Elsevier

Vaccine

Volume 26, Issue 44, 16 October 2008, Pages 5554-5561
Vaccine

Lipid A mimetics are potent adjuvants for an intranasal pneumonic plague vaccine

https://doi.org/10.1016/j.vaccine.2008.08.007Get rights and content

Abstract

An effective intranasal (i.n.) vaccine against pneumonic plague was developed. The formulation employed two synthetic lipid A mimetics as adjuvant combined with Yersinia pestis-derived V- and F1-protective antigens. The two nontoxic lipid A mimetics, classed as amino-alkyl glucosaminide 4-phosphates (AGPs) are potent ligands for the Toll-like receptor (TLR) 4. Using a murine (BALB/c) pneumonic plague model, we showed a single i.n. application of the vaccine provided 63% protection within 21 days against a Y. pestis CO92 100 LD50 challenge. Protection reached 100% by 150 days. Using a homologous i.n. 1°/2° dose regimen, with the boost administered at varying times, 63% protection was achieved within 7 days and 100% protection was achieved by 21 days after the first immunization. Little or no protection was observed in animals that received antigens alone, and no protection was observed when the vaccine was administered to BALB/c TLR4 mutant mice. Vaccine-induced serum IgG titers to F1 and V-antigen were reflected in high titers for IgG1 and IgG2a, the latter reflecting a bias for a cell-mediated (TH1) immune response. This intranasal vaccine showed 90% protection in Sprague–Dawley rats challenged with 1000 LD50. We conclude that lipid A mimetics are highly effective adjuvants for an i.n. plague vaccine.

Introduction

The plague bacterium, Yersinia pestis, is classed as a bioterrorism Category A agent. This Center for Disease Control classification has been assigned because the organism can be spread by aerosol, has a high mortality rate [1], [2] and its illicit release could have a major impact on public health (http://www.bt.cdc.gov/agent/agentlist-category.asp). Previously U.S.-licensed Y. pestis whole cell vaccines only provided protection against the bubonic form of the disease that is acquired from the bite of the rodent flea vector and were not protective against pneumonic exposure [3], [4]. For this reason, the whole cell vaccine was removed from the market and efforts to develop an efficient vaccine for pneumonic plague have been an important focus in recent years.

It is now well established that immunity generated against two Y. pestis antigens, F1 (capsule protein) and V-antigen (type III secretin component), are protective against pneumonic plague [5], [6], [7], [8]. Both of these antigens are virulence factors produced by Y. pestis at 37 °C; F1-capsules inhibit phagocytosis, and the V-antigen forms the distal tip of the type III secretin structure [9], [10], [11], [12], [13], [14]. Subunit vaccines employing purified F1 antigen (F1) and V-antigen proteins, or a recombinant fusion protein combining epitopes of each antigen, administered with classical adjuvants such as alum (alhydrogel), can provide up to 100% protection in a murine or non-human primate pneumonic plague model [6], [15], [16], [17]. Other pneumonic plague vaccine candidates have utilized V-antigen alone, which stimulates humoral protection in challenge studies with virulent Y. pestis and a Y. pestis F1 (caf1) mutant [9], [15], [18], [19]. Most of these successful vaccination studies employ a traditional alum-based adjuvant, require multiple subcutaneous injections, and do not provide protection from a Y. pestis aerosol challenge until 35 or more days post-immunization [6], [9], [20]. Presently alum is the only approved vaccine adjuvant in the U.S. and while effective, it has a humoral immune response bias (stimulating TH2 cells) vs. promoting a cellular response (stimulating TH1 cells)[21]. As Smiley recently suggests [22], a more effective plague vaccine should be one that fosters a TH1 response because Y. pestis can survive and replicate within macrophages.

Several immunization studies have also been conducted using intranasal (i.n.) vaccine applications. Jones et al. [23] established that an intranasal vaccine comprised of F1 and V-antigen mixed with the mucosal adjuvant Protollin™ could provide high-level protection in a pneumonic plague model when mice were challenged with Y. pestis CO92 no sooner than 35 days post-immunization [23]. Other investigators have utilized a subunit vaccine against pneumonic plague in a 1°/2° dose regimen that evaluated intranasal application and injection of the vaccine preparations, but only two dose regimens that included an injected dose provided 100% protection [24], [25].

