A systematic review and meta-analysis of preclinical trials testing anti-toxin therapies for B. anthracis infection: A need for more robust study designs and results
A systematic review and meta-analysis of preclinical trials testing anti-toxin therapies for B. anthracis infection: A need for more robust study designs and results
Wanying Xu 0 1 2
Lernik Ohanjandian 0 1 2
Junfeng Sun 0 1 2
Xizhong Cui 0 1 2
Dante Suffredini 0 1 2
Yan Li 0 1 2
Judith Welsh 0 1
Peter Q. Eichacker 0 1 2
0 Editor: Nicholas J Mantis, New York State Department of Health , UNITED STATES
1 5 agents; LFI , AIG, AVP-21D9, Raxibacumab, and ETI-204. Only five experiments were
2 Critical Care Medicine Department, Clinical Center, National Institutes of Health , Bethesda , Maryland, United States of America, 2 National Institutes of Health Library, National Institutes of Health , Bethesda, Maryland , United States of America
B. anthracis anti-toxin agents are approved and included in the Strategic National Stockpile based primarily on animal infection trials. However, in the only anthrax outbreak an approved anti-toxin agent was administered in, survival did not differ comparing recipients and non-recipients, although recipients appeared sicker. Employ a systematic review and meta-analysis to investigate preclinical studies supporting Compared survival with an anti-toxin agent versus control in B. anthracis challenged, antibiExamine model and study design and the effect of anti-toxin agents on relative risk of death From 9 studies, 29 experiments were analyzed which included 4 species (748 animals) and blinded and no experiment included the cardiopulmonary support sick B. anthracis patients
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
Funding: This research was supported by the
Intramural Program of the NIH, Clinical Center,
Critical Care Medicine Department. The funders
had no role in study design, data collection and
analysis, decision to publish, or preparation of the
anthrax anti-toxin agents.
PubMed, EMBASE, and Scopus.
otic treated animals.
Competing interests: One author's (PQE)
laboratory received Raxibacumab from Human
Genome Sciences and Anthrax Immune Globulin
from the CDC for experimental studies. This
laboratory has also received funding from the
Trans-NIH/FDA Biodefense Program and the FDA
Medical Countermeasures Division. There are no
patents, products in development or marketed
products to declare. This does not alter our
adherence to all the PLOS ONE policies on sharing
data and materials, as detailed online in the guide
receive. Only one agent in a single un-blinded experiment reduced RR significantly [0.45
(0.22,0.940]. However, in six studies testing an agent in more than one experiment in the
same species, agents had consistent survival effects across experiments [I2 = 0, p 0.55 in
five and I2 = 42%, p = 0.16 in one]. Within each species, agents had effects on the side of
benefit; in one study testing AVP-21D9 in mice [0.11(0.01,1.82)] or guinea pigs [0.70
(0.48,1.03)]; across eight rabbit studies testing LFI, Raxibacumab, AIG or ETI-204 [0.62
(0.45,0.87); I2 = 17.4%, p = 0.29]; and across three monkey studies testing Raxibacumab,
AIG or ETI-204 [0.66(0.34,1.27); I2 = 25.3%, p = 0.26]. Across all agents and species,
agents decreased RR [0.64(0.52,0.79); I2 = 5.3%, p = 0.39].
Incidence of selective reporting not identifiable.
Although overall significant, individually anti-toxin agents had weak beneficial effects.
Lack of study blinding and relevant clinical therapies further weakened studies. Although difficult, preclinical studies with more robust designs and results are warranted to justify the resources necessary to maintain anti-toxin agents in national stockpiles.
Due to the relative facility with which B, anthracis spores can be produced and widely deliv
ered and the lethality of B. anthracis infection itself, this bacterium is considered a Biodefense
Category A pathogen presenting a high risk to the US public.[
] This risk was highlighted by
the outbreak of B. anthracis infection that occurred in the US in 2001 related to intentionally
] During that outbreak, 5 of 11 patients with inhalational disease died
despite receiving aggressive therapy with antibiotics the bacteria was sensitive to, together with
intensive supportive measures. Also, while the experience was small, mortality among those
developing evidence of septic shock appeared much higher than with more commonly
encountered bacteria. This outbreak along with other national and international events at the
time, greatly accelerated research directed at developing adjunctive treatments that would add
to or increase the efficacy of conventional ones for the treatment of B. anthracis. Because prior
experience pointed to the production of lethal and edema toxins (LT and ET respectively) as
central to the pathogenesis of B. anthracis infection, much of this research focused on
developing agents to neutralize these toxins. Several potential therapies have resulted from this
intensive research effort and the US Centers for Disease Control and Prevention (CDC) guidelines
now recommend that patients with evidence of systemic and severe infection receive an
antitoxin agent combined with antibiotics and other supportive measures[
Three anti-toxin agents have now received US Food and Drug Administration (FDA)
approval for use during an outbreak of anthrax infection including; anthrax immune globulin
(AIG), a polyclonal antibody; Raxibacumab, a monoclonal antibody (mAb) and; and ETI-204,
another monoclonal antibody.[
] Each of these agents is directed at inhibiting protective
antigen (PA) mediated uptake of toxin by host cells. Because the naturally occurring invasive
and highly lethal forms of B. anthracis infection are rare, these new agents have not been tested
clinically. Instead, the FDA approved them based on the results of trials in animal models of B.
