Research Article: The Role of Immune Correlates and Surrogate Markers in the Development of Vaccines and Immunotherapies for Plague

Date Published: September 29, 2012

Publisher: Hindawi Publishing Corporation

Author(s): E. D. Williamson.


One of the difficulties in developing countermeasures to biothreat agents is the challenge inherent in demonstrating their efficacy in man. Since the first publication of the Animal Rule by the FDA, there has been increased discussion of potential correlates of protection in animal models and their use to establish surrogate markers of efficacy in man. The latter need to be relatively easy to measure in assays that are at least qualified, if not validated, in order to derive a quantitative assessment of the clinical benefit conferred. The demonstration of safety and clinical benefit is essential to achieve regulatory approval for countermeasures for which clinical efficacy cannot be tested directly, as is the case for example, for biodefence vaccines. Plague is an ancient, serious infectious disease which is still endemic in regions of the modern world and is a potential biothreat agent. This paper discusses potential immune correlates of protection for plague, from which it may be possible to derive surrogate markers of efficacy, in order to predict the clinical efficacy of candidate prophylaxes and therapies.

Partial Text

The ancient disease of plague is still present in endemic regions of the modern world and results in approximately 3,000 reported cases each year [1]. Plague is a flea-vectored infection caused by the Gram-negative bacterium Yersinia pestis, a potential biothreat agent. Originally an enteric pathogen, Y. pestis is thought to have evolved from the enteropathogen Y. pseudotuberculosis [2] as a flea-vectored, enzootic infection. Fleas feed on infected rodents and then transmit bacteria to a susceptible mammal by flea bite. Man is an accidental host in this cycle, but if bitten can contract bubonic plague, a serious infection if not treated promptly before the individual becomes symptomatic. A secondary pneumonic plague can develop in an individual suffering from bubonic plague, and this is of even greater concern, since Y. pestis bacteria are highly transmissible in aerosolised form between unprotected individuals in close contact, with the potential for epidemic spread [3].

Y. pestis produces a range of antigens and virulence factors, three of which have known protective efficacy as candidate subunit vaccines: F1-antigen [4], V-antigen [5], and Yersinia secretory factor F (YscF) [6]. These three proteins are virulence factors when secreted by Y. pestis during infection. F1 antigen is a capsular protein with antiphagocytic properties [7], whilst the V-antigen is a regulatory protein in the type three secretion system (TTS) utilised by the bacterium to gain access to and deliver other cytotoxic and anti-phagocytic Yersinia outer proteins (Yops) to host cells [8]. V-antigen occurs both within the bacterium, where it has some regulatory function in the initiation of the TTS process, and also at the tip of the injectisome [9]. The columnar structure of the injectisome is comprised of YscF [6].

Vaccination and postexposure therapy are both options to protect individuals against exposure to Y. pestis. There has been a series of killed whole cell vaccines (KWCVs) for plague, starting with Haffkine’s vaccine in the late 1800s [21], through to the currently available KWCV produced by the Central Serum Laboratories (CSL), which comprises a suspension of heat-killed Y. pestis (>109/mL). Whilst KWCVs are efficacious against bubonic plague, epidemiological evidence suggests that they have little protective efficacy against pneumonic plague [15, 20, 22, 23]. F1 appears to be the key protective antigen in KWCV formulations, which thus do not protect against F1−  Y. pestis [4, 5]. Unlike the KWCVs, the rF1-/V-antigen combination has been demonstrated to protect both mice and macaques against pneumonic plague [24–27], representing a significant advance in candidate countermeasures for plague infection. Different presentations of F1/V have been studied including DNA vaccines [28], oral formulations [29], and live vaccine-vectored expression from, for example, salmonella strains [30].

The early detection and administration of antibiotic therapy within 18–24 hours following suspected exposure to Y. pestis and before the appearance of symptoms, is critical for the successful treatment of plague. The recommended antibiotic regimen comprises a high dose of gentamicin intravenously (5 mg/kg intravenously once a day) or the equivalent dosage of streptomycin, ciprofloxacin, gentamicin, or doxycycline for 10 days [35]. Chloramphenicol may also be used if plague meningitis is suspected [36]. As the patient responds to treatment, it may be possible to change to the oral route of administration of the preferred antibiotic. It is essential that antibiotic treatment is adjusted dependent on the antibiotic susceptibility of the infecting organism in culture, particularly if deliberate use of an antibiotic- resistant strain is suspected.