More recently, alternative adjuvants including Sigma adjuvant system (an oil-in-water emulsion containing generic monophosphoryl lipid A plus trehalose dicorynomycolate), CpG (synthetic unmethylated dinucleotides), or ADP-ribosylating enterotoxins, have been examined [7]. The former two rely on stimulation of Toll-like receptors (TLRs); TLR4 for lipid A and TLR9 for CpG DNA, both of which induce TH1 immune responses. The ADP-ribosylating enterotoxins appear to elicit a more balanced TH1 and TH2 response. These studies demonstrate that adjuvant selection is important in directing the type of immune response elicited.

The ability of Y. pestis to evade the innate immune response is well established [26]. Temperature induced modulation of Y. pestis lipid A from hexa-acylated (25 °C) to tetra-acylated (37 °C) is an important aspect of this process because tetra-acylated lipid A does not activate TLR4 and Y. pestis mutants engineered to express hexa-acylated lipid A at 37 °C are attenuated [27], [28]. In this report, we examined the efficacy of using two amino-alkyl glucosaminide 4-phosphates (AGPs) as adjuvant for an intranasal pneumonic plague vaccine. These synthetic compounds, designated CRX-524 and CRX-527, are immunostimulatory ligands for TLR 4, but lack the highly toxic properties of bacterial-derived lipid A [29], [30]. A variety of AGP lipid A mimetics have been synthesized with varying lengths of secondary acyl side chains and the functional group on the aglycon component. The AGPs employed in this study (CRX-524 and CRX-527) have acyl side chains of 10 carbons and contain either H or a carboxyl group respectively at the aglycon unit [31]. We have shown these compounds, when administered intranasally, induce high levels of TNF-α, IL-12p70, and IFN-γ in murine lung tissue [32]. Using a murine model of pneumonic plague, we tested an intranasal vaccine using AGPs as adjuvant with F1 and/or V-antigen to determine (i) the best concentrations of AGPs; (ii) the effect of primary and secondary vaccine regimens; (iii) the shortest duration to protection; (iv) the requirement for TLR4 stimulation; (v) protection in rats.

Section snippets

Bacteria and growth conditions

Y. pestis CO92 (CDC, Ft. Collins, CO) was grown in Brain Heart Infusion (BHI) broth overnight at 30 °C, diluted 1:200, and incubated for 36 h at 30 °C with aeration. For use in experiments, aliquots of a single stock culture (∼1 × 108 CFU/mL) were mixed with 20% (v/v) glycerol, and stored at −80 °C. Challenge doses for each experiment were quantified by plate counts. All experiments were performed under CDC-certified BSL-3 conditions at the University of Idaho.

Vaccine preparation

F1 was purified from Y. pestis as

Results

AGPs were evaluated as adjuvants in vaccine preparations containing F1 capsule protein and/or V-antigen from Y. pestis to protect against pneumonic plague. Vaccination regimens that varied the amount, the route, the timing, and the number of administrations were tested in a mouse model of pneumonic plague. The most effective vaccine regimen was tested in rats and the mechanism of protection was analyzed using TLR4 mutant mice. When determining vaccine efficacy, the Food and Drug

Discussion

At present, plague is a modest contributor to human morbidity and mortality. A total of 36,876 cases of plague were reported by the WHO between 1987 and 2001, the majority being the bubonic form of the disease [39]. Of this total, 2847 cases were fatal. Nevertheless, because of the potential application of Y. pestis as an aerosolized bioterrorist agent, plague remains a significant concern. This concern is highlighted by the lack of a fast acting vaccine that provides protection against the

Acknowledgements

This work was supported, in part, by the Idaho Agriculture Experiment Station and Public Health Service grants U54-AI-57141, P20-RR16454, and P20-RR15587 from the National Institutes of Health. The authors thank Dr. Jay Evans (GlaxoSmithKline, Hamilton, MT) for helpful discussions and for providing the AGPs used in this study. Drs. Sam Miller and Shawn Skerrett provided helpful discussion during the course of this work.

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