2 / 17
anthracis infection under the animal rule together with safety studies in healthy humans. This
experience has also provided the basis for including AIG and Raxibacumab in the Strategic
National Stockpile (SNS) and for consideration of such inclusion for ETI-204. However, while
these agents may be safe, whether they are efficacious in humans is unknown.
At this time there has only been one clinical experience with an approved anti-toxin agent
during an outbreak of invasive B. anthracis infection. Anthrax immune globulin (AIG) was
administered to 15 of 47 patients with B. anthracis soft-tissue infection during the 2009 to
2010 outbreak in Scotland related to the use of contaminated heroin.[
] Notably, review of 43
patients from that outbreak including all AIG recipients, showed that mortality in this
uncontrolled experience did not differ comparing recipients and non-recipients. However,
comparisons between these recipients and non-recipients are difficult to make. Related possibly to
higher bacterial loads, more extensive tissue injury or underlying co-morbidities, patients
receiving AIG had higher sequential organ failure scores and were sicker than non-recipients
at presentation in this outbreak. Also, AIG was used off label for the soft tissue infections
comprising this outbreak since all antitoxins licensed thus far, including AIG, have been developed
for inhalational infection alone.
However, these findings from the UK outbreak combined with the substantial resources
necessary to supply and maintain B. anthracis anti-toxin agents in the SNS, prompted us to try and
better understand the strength of evidence derived from preclinical animal models of infection
supporting such agents. To do this, we performed a systematic review and meta-analysis. We
searched the literature looking specifically for preclinical trials that compared the effect of a B.
anthracis anti-toxin agent to a control treatment in animal models employing live B. anthracis
challenges. Because antibiotics are an effective therapy for B. anthracis infection if used early
enough and are routinely administered to patients, we also required that the trials analyzed were
conducted in antibiotic treated animal models. While a prior systematic review examined a
similar question, the present investigation includes almost double the number of animals as this other
] It also includes a meta-analysis of the retrieved studies, which the prior review did not.
Materials and methods
Literature search and study selection
PubMed, EMBASE, and Scopus databases were searched to retrieve relevant studies on the
preclinical assessment of B. anthracis toxin associated inhibitors. The search was not limited
by year or language and was conducted through July 31, 2016. Principle keywords included B.
anthracis, anthrax, antitoxins, immunoglobulins, antibodies, inhibitors, blockers, and
neutralizers. The search strategies reported in S1 Text were adapted to accommodate the unique
searching features of each database, including database specific MESH and EMTREE controlled
vocabulary terms as appropriate (S1 Text). Data was also obtained from FDA briefing
documents related to specific agents.
Studies meeting the following criteria were analyzed: employed an in vivo animal model
challenged with a strain of live B. anthracis; compared the effects of a therapy designed to
inhibit B. anthracis LT or ET or both to a control agent on survival; and all animals, besides
receiving either an anti-toxin or control agent, received similar antibiotic treatment. Three
authors, W.X., L.O. and P.Q.E. identified relevant studies. Included studies were searched for
additional references. Author consensus resolved uncertainty regarding study inclusion.
Outcomes examined and data extracted
Data was extracted from included studies using a standardized tool. Authors of included studies were contacted when data required clarification. The primary outcome examined was the
3 / 17
effect of anti-toxin therapy on the risk ratio of death based on the duration of survival
stipulated in the study. Characteristics of each study extracted included; the species, weight and age
of animals studied; the strain, route and dose of B. anthracis challenge; the type, route, dose,
timing and frequency of anti-toxin and control treatment; the type, route, dose, timing and
frequency of antibiotic treatment. Other aspects of trial design and conduct extracted included;
randomization; blinding; prospective power analyses to determine study group sizes; exposure
dose analysis; prospective observation schedules and euthanasia criteria; use of
cardiopulmonary supportive measures; secondary endpoints; and industry involvement with a trial.
Investigators of analyzed studies were contacted to obtain data not available from the published
The relative risk (RR) of death for anti-toxin treatment versus control was estimated using a
] Heterogeneity among studies was assessed using the Q statistic and
] We first analyzed repeated experiments in each species within each paper, and
pooled the data when appropriate. The pooled data were then analyzed by species and by
antitoxin agents to assess their effects. An overall treatment effect was estimated if there was no
evidence of species or agent effect. In two groups of experiments, meta-regression was used to
assess the relationship between time and treatment effects. We first tested to determine
whether there was a significant relationship between treatment time and effect within each
study. Then, we pooled the data from both studies and fitted a meta-regression model with a
common slope but different intercepts. Sample size calculation was done using STPLAN
version 4.5 (https://biostatistics.mdanderson.org/softwaredownload/SingleSoftware.aspx?
Software_Id=41) for two-sample binomially distributed outcomes with equal sample sizes in
both groups. All analyses were done using R (version 3.3.1) packages meta (version 4.3±2) and
metafor (version 1.9±8).[
] Two-sided p-values 0.05 were considered significant.
