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Comparative Study
. 2016 Mar 23;11(3):e0148713.
doi: 10.1371/journal.pone.0148713. eCollection 2016.

A Comparison of the Adaptive Immune Response between Recovered Anthrax Patients and Individuals Receiving Three Different Anthrax Vaccines

Thomas R Laws  1 , Tinatin Kuchuloria  2   3 , Nazibriola Chitadze  4 , Stephen F Little  5 , Wendy M Webster  5 , Amanda K Debes  5 , Salome Saginadze  4 , Nikoloz Tsertsvadze  4 , Mariam Chubinidze  4 , Robert G Rivard  5 , Shota Tsanava  2   4 , Edward H Dyson  1 , Andrew J H Simpson  1 , Matthew J Hepburn  5 , Nino Trapaidze  3   4
Affiliations
Comparative Study

A Comparison of the Adaptive Immune Response between Recovered Anthrax Patients and Individuals Receiving Three Different Anthrax Vaccines

Thomas R Laws et al. PLoS One. .

Abstract

Several different human vaccines are available to protect against anthrax. We compared the human adaptive immune responses generated by three different anthrax vaccines or by previous exposure to cutaneous anthrax. Adaptive immunity was measured by ELISPOT to count cells that produce interferon (IFN)-γ in response to restimulation ex vivo with the anthrax toxin components PA, LF and EF and by measuring circulating IgG specific to these antigens. Neutralising activity of antisera against anthrax toxin was also assayed. We found that the different exposures to anthrax antigens promoted varying immune responses. Cutaneous anthrax promoted strong IFN-γ responses to all three antigens and antibody responses to PA and LF. The American AVA and Russian LAAV vaccines induced antibody responses to PA only. The British AVP vaccine produced IFN-γ responses to EF and antibody responses to all three antigens. Anti-PA (in AVA and LAAV vaccinees) or anti-LF (in AVP vaccinees) antibody titres correlated with toxin neutralisation activities. Our study is the first to compare all three vaccines in humans and show the diversity of responses against anthrax antigens.

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Conflict of interest statement

Competing Interests: T. Kuchuloria’s employment via TMC does not alter the authors’ adherence to PLOS ONE policies on sharing data and materials. The authors of the manuscript declare that they have no interests that that interfere with, or could reasonably be perceived as interfering with, the full and objective presentation of the data shown here.

Figures

Fig 1
Fig 1. The characteristics of individuals enrolled into this study including: age, number of exposures to B. anthracis antigens and time since exposure in relation to the type of exposure to B. anthracis antigens.
Panel A shows the distribution of age across groups in study including individuals with no history of Anthrax, recent Anthrax infection, or one of three different vaccines. Each symbol denotes a single participant, the line is the mean and the error bars 95% Confidence Intervals. Statistical analysis is shown where Levene’s test validated GLM has been performed with Bonferroni’s post-tests. Panel B shows the distribution of time since exposure to vaccine or onset of anthrax symptoms across groups in the study. Each symbol denotes a single participant, the line is the median and the error bars the quartile range. The data were analysed by Kruskal-Wallis test (excluding naïve controls), which indicated differences between groups with Dunn’s individual comparisons. Panel C shows the distribution of number of anthrax-based immunological stimuli (doses) across groups in the study including percent people who had had Anthrax infection, or one of three different vaccines. Each symbol denotes a single participant, the line is the median and the error bars the quartile range. The data (excluding naïve and naturally infected controls) were analysed by Kruskal-Wallis test, which indicated differences between groups with Dunn’s individual comparisons. Significance markers indicate either P-values associated with either Bonferroni’s (For GLM) or Dunn’s (for Kruskal-Wallis) multiple comparisons where * indicates P < 0.05, ** indicates P < 0.01 and *** indicates P < 0.001).
Fig 2
Fig 2. Correlations between immune response to B. anthracis antigens and individual’s age and the number of vaccine doses.
Panel A shows the correlation between age and antibody response to EF, in participants of a study which included individuals who had suffered from cutaneous anthrax previously, been vaccinated with either AVA, LAAV or AVP, or have no history of exposure to anthrax antigens. Antibody responses were measured by ELISA estimating antigen specific antibody in μg / ml. Panels B-D show the correlation between the numbers of doses of vaccine received and antibody response. Antibody responses were measured by ELISA estimating antigen specific antibody against PA (panel B), LF (panel C) and toxin neutralisation activity (Panel D). Partial correlation analyses indicated a significant negative correlation between the parameters (marked on the graphs).
Fig 3
Fig 3. The memory immune responses towards parts of the Anthrax toxin systems in individuals who had suffered from cutaneous anthrax previously, been vaccinated against anthrax with either AVA, LAAV or AVP, or have no history of exposure to anthrax antigens.
Antibody responses were measured by ELISA, estimating antigen specific antibody in μg / ml or a dilution end point. IFN-γ T cell responses were estimated by ELISPOT and numerated as fold induction in IFN-γ producing cells by exposure to anthrax antigen, when compared to unstimulated cells. Toxin neutralisation was measured in the ED50 of cells exposed to anthrax toxin in the presence of the serum. ULQ signifies the upper limit of quantification, due to confluence of spots on the ELISPOT plate, which was regarded as 25 fold. An additional line has been included which is indicative of the 90 percentile of the naïve group. The purpose of this is to give an approximate indication of the number of individuals with greater than background antigen recognition. Statistical analysis is shown. First, Levene’s tests of unequal variances were performed to establish suitability for parametric analysis. Unsuitability necessitated Kruskal-Wallis tests with Dunn’s post-tests. Suitability allowed for GLM analysis which included any potential covariates suggested previously, with Bonferroni’s multiple comparisons. The significance markers show results post-test comparisons: *** = P < 0.001, ** = P <0.01 * = P <0.05.
Fig 4
Fig 4. The relativity of different antigen specific responses in groups exposed to B. anthracis antigens in different ways.
The panels show the five different groups investigated in the course of this study. Participants included individuals who had suffered from cutaneous anthrax previously, been vaccinated with either AVA, LAAV or AVP, or had no history of exposure to anthrax antigens. Each line represents an individual with 7 different immune measurements take. Antibody responses were measured by ELISA estimating antigen specific antibody in μg / ml or a dilution end point. IFN-γ T cell responses were estimated by ELISPOT and numerated as fold induction in IFN-γ producing cells by exposure to anthrax antigen, when compared to unstimulated cells. Toxin neutralisation was measured in the ED50 of cells exposed to anthrax toxin in the presence of the serum. The analyses are Pearson Correlations and the R values are given with the significance markers of *** = P < 0.001, ** = P <0.01 * = P <0.05.
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Grants and funding

Funding for this project was provided by the UK Ministry of Defence and US Department of Defense Cooperative Threat Reduction Program, implemented by the US Defense Threat Reduction Agency. The views of the authors do not purport to reflect the positions of the US Department of Defense. During this study, author T. Kuchuloria worked as a sub-contractor for the commercial entity Technology Management Company (TMC), on behalf of the US Defense Threat Reduction Agency via USAMRIID. TMC administered a salary for T. Kuchuloria but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.