1. Introduction
Gram-positive
Bacillus anthracis forms highly stable spores even after excessive UV, heat, or toxic chemical introductions that affect its survival. Exposure to these spores by cutaneous, gastrointestinal, or aerosol routes results in lethal anthrax infection
[1]. Poly-D-glutamic acid capsule and anthrax toxin are the two factors that are related to the toxicity of
B anthracis in living organism
[2]. The expression of poly-D-glutamic acid capsule, coded on pX02 plasmid, is controlled by
acpA gene and the capsule itself is in charge of protecting bacteria from the immune system, especially from phagocytosis
[2,
3]. There are three different protein types of anthrax toxin; protective antigen (PA), lethal factor (LF), and edema factor (EF)
[4]. These proteins are coded on pX01 plasmid as
pag, lef, and
cya genes respectively, and their expression is under the control of
atxA gene
[3]. Among these anthrax toxins, 83 kDa of PA cleaves into 63 kDa as it attaches to the surface of a host cell, such as a macrophage, and becomes a hydrophobic heptamer
[4]. LF or EF binds on this hydrophobic surface to enter the host cell for expressing own toxicity
[5]. When the concentration of EF, which is a kind of calmodulin-dependent adenylate cyclase, increases, it raises the concentration of cyclic adenosine monophosphate
[6], and, therefore, chloride ions and water molecules flow out of the cell body finally causing edema of the tissue
[7]. On the contrary, LF reduces the mitogen-activated protein kinase signal transduction and strengthens the level of cytokinesis tumor necrosis factor-α and interleukin-1β in macrophage cells
[8]. It also elevates the amount of oxygen radicals
[7] in the host cell resulting in macrophage targeted cytotoxicity, which can cause the actual cell and tissue destruction, and, ultimately, death of the organism.
Interfering with the binding of PA to LF or EF provides effective protection from anthrax infection in immunized animals
[9]. Under this concept, the current trend in developing anthrax vaccine ismainly focused onPArather than LF or EF. For example, the anthrax vaccines approved in theUSAandUKare adsorbed formof purified PA obtained from the culture supernatant of nontoxigenic
B anthracis
[9]. Anthrax Vaccine Adsorbed-Biothrax (AVA-Biothrax, formerly identified as AVA and MDPH-PA; BioPort Corporation, Lansing, MI, USA) is a widely-used anthrax vaccine in the USA which is in a form of aluminum hydroxide-adsorbed highly purified recombinant PA (rPA). However, the need for developing new vaccine is still exist as AVA-Biothrax has some drawbacks such as partial side-effects in some species and inconvenience of multiple-vaccination to achieve a decent level of immunization
[10].
In this study, adsorbed form of rPA to adjuvant was tested in a guinea-pig model and their protection rate was determined to set up a reliable surrogate marker of anthrax vaccination. After single or double immunization in 4-week intervals, serum samples from immunized animals were analyzed and compared to the survival rate after virulent B anthracis H9401 spore challenge.
4. Discussion
The correlation between neutralizing-antibody titers and protection against
B anthracis infection in different animal species vaccinated with rPA provides surrogate markers to evaluate the protective efficiency of rPA vaccines in humans
[19-
27]. Because of the cost associated with conducting such studies with a large enough number of nonhuman primates to achieve statistically significant results, alternative animal models have been examined. McBride et al
[26] were unable to identify a correlate of protection in guinea pigs that had received three inoculations of rPA (250, 2.5, 0.25, or 0.025 μg/dose) formulated with Alhydrogel adjuvant at
Table 2.
Survival against an i.m. challenge and antibody response of guinea pigs after two injections of rPA vaccine
PA(μg) dose |
Survival percentage (alive/total) |
Geometric mean ELISA titer (standard error) |
Geometric mean ED50 TNA assay titer (standard error)a
|
|
50 |
100 (9/9) |
43890.9 (1.12) |
4063.8 (1.26) |
25 |
100 (9/9) |
25600.0 (1.22) |
3225.4 (1.26) |
12.5 |
88.9 (8/9) |
16126.9 (1.29) |
1097.3 (1.35) |
6.3 |
80.0 (8/10) |
5198.4 (1.26) |
685.9 (1.18) |
3.1 |
33.3 (3/9) |
696.4 (1.72) |
278.6 (1.38) |
1.6 |
30.0 (3/10) |
459.5 (1.09) |
113.1 (1.37) |
0 (control) |
0.0 (0/10) |
1.0 (1.00) |
1.0 (1.00) |
0, 2, and 4 weeks and challenged at 6 weeks by the aerosol route with the Ames strain of
B anthracis. Pitt et al
[27] reported that survival of guinea pigs that had been challenged 3 months after being inoculated with two injection of rPA (50, 5, or 0.5 μg/dose) formulated with alhydrogel at 0 and 4 weeks was not dose dependent. Reuveny et al
[18] argued against multiple
Figure 2.