Since standard Phase III clinical efficacy studies are not feasible to carry out with plague and other serious human diseases, on both ethical and practical grounds (too few naturally occurring cases as well as outbreaks which are spasmodic), it is essential to establish satisfactory animal models of the disease. These, in turn, can be used to assess the efficacy of candidate vaccines and therapies and to identify correlates of protection. Robust animal models of plague infection which authentically represent the human disease syndrome are the objective and models have been established in standard laboratory animal species (mouse, rat, rabbit, and macaque), as well as nonstandard species such as the black-footed ferret (reviewed in [17]).

Immunisation of mice with either [4, 5] or both F1 and V proteins [15, 16] was protective against plague and a titre of specific antibody correlated with protection. Whilst the development of an IgG titre to these proteins correlates with protection as observed in mice [46], guinea pig [39], nonhuman primate (NHP) [40] and inferred from passive transfer studies with clinical trial serum [41], neutralizing antibody alone does not describe the entire mechanism of protection against this virulent pathogen [17]. Researchers from several groups have reported a strong CMI response to be operating [38, 47, 48] and in response to an alhydrogel-adsorbed formulation of the rF1 + rV vaccine, this generally has been observed to be a CD4+ Th2-biased CMI response [46]. However, alternative formulations of the rF1 + rV vaccine in which different adjuvants have been substituted for alhydrogel have also been demonstrated to induce protective immunity in a CD4+ Th1-biased setting [49, 50]. Additionally, strains of mice with targeted gene deletions affecting antibody production by B cells (μMT B cell knockouts or SCID/beige) or the nature of the Th cell response including Stat 4/Stat 6 knockouts and IL4/IL10 knockouts have been studied [17, 49–51]; rF1 + rV-immunised Stat-4-deficient mice, which have low levels of IFNγ production, were found to be poorly protected from Y. pestis challenge, despite producing similar antibody titres to rF1 + rV as the intact controls [49]. Moreover, the rF1 + rV vaccine was able to induce protective immunity in IL4 knockout mice despite a Th1-biased environment operating in these animals [50]. Indeed, Stat-4-mediated immune mechanisms leading to a Th1 response were found to be essential for protection, whereas Stat 6/Th2-mediated responses were not [49]. Thus for the rF1 + rV vaccine, the induction of specific antibody neutralising the F1 and V antigens is a significant immune correlate of protection; however the supporting CMI response is not necessarily Th2-polarised and indeed the operation of Th1 mechanisms during infection appears to be essential for full protection and recovery [17].

Based on these data on immune correlates and on the immunoanalysis data published to date on samples from clinical trial volunteers immunised with the rF1/V subunit vaccine [41], it is possible to identify several serological surrogate markers of efficacy. These may include the inhibitory-activity of human immune serum on the cytotoxicity of V-antigen secreted from Y. pseudotuberculosis. Qualitative data from this assay have been published [40], however, the assay has subsequently been improved and made quantitative [54]. It has been demonstrated that decreased caspase-3 activity in macrophages exposed to immune NHP serum correlated with increased survival of those NHP to Y. pestis infection.

Much work is ongoing to identify statistically valid immune correlates of protection for plague, particularly since a clinical demonstration of efficacy is not possible. This has required the development of nonclinical models which authentically represent the human infection. As far as possible, the immune correlate should be demonstrated in more than one nonclinical model. Whilst the immune correlate(s) may not describe all the immune mechanisms operating in protection against a pathogen, they should be reproducibly consistent between the selected nonclinical models and the clinic and should be quantitative, to assess the likely benefit to be conferred on the vaccinee. With an increasing understanding of the molecular basis of pathogenicity and of the innate and adaptive immune response mechanisms required to counter Y. pestis, immune correlates of protection are being identified and reported and this in turn will expedite the development of next-generation vaccines and immunotherapies.




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