Elsevier

Vaccine

Volume 28, Issue 6, 10 February 2010, Pages 1655-1660
Vaccine

Comparison of mouse, guinea pig and rabbit models for evaluation of plague subunit vaccine F1 + rV270

https://doi.org/10.1016/j.vaccine.2009.02.078Get rights and content

Abstract

In this study, a new subunit vaccine that comprised native F1 and recombinant rV270 was evaluated for protective efficacy using mouse, guinea pig and rabbit models in comparison with the live attenuated vaccine EV76. Complete protection against challenging with 106 colony-forming units (CFU) of virulent Yersinia pestis strain 141 was observed for mice immunized with the subunit vaccines and EV76 vaccine. In contrast, the subunit vaccine recipes VII (F1-20 μg + rV270-10 μg) and IX (F1-40 μg + rV270-20 μg) and EV76 vaccine provided 86%, 79% and 93% protection against the same level of challenge in guinea pigs and 100%, 83% and 100% protection in rabbits, respectively. The immunized mice with the vaccines had significantly higher IgG titres than the guinea pigs and rabbits, and the immunized guinea pigs developed significantly higher IgG titres than the rabbits, but the anti-F1 response in guinea pigs was more variable than in the mice and rabbits, indicating that guinea pig is not an ideal model for evaluating protective efficacy of plague subunit vaccine, instead the rabbits could be used as an alternative model. All the immunized animals with EV76 developed a negligible IgG titre to rV270 antigen. Furthermore, analysis of IgG subclasses in the immunized animals showed a strong response for IgG1, whereas those receiving EV76 immunization demonstrated predominant production of IgG1 and IgG2a isotypes. The subunit vaccine and EV76 vaccine are able to provide protection for animals against Y. pestis challenge, but the subunit vaccines have obvious advantages over EV76 in terms of safety of use.

Introduction

Plague is a zoonotic disease caused by Gram-negative bacterium Yersinia pestis, which is usually transmitted to humans from infected rodents via the bite of an infected flea [1]. Three human plague pandemics have been responsible for more deaths than any other infectious agents, as far as we know, in human history. Recently plague has attracted a considerable attention because of its potential use as an agent of biological warfare or bioterrorism. Plague represents three main clinical forms, bubonic, septic and pneumonic, depending on how the bacteria enter into and spread within the body [2]. Patients with bubonic plague can develop highly infectious pneumonic form that can be spread from person-to-person via respiratory droplets generated from sneezing and coughing of the patients. Due to the high infectivity and mortality of plague, there is urgent need to develop vaccines for protecting people.

The plague vaccine USP [3], a formaldehyde-killed whole-cell vaccine derived from the virulent 195/P strain of Y. pestis, was previously licensed in the U.S.A., but its production was discontinued in 1999 because it was not effective against pneumonic plague. The only currently available plague vaccine CSL, a heat-killed whole-cell vaccine derived from the 195/P strain of Y. pestis, was licensed for use in Australia, which appears to have similar properties to the USP vaccine [4]. Y. pestis live attenuated vaccine EV76, a mutant of the virulent strain that has lost the pgm locus, was developed from a virulent strain isolated from a plague patient in Madagascar by five-year's subculture, and protected against higher challenge doses than the killed vaccine USP, but this vaccine showed side effects of varying severity and was not currently used in the Western world [3], [5], [6]. The DNA vaccine based on Y. pestis F1 and LcrV antigen alone or in combination was efficacious against both bubonic and pneumonic plague [7], [8], [9]. However, immunization with plasmid DNA causes novel safety issues [10]. In contrast, subunit vaccines have obvious advantages over the live attenuated vaccine and DNA vaccine in terms of safety of use. In this regard, a 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 [11].

At present, plague subunit vaccines are being developed mainly based on the combination of F1 and LcrV or a fusion protein of these two antigens [12], which have been reported widely for their efficacy to protect mice [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], guinea pigs [24] and primates [25], [26]. It has been shown that LcrV antigen suppresses host innate immune functions by stimulating the release of interleukin 10 (IL-10) and preventing the release of proinflammatory cytokines, such as gamma interferon (IFN-γ) or tumor necrosis factor alpha (TNF-α) [27], [28], [29], [30]. Recently, an LcrV variant lacking amino acid residues 271–300 (rV10), compared with full-length LcrV, displayed a reduced immunosuppressive function [31]. In order to develop a safe and effective subunit vaccine for plague, a recombinant protein lacking amino acids 271–326 of LcrV (referred to as rV270 in this paper) was expressed in Escherichia coli BL21 [32] and the high-purity native F1 antigen was also prepared from Y. pestis EV76 [33].

In this study, the protective efficacy of the F1 + rV270 vaccines in protecting mice against subcutaneous challenge with a virulent Y. pestis 141 strain was evaluated in comparison with that of the EV76 vaccine. The antibody titres induced by different weight ratio of F1 + rV270 vaccines in BALB/c model were determined and compared with each other. An optimal formulation of F1 + rV270 was screened based on the titres elicited by candidate subunit vaccines in Balb/c model. Then, the optimal formulation of F1 + rV270 was tested in guinea pigs and New Zealand White rabbits by determining their antibody titres and protective efficacy against subcutaneous challenge with a virulent Y. pestis 141 strain.

Section snippets

Animals

Female BALB/c mice of 6–8 weeks, female guinea pigs of 6–8 weeks and New Zealand White rabbits of 1.5–2 kg raised under specific-pathogen-free conditions were used in this study. All the animals were group-housed and provided with food and fresh water ad libitum during the study. All animal experiments were conducted in accordance with the Guidelines for the Welfare and Ethics of Laboratory Animals of China.

Vaccines

The native F1 antigen was prepared from Y. pestis EV76 by physical disruption, followed

Antibody responses to the vaccines in different animals

The titres of anti-F1 and anti-rV270 specific antibodies in all immunized and control mice were determined on weeks 6, 8, 10, 12, 14, 16 and 18 after primary immunization. The geometric mean of antibody titres from each group of 10 mice and the standard error of the mean were calculated from the data obtained by ELISA (Supplementary Table S1). The immunized mice with subunit vaccines I to VIII developed higher titres of circulating IgG to both F1 and rV270 than those immunized with EV76

Discussion

In this study, we examined one-dose and two-dose of immunization regimens of native F1 plus recombinant rV270 vaccine at the same dosage level by using mouse, guinea pig and rabbit model. Groups of mice receiving two-dose of subunit vaccines had a significant stronger IgG response than those given one-dose of them. However, there was no significant IgG titre difference between the mice given a single dose of EV76 vaccine and those receiving two-dose of it. These results showed that subunit

Acknowledgments

Financial support for this study came from the National High Technology Research and Development Program of China (863 program) (contract no. 2006AA02Z438). We are grateful to Dr. Wei Liu for her assistance in statistical analysis.

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    Both contributed equally to this work.

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