The literature search identified 7,782 reports for review (Fig 1). Of these, 4,495 were duplicate
studies and 2,917 were excluded based on title and abstract review. After full review of the
remaining 370 reports, 7 studies were included for analysis (cited in Table 1 and designated in
tables by the first author and year of publication). Two FDA briefing documents were also
identified which provided data on additional experiments (designated in tables as ªFDAº
along with the anti-toxin agent tested). Therefore, a total of 9 studies were included in the final
Study characteristics and designs
In the 9 studies reviewed there were a total of 29 individual experiments (i.e., 29 comparisons
between an anti-toxin agent and a control treatment under the same conditions), each of
which examined 1 of 5 anti-toxin agents including; a hydroxamate lethal factor inhibitor
(LFI)± 1 experiment; AVP-21D9, a PA directed monoclonal antibody± 5 experiments;
Raxibacumab± 3 experiments; AIG± 11 experiments; and ETI-204±9 experiments (Table 1; for
reference, experiments are numbered based on chronology and anti-toxin type). LFI was studied in
a rabbit model, AVP-21D9 in mouse and guinea pig models, and the other three agents in
both rabbit and cynomolgus monkey models. All models employed the Ames strain of B.
anthracis as the bacteria challenge. These challenges were administered at varying doses and
4 / 17
Fig 1. Flow diagram that summarizes results of the literature search.
via subcutaneous (SC), intra-nasal (IN) or inhalational (INH) routes. The antibiotic regimens
employed in the models included ciprofloxacin, levofloxacin or doxycycline administered via
SC, intraperitoneal (IP), intravenous (IV), gastric (GI) or oral routes. Antibiotics were
administered anywhere from the time of to 96h following B. anthracis challenge. Antitoxin agents
were delivered via SC, IP, or IV routes and were also administered anywhere from 0 to 96h
after bacteria challenge. In some cases, the timing of antibiotic and anti-toxin treatment was
scheduled and in others it was based on a change in body temperature or detection of PA in
the blood. In Experiment 21, antitoxin was administered at 30h if PA had not yet become
detectable. In two Experiments 28 and 29, PA was detected at 48h in animals, and this was the
time treatment was administered.
5 / 17
Type Route Dose T(ihm)e Trigger
LFI SC m1g0/0kg 66 NT
AVP SC 500 ug 24 NT
AVP IP 5 mg/kg 24 NT
AVP IP 50kmg g/ 24 NT
AVP IP 50kmg g/ 24 NT
AVP IP 50kmg g/ 24 NT
RAXI IV 40kmg g/ NR PA det
RAXI IV 40kmg g/ NR PA det
RAXI IV 40kmg g/ 84 NT
AIGIV IV 15 U/kg 30 NT
AIGIV IV 15 U/kg 36 NT
AIGIV IV 15 U/kg 48 NT
AIGIV IV 15 U/kg 60 NT
AIGIV IV 15 U/kg 60 NT
AIGIV IV 15 U/kg 72 NT
AIGIV IV 15 U/kg 84 NT
AIGIV IV 15 U/kg 96 NT
AIGIV IV 15 U/kg 96 NT
AIGIV IV 15 U/kg 64 NT
Monkey M/F Ames INH
*If no PA detected then treatment given at 30h post-exposure
**PA detection equivalent to 48h post-exposure
AIGIV- Anthrax Immune Globulin Intravenous; AVP-human monoclonal antibody; c-Colony Forming Units; Cipro-Ciprofloxacin; Doxy- Doxycycline;
ETI-ETI-204; Exp #-experiment number; GI-gastrointestinal; IM-intramuscular; IN-intranasal; IP-intraperitoneal; IV-intravenous; LD-lethal dose;
LevoLevofloxacin; LFI-lethal factor inhibitor; NR-not recorded; NT-no trigger; PA det- protective antigen detection; RAXI-Raxibacumab; s-spores;
The treatments that control and anti-toxin animals received are shown in Table 2. In some
studies, whether and what placebo was used in controls to match the antitoxin agent studied
was not available. Table 2 also shows the number of animals that were originally infected in
studies and the number that were ultimately randomized or assigned to control and anti-toxin
groups and included in analysis for each of the 29 experiments. Altogether, there were 1,283
animals infected with bacteria, of which 748 animals survived to be randomized or assigned;
377 to anti-toxin groups and 371 to a control groups. These 748 animals were included in the
present analysis. Finally, Table 2, shows whether the data employed for analysis was available
from published reports in a scientific journal alone, from an FDA briefing document alone, or
from both. In all cases where data was available from both sources, it was the same.
While all 29 experiments except 1 were randomized, only 5 were blinded ones (Table 3).
Twenty-three experiments reported using prospective sample size determinations, 20 used
prospective observation schedules and 20 used prospective euthanasia criteria. No study
provided hemodynamic or respiratory support for animals. The primary endpoint was survival in
all studies and no study included secondary endpoint data such as the effects of anti-toxin
treatment on blood pressure, oxygen exchange or other measures of organ function. All
experiments accept one (Experiment 21) were conducted by or in association with industry
producers of the agents being tested.
Effect of anti-toxin treatments on mortality
Survival data with numbers of non-survivors and total numbers of animals in control and
anti-toxin groups, percentage mortality rates and relative risks of death (95% CI) are shown
for each experiment within their respective studies in Fig 2. In only one experiment did an
anti-toxin agent (ET-204 in cynomolgus monkeys, experiment 28) demonstrate a beneficial
effect on survival that reached statistical significance, and this experiment was not blinded.
