challenge with 4000 MLD) and several of the vaccinated animals suffered sub-lethal, plague-related sequelae to the challenge.

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.

An IgG1 titre to the F1 and V antigens correlates with protection against plague in the mouse model

The objective of this study was to identify an immunological correlate of protection for a two‐component subunit vaccine for plague, using a mouse model. The components of the vaccine are the F1 and V antigens of the plague‐causing organism, Yersinia pestis, which are coadsorbed to alhydrogel and administered intramuscularly. The optimum molar ratio of the subunits was determined by keeping the dose‐level of either subunit constant whilst varying the other and observing the effect on specific antibody titre. A two‐fold molar excess of F1 to V, achieved by immunizing with 10 μg of each antigen, resulted in optimum antibody titres. The dose of vaccine required to protect against an upper and lower subcutaneous challenge with Y. pestis was determined by administering doses in the range 10 μg F1 + 10 μg V to 0.01 μg F1 + 0.01 μg V in a two‐dose regimen. For animals immunized at the 1‐μg dose level or higher with F1 + V, an increase in specific IgG1 titre was observed over the 8 months post‐boost and they were fully protected against a subcutaneous challenge with 105 colony‐forming units (CFU) virulent Y. pestis at this time point. However, immunization with 5 μg or more of each subunit was required to achieve protection against challenge with 107 CFU Y. pestis. A new finding of this study is that the combined titre of the IgG1 subclass, developed to F1 plus V, correlated significantly (P < 0.05) with protection. The titres of IgG1 in vaccinated mice which correlated with 90%, 50% and 10% protection have been determined and provide a useful model to predict vaccine efficacy in man.

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.

Local and systemic immune response to a microencapsulated sub-unit vaccine for plague.

Microencapsulated Fl and V sub-unit antigens of Yersinia pestis were used to immunize mice intraperitoneally with a combination of 25 micrograms of each of the microencapsulated sub-units. The combined microsphere formulation induced both mucosal and systemic immunity. There was an additive effect in combining sub-units and the protection afforded by the combined microencapsulated antigens was superior to that provided by the administration of any single encapsulated antigen and by the existing whole cell vaccine. The protective efficacy of the combined microencapsulated sub-units was further enhanced by co-administering cholera toxin B sub-unit. Microencapsulation of the sub-units offered advantages which included depot release of the vaccine in vivo and the facilitation of oral, intranasal or inhalational delivery. Therefore, immunization with microencapsulated sub-unit antigens was an effective means of generating humoral and cellular responses which endowed protective immunity.