Relationship between survivals of guinea pig inoculated with two injection of rPA vaccine after i.m. challenge with B. anthracis H9401 spore and Week 7. (A) Anti-PA IgG titer; (B) ED50 TNA assay. Guinea pigs (◆) surviving or (◇) dying after i.m. injection.
inoculations and favored a single inoculation of vaccine. Their group demonstrated that their neutralizing antibody titers could serve as an effective correlate of protection for guinea pigs that were challenged intradermally with spores of the Vollum strain spore of
B anthracis after a single subcutaneous inoculation of rPA (25, 12.5, 6.3, 3.1, 1.6, or 0.8 μg) adsorbed to Alhydrogel. The anti-PA IgG titers, however, proved to be of limited value in serving as a surrogate marker
[18].
Our present study was designed to find correlations between protection level and either anti-PA IgG IgG titers or neutralizing-antibody titers. To eliminate
Figure 3.
Correlation between TNA titers and protection after two injections. Relationship between serological titer and protection of guinea pigs with a double vaccination serially diluted of rPA vaccine at a 4-week interval. After 4 weeks after the second vaccination, guinea pigs were challenged i.m with B anthracis H9401 spores. Each point in the group represents the average of duplicate determinations performed with pooled sera derived from 9 or 10 guinea pigs. TNA titers of serum samples from individual animals in each group were compared to the survival data of the animals (inset). Linear regression was performed using data from experimental cohorts with neutralizing-antibody titers of 1176 or less. The calculated R2 value is 0.96.
potential effects of other B anthracis derived components, we have used highly purified rPA adsorbed to alhydrogel as a vaccine. For the first step of establishing a surrogate marker for anthrax vaccine, suitable immunization intervals (4 week) were determined (data not shown). Then we tested the number of vaccinations to obtain adequate level of protection efficacy for testing rPA-alhydrogel vaccine. In the case of the single injection experiment, we obtained just 50% protection with the highest rPA dose and antibody titers were not high enough to guarantee the reproduction of this result, especially in TNA (titers < 100). Because of these reasons, single injection was not the optimal condition to examine vaccine efficacy.
With the information that a single injection is not sufficient to establish serological marker, we performed another set of experiments using two injections with a 4- week interval. In this case, it would obviously obtain higher serological titers in both assays compared to those of single injection schedule. Overall, animals that received two injections could survive more than animals with single injection and serological titers rapidly increased after the second injection. These results clearly support the classical concept that boosting brings higher serological titer and a higher protection rate to immunological challenge
[17]. We could obtain relatively high correlation coefficients from anti-PA IgG and neutralizing-antibody titers with serum samples from 4 weeks injection schedule (
R2 = 0.18 in anti-PA IgG ELISA,
R2 = 0.96 in TNA). These correlation coefficients ensured that a two-injection schedule with a 4 week interval might be able to serum as a good surrogates about vaccine efficacy.
Our findings support the notion that antibodies involved in neutralization are also involved in protection against the full course of infection. This is in agreement with previous observations in which monoclonal antibodies capable of neutralizing the cytotoxic effect were shown to delay death in guinea pigs
[28]. The results indicate that neutralizing-antibody titers can be used as a reliable correlate for protection in guinea pigs. A correlation between protection and neutralization titers was recently observed in a rabbit model
[15], suggesting that this phenomenon is not species specific. Application of such markers to human studies therefore appears to be feasible, even though similar experiments in nonhuman primates would be required to gain more confidence.
In conclusion, we have produced a series of experimental data to establish a surrogate marker that can indicate rPA vaccine efficacy. According to our results, the best correlation between serological titers and animal survival rates was obtained with double immunization using a 4-week interval schedule. Under this condition, antibody titers obtained by TNA were high enough to be reproduced and R2 values higher than 0.95, which indicates that TNA can be a reliable experimental technique for estimating animal survival. Therefore, to test the efficacy of an anthrax PA vaccine, two injections in combination of appropriate adjuvant at a 4-week interval and TNA for serological assay are recommended.