7 / 17
*Placebo noted but not described
**No Placebo described
#Animals that had not died but were bacteremic at 64h
##Number of animals infected at the outset of the experiment, from which some expired prior to later treatment in several experiments
AVP-human monoclonal antibody; Cipro-ciprofloxacin; FDA-BD-Food and Drug Administration briefing document; IGIV- Human Immune Globulin
Intravenous; Levo-levofloxacin; LFI-lethal factor inhibitor; PR-published results; Raxi-Raxibacumab.
However, a total of 20 experiments had effects on the side of benefit and only 4 had effects on the side of harm.
Notably, in five of the six studies testing an agent in more than one experiment in the same
species, anti-toxin agents had highly consistent effects on the side of decreasing the overall
relative risk of death (95%CI) across experiments as follows; 8 experiments with AIG in rabbits,
[0.79 (0.51, 1.24), I2 = 0, p = 0.69]; 2 experiments with AIG in cynomolgus monkeys, [0.77
(0.27, 2.21); I2 = 0, p = 0.82]; 2 experiments with ETI-204 in cynomolgus monkeys, [0.53(0.32,
0.88); I2 = 0, p = 0.55]; 4 experiments with ETI-204 in rabbits, [0.76(0.43, 1.32); I2 = 0,
8 / 17
Blind.±blinding; CP sup±cardiopulmonary support; Euth. crit.±prospective euthanasia criteria; Exp. #Ðexperiment order number; NR±not reported; Prim.
endpt.±primary endpoint; Pro. obs. sched.±prospective observation schedule; Pro. samp. size±Prospective sample size analysis; Random.±randomization;
Sec. endpt.±secondary endpoint; Surv.Ðsurvival
p = 0.59]; and 2 other experiments with ETI-204 in rabbits, [0.13(0.03, 0.61); I2 = 0, p = 0.64]
(Figs 2 and 3). In the remaining study with four experiments testing AVP-21D9 in guinea pigs,
the relative risk of death (95%CI) was also on the side of decreasing mortality [0.79 (0.48,
1.29)] but the I2 value was moderate although not significant (I2 = 42%, p = 0.16).
Based on the similarity of the effects of individual anti-toxin agents across experiments in
the same species, we further analyzed the overall effects of the agents across studies within the
same species (Fig 4). In this analysis, all agents were associated with a decrease in the relative
risk of death (95%CI) as follows; AVP-21D9 in one study in mice [0.11(0.01, 1.82)];
AVP21D9 in one study in guinea pigs [0.70(0.48, 1.03)]; LFI, Raxibacumab, AIG and ETI-204 in a
total of eight studies in rabbits [0.62(0.45, 0.87); I2 = 17.4%, p = 0.29]; and Raxibacumab, AIG
and ETI-204 in a total of three studies in cynomolgus monkeys [0.66(0.34, 1.27); I2 = 25.3%,
p = 0.26].
9 / 17
Fig 2. This figure shows the anti-toxin agent and species studied, the number of animals initially infected, the numbers of total and
nonsurviving animals in treatment and control groups with respective mortality rates, and the effects of the anti-toxin agent (i.e. agents) on the
relative risk of death (95%CI) for 29 individual experiments from 9 studies. The anti-toxin agents studied included: lethal factor inhibitor (LFI),
AVP21D9 (AVP), Raxibacumab (Raxi), Anthrax Immune Globulin (AIG), and ETI-204 (ETI). Control treatments were an antibiotic + the following; *a placebo but
the placebo was not described; **no placebo was described; #anti-toxin buffer; ##human intravenous globulin; @saline (see Table 2).
Because the effects of the anti-toxin agents were consistent across these studies in the same
species (I2 25.3%), we then examined the effects of anti-toxin treatments across all four agents
and all four species (Fig 4). In this analysis, antitoxin agents decreased the overall relative risk
of death (95%CI) significantly [0.64(0.52, 0.79)] and in a pattern that was consistent across
agents and species (I2 = 5.3%, p = 0.39).
The prior systematic review observed that anti-toxin agents demonstrated greater benefit
when administered later in antibiotic treated models.[
] Consistent with that observation, in
the two studies here which administered anti-toxin agents earlier in some experiments and
later in others in the same species (AIG in experiments 10 to 17 and ETI-204 in experiments
24 to 27, both in rabbits) later treatment appeared associated with greater decreases in the
relative risk of death. The windows of treatment examined within each set of experiments
extended from 30 to 96h following challenge. Although these relationships between time and
the effects of anti-toxin agents on the log relative risk of death [slope in log(RR) (95% CI)] did
not reach significance for either AIG [-0.023 (-0.060, 0.015), p = 0.24] or ETI-204 [-0.042
10 / 17
Fig 3. This figure shows data from the six studies testing an anti-toxin agent in more than one experiment in the same species, as well as the
overall effects anti-toxin agents had on the relative risk of death (95%CI) (RR) across each of these 6 groups of experiments and the I2 and its
level of significance for the consistency of these overall effects. Individual RRs for experiments are shown by the squares and overall RRs are shown
by the inverted triangles. In the six studies shown, AVP-21D9 (AVP) was studied in four experiments in the guinea pig (Gpig); Anthrax Immune Globulin
(AIG) in eight experiments in the rabbit and two experiments in the monkey; and ETI-204 (ETI) in two experiments in the rabbit and two in the monkey. In the
five studies testing AIG or ETI, these agents had very consistent effects on the side of benefit across experiments (I2 = 0, p 0.56) in the same species. In
the four experiments testing AVP-21D9 in guinea pigs, the RR was also on the side of decreasing mortality but the I2 value was moderate although not
significant. Control treatments were antibiotics + the following; *no placebo was described; **human intravenous globulin (see Table 2).
