Comparative efficacy of two leading candidate ricin toxin a subunit vaccines in mice

Abstract

The two leading ricin toxin vaccine candidates, RVEc and RiVax, are recombinant derivatives of the toxin's 267-amino-acid enzymatic A chain (RTA). RVEc is truncated at the C terminus (residues 199 to 267) to improve protein thermostability, while RiVax has two point mutations (V76M and Y80A) that eliminate the RNA N-glycosidase activity of RTA, as well as its ability to induce vascular leak syndrome. The two vaccines have never been directly compared in terms of their ability to stimulate RTA-specific antibodies (Abs), toxin-neutralizing activity (TNA), or protective immunity. To address this issue, groups of female BALB/c mice were immunized two or three times with Alhydrogel-adsorbed RiVax or RVEc at a range of doses (0.3 to 20 μg) and then challenged with 10 50% lethal doses (LD(50)s) of ricin. We found that the vaccines were equally effective at eliciting protective immunity at the doses tested. There were, however, quantitative differences in the antibody responses. RVEc tended to elicit higher levels of ricin-specific RTA IgG and TNA than did RiVax. Pepscan analysis revealed that serum Abs elicited by RVEc were skewed toward a solvent-exposed immunodominant α-helix known to be the target of potent toxin-neutralizing Abs. Finally, immunodepletion experiments suggest that the majority of toxin-neutralizing Abs elicited by RiVax were confined to residues 1 to 198, possibly explaining the equal effectiveness of RVEc as a vaccine.

Fig 1

RTA-specific serum IgG antibody titers and TNA in mice immunized with RiVax or RVEc. RTA-specific serum IgG titers after the second (A) or third (B) immunizations with indicated doses (10, 3, or 1 μg) of Alhydrogel-adsorbed RiVax or RVEc. (C) Prechallenge TNA EC₅₀s in sera collected after three immunizations with RiVax or RVEc, as shown in panel B. (D) RTA-specific serum IgG GMTs after two immunizations with 0.3 μg of Alhydrogel-adsorbed RiVax or RVEc. An unpaired t test was used to compare the differences in serum IgG GMTs or TNA elicited by the same doses of RiVax and RVEc. *, P < 0.05, and ***, P < 0.001.

Fig 2

RTA pepscan analysis of sera from RiVax- and RVEc-immunized mice. Individual serum samples (n = 6 per group) from mice immunized three times with 20 μg RiVax or RVEc were applied to ELISA plates coated with an overlapping 18-mer peptide array spanning the length of RTA. The cumulative reactivities of the individual samples (y axis) are plotted versus each RTA peptide (x axis). The final peak (far right) represents antibody reactivity with RTA. RVEc antisera displayed little to no reactivity with peptides A04 to A06 or B11 to C05, which correspond to regions of RTA that were deleted in the construction of RVEc. OD₄₅₀, optical density at 450 nm.

Fig 3

A11 peptide (RTA residues Y91 to F108) reactivities associated with serum antibodies from RiVax- and RVEc-immunized mice. Sera from mice immunized three times with 10 μg, 3 μg, or 1 μg of Alhydrogel-absorbed RiVax or RVEc (x axis) were subjected to BIAcore analysis using an A11 peptide-coated chip. Shown on the y axis are relative resonance units (RU). Error bars represent the standard errors of the means (SEM). An unpaired t test with Welch's correction was used to determine the differences in A11-specific reactivities between sera from RiVax- and RVEc-immunized mice. *, P < 0.05, and **, P < 0.01.

Fig 4

TNA associated with FD3-specific Abs. Shown is the TNA associated with pooled RiVax immune sera (FD1 + FD2 + FD3), the eluate from an RVEc affinity column (FD1 + FD2), or the RVEc affinity column flowthrough (FD3) fraction. The eluate is enriched for FD1- and FD2-specific Abs, whereas the flowthrough is enriched in FD3-specific Abs. The amount of RTA-specific Abs in the pooled sera, eluate, and flowthrough were normalized to 50 μg/ml prior to being tested in the Vero cell cytotoxicity assay. Error bars represent the SEM.