(-0.104, 0.021), p = 0.19] alone, the trend for the two agents when combined approached
significance [-0.028 (-0.060, 0.005), p = 0.09].
Study sizes necessary to confirm the beneficial trends noted with
raxibacumab in experiment 9 and AIG in experiments 18
The two largest experiments testing Raxibacumab and AIG (180 and 336 initially infected ani
mals respectively), showed that these agents when combined with antibiotics during
established B. anthracis infections produced beneficial trends but not significant increases in
survival. Based on the numbers of animals that were originally infected, the number available
at the time of randomization, and the treatment effects observed in these prior experiments
(Table 2 and Fig 2), we determined the study sizes necessary to independently show that each
11 / 17
Fig 4. Based on the similarity of the effects of individual anti-toxin agents across experiments in the same species (shown in Fig 3), this figure
shows the overall effects of the anti-toxin agents on the relative risk of death (95%CI) (RR) across studies within the same species and the I2 and
its level of significance for the consistency of these overall effects. Individual RRs for studies are shown by the squares and overall RRs are shown by
the inverted triangles. AVP-21D9 in one study in mice and one study in guinea pigs, LFI, Raxibacumab, AIG and ETI-204 in eight studies in rabbits and
Raxibacumab, AIG and ETI-204 in three studies in monkeys were all associated with reductions in RR. Because the effects of the anti-toxin agents were
consistent across these studies in the same species (I2 25.3%), the effects of treatment on the RR averaged across all four anti-toxin agents and all four
species is shown by the diamond at the bottom of the figure. Control treatments were antibiotics + the following; *no placebo was described; **placebo was
noted but not described; #anti-toxin buffer; ##human intravenous globulin; @saline (see Table 2).
of these agents would result in a significant improvement in survival. In the case of
Raxibacumab, 104 animals per group would need to be randomized to control and anti-toxin groups to
have 80% power to detect a difference of 17% in mortality (35% vs 18%) at a 2-sided
significance level of 0.05. Based on the 58% mortality rate observed prior to randomization and
treatment in Experiment 9, in this proposed study 496 animals would therefore have to be initially
infected. Applying data similarly from Experiment 18 with AIG (81% mortality prior to
randomization), 1138 animals would need to be infected to provide 108 animals per group to be
randomized to control and anti-toxin groups. This number of randomized animals would
have an 80% power to detect a difference of 19% in mortality (61% vs 42%) at a 2-sided
significance level of 0.05.
12 / 17
In this analysis, only one B. anthracis anti-agent in a single experiment had a beneficial effect
on survival that reached significance (i.e., ETI-204 in a cynomolgus monkey model,
experiment 28). However, in 20 other experiments anti-toxin agents had effects on the side of benefit,
while in only 4 experiments were these effects on the side of harm. Furthermore, when
examined within species and across anti-toxin agents, treatment had consistent beneficial effects in
rabbits and cynomolgus monkey models in patterns that were significant or had a significant
trend. Finally, when examined across all agents in all species, anti-toxin agents reduced the
relative risk of death significantly and consistently.
On the one hand, these findings in antibiotic treated animal models employing live B.
anthracis challenge provide a basis for a therapeutic approach for this infection that
incorporates anti-toxin agents. They also add support to the CDC's recommendation to administer
approved anti-toxin agents along with conventional therapies in patients with evidence of
severe B. anthracis infection.[
] Failure to achieve significance in all but one of the individual
experiments analyzed may relate in part to the difficult conditions under which virulent B.
anthracis strains must be studied and the limitations those constraints put on sample sizes. In
addition, antibiotics can be effective treatment for B. anthracis infection and demonstrating
that anti-toxin agents add to those benefits also increases the size and complexity of the studies
needed to show significant benefit. Trends towards increased survival with individual agents
in this meta-analysis are consistent with some clinical observations that have been made with
anti-toxin agents. In contrast to the experience with AIG during the outbreak of soft tissue
infection in injection drug users, it has been noted that two of three isolated cases of
inhalational disease receiving AIG survived. This survival rate appears greater than might be
expected from past experience in patients not receiving anti-toxin therapy. Also, a historical
review of inhalational cases presenting in the US since 1900 suggested that use of anti-toxin
preparations was protective.[
] In both of these retrospective reports though, authors noted
that there were confounding factors potentially contributing to the apparent beneficial effects
of anti-toxin agents. It is possible though that anti-toxin agents are more effective in the
inhalational form of anthrax infection than in soft tissue disease. As noted in the introduction, FDA
approval of anti-toxin agents has been for inhalation infection only.
On the other hand, several aspects of the studies analyzed here weaken conclusions
regarding the benefits of this therapeutic approach. As with most of the experiments analyzed, the
one individual experiment in which an anti-toxin agent was significantly beneficial was
unblinded. In the five experiments that were blinded, only two were powered to possibly show a
convincing treatment effect (n = 76 for Raxibacumab in experiment 9, and n = 64 in
experiment 18 with AIG) and in both of these experiments treatment was only associated with trends
in improved survival (p = 0.087 and p = 0.135 respectively). Also, different from clinical trials,
none of the animal studies provided secondary endpoint data such as serial measures of
cardiopulmonary or other organ function to provide a basis and support for survival effects that
trended towards but did not reach significance. Although all of the studies analyzed included
antibiotic therapy, none of them employed the type of cardiopulmonary support with titrated
fluid, vasopressor, oxygen and ventilation therapies that critically ill patients receive. While
some animal studies have suggested that anti-toxin agents can augment hemodynamic support
in B. anthracis toxin challenged models, such data is not available in animal models of B.
] Failure to include these supportive therapies in infection models
probably relates to the fact that just as the virulence of B. anthracis limits the size of animal
experiments testing anti-toxin agents, it also constrains the use of these types of titrated
therapies that require invasive interventions and frequent and close measurements to adequately
13 / 17
administer. It is known however, that antibiotics and hemodynamic support have synergistic
beneficial survival effects during severe bacterial infection.[
] Such synergistic effects
could lessen or negate the benefit of anti-toxin agents. It has been noted that one of the reasons
selective anti-inflammatory agents (e.g., anti-TNF antibodies) were beneficial in preclinical
animal studies but not in clinical trials was because the preclinical experience did not account
for the types of cardiopulmonary support patients receive.[
] Finally, the results of analysis
here, although not reaching statistical significance, suggest that delaying administration of
anti-toxin agents and antibiotics following bacterial challenge in these models increased the
effectiveness of treatment. Presumably this delay allowed bacteria to replicate and toxin
concentrations to increase to pathogenic levels that anti-toxin exerted a beneficial effect on.
During an outbreak of B. anthracis infection, some patients will likely present early, when
antibiotics alone are sufficient therapy, while others will present so late that no therapy will be
effective. Although it has been proposed that circulating PA or toxin levels may predict
patients most likely to benefit from anti-toxin agents, proven methodology to reliably make
this prediction is not available.
Ultimately, absence of a clear and significant beneficial effect of anti-toxin agents in most
individual experiments in the present analysis may relate to the fact that while toxin
production is important in the pathogenesis of B. anthracis infection, other bacterial components
contribute as well. Growing evidence indicates that B. anthracis cell wall and its peptidoglycan
component can produce an injurious and lethal host inflammatory response.[
Proteases other than LT may also contribute to tissue injury and organ dysfunction during
] Therefore, adjunctive therapies targeting toxin production alone, while potentially
beneficial, may not have as great an impact on overall outcome as expected. Larger
experiments than the ones available for analysis here appear necessary to show conclusively that
targeting toxin during active infection improves survival.
Three of the anti-toxin agents we examined including AIG, Raxibacumab and ETI-204,
have received FDA approval. AIG and Raxibacumab have also been included in the Strategic
National Stockpile, and ETI-204 may be as well. The FDA publishes criteria that must be met
during testing to support its approval of agents that can only be investigated in animal models
of disease because human efficacy studies are not ethical or feasible, as was the case with these
] These criteria include among others randomization, blinding and prospective
statistical plans, endpoints, euthanasia criteria, and observation schedules. Although many of
these criteria were addressed in individual experiments, based on published methods and
correspondence with investigators, only one experiment testing AIG in rabbits (Experiment 18),
and none of those testing ETI-204 were blinded (Table 3). Absence of blinding weakens the
results of studies even when other design criteria are met.
Compared to a prior review of studies examining the effects of antitoxin agents in B.
anthracis challenged and antibiotic treated preclinical models which included 5 studies and
424 animals, the present one analyzed 9 studies and a total of 748 animals.[
] This difference
was due to our inclusion of studies assessing LFI and AVP-21D9 and of more recent studies
testing ETI-204. Different from the prior analysis, the present one also included a
meta-analysis of the retrieved studies.
It has been suggested that with a biologically plausible basis and the limited adverse effects
of anti-toxin agents in healthy volunteer safety studies, the beneficial trends anti-toxin agents
have demonstrated in animal B. anthracis infection models provide a basis for their
] Furthermore, a pressing need to rapidly establish a national stockpile of
adjunctive therapies for use during an outbreak of B. anthracis, may have justified the approval
and inclusion of AIG and Raxibacumab based on the studies analyzed here. At present,
approved anti-toxin agents should be made available for use in patients with invasive B.
14 / 17
anthracis infection that are not responding to conventional treatment. Although not examined
here, there is also data and a rational for the FDA indicated use of anti-toxin agents when the
underlying B. anthracis strain being treated is resistant to available antibiotic therapy or for
patients in whom antibiotic therapy is not available or contra-indicated[
However, for agents that can be tested clinically, it is expected that regardless of biologic
plausibility or safety data, they show statistical significance in clinical trials to justify their
approval and use. Stocking and maintaining anti-toxin agents in the SNS is costly.[
Given the weak effects anti-toxin agents demonstrated and the limitations of the studies we
have analyzed, it seems very important that additional testing of these agents in adequately
powered and designed studies be considered to clearly document their efficacy and support
their continued inclusion in the SNS. Ideally, although possibly not practical due to associated
risks, these studies would include in addition to antibiotics, the types of conventional
cardiopulmonary support and monitoring patients with severe B. anthracis infection would typically
receive. Such studies would be analogous to the phase 4 type testing often required of agents
approved based on clinical testing. Clearly such studies would be difficult and expensive. As
shown with the sample size calculation in the results, 496 and 1138 animals would need to be
challenged to provide sufficient numbers of subsequently randomized animals to confirm that
Raxibacumab and AIG respectively would produce significant improvements in survival when
added to antibiotics during life-threatening infection. To also test the effects of an anti-toxin
agent in groups receiving some sort of titrated cardiopulmonary support would add further to
these animal requirements. Despite the expense of such additional studies however, this cost
would not be as great as the continued maintenance of a national stockpile of anti-toxin agents
that are later found to be ineffective when administered to patients during a widespread
outbreak of B. anthracis infection.
S1 Text. Search terms and strategies.
S2 Text. PRISMA checklist for anthrax meta-analysis.
We thank Ms. Kelly Byrne for her editorial assistance with this manuscript.
Disclaimer: The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the National Institutes of Health.
Conceptualization: Peter Q. Eichacker.
Data curation: Wanying Xu, Lernik Ohanjandian, Junfeng Sun, Xizhong Cui, Judith Welsh,
Peter Q. Eichacker.
Formal analysis: Junfeng Sun, Peter Q. Eichacker.
Funding acquisition: Peter Q. Eichacker.
Investigation: Wanying Xu, Lernik Ohanjandian, Peter Q. Eichacker.
Methodology: Wanying Xu, Lernik Ohanjandian, Junfeng Sun, Peter Q. Eichacker.
15 / 17
Project administration: Peter Q. Eichacker.
Resources: Peter Q. Eichacker.
Supervision: Peter Q. Eichacker.
Validation: Wanying Xu, Lernik Ohanjandian, Junfeng Sun, Xizhong Cui, Peter Q. Eichacker.
Visualization: Wanying Xu, Lernik Ohanjandian, Xizhong Cui, Peter Q. Eichacker.
Writing ± original draft: Wanying Xu, Lernik Ohanjandian, Junfeng Sun, Peter Q. Eichacker.
Writing ± review & editing: Wanying Xu, Lernik Ohanjandian, Junfeng Sun, Xizhong Cui,
Dante Suffredini, Yan Li, Peter Q. Eichacker.
16 / 17
canines. Intensive Care Med Exp. 2015; 3: 9 https://doi.org/10.1186/s40635-015-0043-4 PMID:
1. Adalja AA , Toner E , Inglesby TV. Clinical management of potential bioterrorism-related conditions . N Engl J Med . 2009 ; 368 : 954 ± 962
2. Jernigan JA , Stephens DS , Ashford DA , Omenaca C , Topiel MS , Galbraith M , et al. Bioterrorism-related inhalational anthrax: the first 10 cases reported in the United States . Emerg Infect Dis 2001 ; 7 : 933 ± 944 https://doi.org/10.3201/eid0706.010604 PMID: 11747719
3. Jernigan DB , Raghunathan PL , Bell BP , Brechner R , Bresnitz EI , Butler JC , et al. Investigation of bioterrorism-related anthrax , United States , 2001 : epidemiologic findings . Emerg Infect Dis 2002 ; 8 : 1019 ± 1028 https://doi.org/10.3201/eid0810.020353 PMID: 12396909
4. Hendricks KA , Wright ME , Shadomy SV , Bradley JS , Morrow MG , Pavia AT , et al. Centers for disease and control prevention expert panel meetings on prevention and treatment of anthrax in adults . Emerg Infect Dis . 2014 ; 20 ( 2 ): e130687
5. Bower WA , Hendricks K , Pillai S , Guarnizo J , Meaney-Delman D , Centers for Disease Control and Prevention (CDC). Clinical framework and medical countermeasure use during an anthrax mass-casualty incident . MMWR Recomm Rep . 2015 ; 64 : 1± 22
6. Ohanjanian L , Remy KE , Li Y , Eichacker PQ . An overview of investigational toxin-directed therapies for the adjunctive management of Bacillus anthracis infection and sepsis . Expert Opin Investig Drugs . 2015 ; 24 : 851 ±865 https://doi.org/10.1517/13543784. 2015 .1041587 PMID: 25920540
7. Greig SL . Obiltoxaximab: First global approval . Drugs . 2016 ; 76 : 823 ±830 https://doi.org/10.1007/ s40265-016 -0577-0 PMID: 27085536
8. Huang E , Pillai SK , Bower WA , Hendricks KA , Guarnizo JT , Hoyle JD , et al. Antitoxin treatment of inhalation anthrax: a systematic review . Health Secur . 2015 ; 13 : 365 ±377 https://doi.org/10.1089/hs. 2015 . 0032 PMID: 26690378
9. Cui X , Nolen LD , Sun J , Booth M , Donaldson L , Quinn CP , et al. Analysis of anthrax immune globulin intravenous with antimicrfobial treatment in injection drug users , Scotland , 2009 ± 2010 . Emerg Infect Dis . 2017 ; 23 : 56 ±65 https://doi.org/10.3201/eid2301.160608 PMID: 27983504
10. DerSimonian R and Laird N. Meta-analysis in clinical trials . Controlled Clinical Trials 1986 ; 7 : 177 ± 188 . PMID: 3802833
11. Higgins JP , Thompson SG . Quantifying heterogeneity in a meta-analysis . Statistics in medicine. Jun 15 2002 ; 21 ( 11 ): 1539 ± 1558 . https://doi.org/10.1002/sim.1186 PMID: 12111919
12. R Core Team ( 2014 ). R: A language and environment for statistical computing . R Foundation for Statistical Computing , Vienna, Austria. URL http://www.R-project. org/.
13. Guido Schwarzer ( 2015 ). meta: General Package for Meta-Analysis . R package version 4 .1±0.http:// CRAN.R-project.org/package=meta
14. Viechtbauer W. ( 2010 ). Conducting meta-analyses in R with the metafor package . Journal of Statistical Software , 36 ( 3 ), 1 ± 48 .
15. Holty JE , Bravata DM , Liu H , Olshen RA , McDonald KM , Owens DK . Systematic review: a century of inhalational anthrax cases from 1900 to 2005 . Ann Intern Med. 2006 ; 144 : 270 ±280 PMID: 16490913
16. Barochia AV , Cui X , Sun J , Solomon SB , Migone TS , Subramanian GM , et al. Protective antigen antibody augments hemodynamic support in anthrax lethal toxin shock in canines . J Infect Dis . 2012 ; 205 : 818 ±829 https://doi.org/10.1093/infdis/jir834 PMID: 22223857
17. Remy KE , Cui X , Li Y , Sun J , Solomon SB , Fitz Y , et al. Raxibacumab augments hemodynamic support and improves outcome during shock with B. anthracis edema toxin alone or together with lethal toxin in
18. Natanson C , Danner RL , Reilly JM , Doerfler ML , Hoffman WD , Akin GL , et al. Antibiotics versus cardiovascular support in a canine model of human sepsis shock . Am J Physiol . 1990 ; 259 : H1440 ±1447 PMID: 2240243
19. Rhodes A , Evans LE , Alhazzani W , Levy MM , Antonelli M , Ferrer R , et al. Surviving sepsis campaign: International guidelines for management of sepsis and septic shock: 2016 . Intensive Care Med . 2017 ; 45 : 486 ± 552
20. Qiu P , Li Y , Ding Y , Weng J , Banks SM , Kern S , et al. The individual survival benefits of tumor necrosis factor soluble receptor and fluid administration are not additive in a rat sepsis model . Intensive Care Med . 2011 ; 37 : 1688 ±95 https://doi.org/10.1007/s00134-011-2324-z PMID: 21922303
21. Iyer JK , Khurana T , Langer M , West CM , Ballard JD , Metcalf JP , et al. Inflammatory cytokine response to Bacillus anthracis peptidoglycan requires phagocytosis and lysosomal trafficking . Infect Immun . 2010 ; 78 : 2418 ±2428 https://doi.org/10.1128/IAI.00170-10 PMID: 20308305
22. Coggeshall KM , Lupu F , Ballard J , Metcalf JP , James JA , Farris D , et al. The sepsis model: an emerging hypothesis for the lethality of inhalation anthrax . J Cell Mol Med . 2013 ; 17 : 914 ±920 https://doi.org/10. 1111/jcmm.12075 PMID: 23742651
23. Qiu P , Li Y , Shiloach J , Cui X , Sun J , Trinh L , et al. Bacillus anthracis cell wall peptidoglycan but not lethal or edema toxins produces changes consistent disseminated intravascular coagulation in a rat model . J Infect Dis . 2013 ; 208 : 978 ±89 https://doi.org/10.1093/infdis/jit247 PMID: 23737601
24. Mukherjee DV , Tony JH , Kim KS , Ramarao N , Popova TG , Bailey C , et al. Bacillus anthracis protease InhA increases blood brain barrier permeability and contributes to cerebral hemorrhage . PLoS One . 2011 ; 6 : e17921 https://doi.org/10.1371/journal.pone. 0017921 PMID: 21437287
25. US Dept. of HHS FDA, Center for Drug Evaluation and Research and Center for Biologics Evaluation and Research. Product development under the animal rule; guidance for industry . October 2015 .
26. US Dept. of HHS FDA . Raxibacumab injection prescribing information . December 2012 . https://www. accessdata.fda.gov/drugsatfda_docs/label/2012/125349s000lbl.pdf. Accessed July 16 , 2017
27. US Dept. of HHS FDA. Anthim (obiltoxaximab) injection prescribing information . March 2016 . https:// www.accessdata.fda.gov/drugsatfda_docs/label/2016/125509lbl.pdf. Accessed July 16 , 2017 .
28. Roos R . Anthrax countermeasures better than in 2001, but work remains . October 18 , 2011 . www. cidrap.umn.edu/news. . ./10/anthrax-countermeasures-better-2001 - work-remains. Accessed March 21 , 2017
29. US Government adds two drugs that treat inhalational anthrax to Strategic National Stockpile . November 14 , 2015 . http://outbreaknewstoday.com /us-government-adds-two-drugs-that-treat-inhalationalanthrax-to-strategic- national- stockpile- 38354 /. Accessed March 21, 2017