Resurgence of pertussis calls for re-evaluation of pertussis animal models

Figures & data

Figure 1. Changes in pertussis epidemiology and evolution of Bordetella pertussis since the introduction of mass vaccination programs.

Pertussis changed from a classical childhood disease in the prevaccination era to a disease that affects all ages except recently vaccinated groups. B. pertussis resurged in well-vaccinated populations largely due to waning of immunity and pathogen adaptation (left panel). Genomic and antigenic shifts in vaccine antigens Ptx, Prn and Fim occurred in time, causing an increased antigenic mismatch between vaccines in use and circulating strains (right panel).

Table 1. Brief summary of features of pertussis animal models compared with human infection and immunity.

Animal species	Model	B. pertussis strain	Infection and shedding	Clinical signs postinfection	Surrogate of protection	Whole cell vaccines	Acellular vaccines	Vaccine candidates	Remarks
Mouse	Kendrick test (ic-MPT) [17]	Only strain 18323 is infectious and virulent clinical isolates are not^†	Intracerebral (10^7–10^8 CFU)	Lethal due to meningitis and no signs of classical disease	Concentration of antibody against outer membrane proteins at day 14 is an indication for survival [102,103]	Mandatory release test. Relationship between WCV efficacy and potency but poor in reproducibility[104]	Not protective, only in suckling mouse test [18]	OMV are protective. 92 kDa/38 kDa OMP ratio is indicative for protectiveness of vaccine under test [95]	In use to demonstrate consistency in production and stability of WCV
Mouse	Respiratory challenge model (RMCM and in-MCM) [20,21]	Laboratory strains [102] and clinical isolates occasionally [34]. 18323 more rapidly cleared than other strains^†	Intranasal or aerosol (10^3–10^7 CFU); shedding in ±6 weeks	No signs of classical pertussis, only weight retardation	No direct correlation of protection between antibody and/or cellular immune responses and clearing of the lungs	PtxA1/Prn2 WCV are more protective than Ptx2/Prn1 WCV [97]	ACV protectiveness in mice related to ACV efficacy in children [22] but could not be confirmed in an international collaborative study	OMV [122–124] and BPZE1 [118–121] are protective. Relationship between anti-Ptx and FHA antibody responses and protection [90]	Mostly used as research model and suggested for preclinical studies (WHO)
Mouse	Immuno-genicity test	Not applicable	Not applicable	Not applicable	Antibody responses are indicative for protection	Low in anti-Ptx, FHA and Prn titers and high in anti-OMP titers [102–104]	Release test for ACV in Europe and America but no direct correlation with protection in man [19–22]	No reports	In use to demonstrate consistency in production and stability of ACV
Rat	CRM [91–94,98]	Mainly 18323 and Tohama 1 embedded in agarose	Intrabronchial (10^5–10^8 CFU); shedding in ±3 weeks	Resembles human disease including paroxysmal coughing	No direct correlation of protection between antibody and/or cellular immune responses and clinical disease	Protects against disease and not infection as in humans	Protects against disease and not infection as in humans	No reports	Interesting as preclinical model
Rabbit	Respiratory infection model [101]	Multiple clinical isolates	Intranasal and intratracheal, (10^6–10^10 CFU); shedding in 2–10 months	Fever but no classical sign of disease. Interference by B. bronchiseptica infections^†	Not established	No reports of vaccination studies			Switch of serotypes during infection observed
Piglet	Respiratory infection and challenge model [88,89]	Tohama 1 embedded in agarose	Intrapulmonary (10^9 CFU); shedding in 3–7 weeks	Resembles classical disease but no paroxysmal coughing or whoops	Not established	Not a suitable model to study parenteral vaccine efficacy. Only newborn piglets younger than 4 weeks are sensitive to pertussis infection. Piglets older than 4 weeks produce host defense proteins that protect against pertussis but not parapertussis infections			Interesting to study maternal immunity and neonatal immunization
Nonhuman primates	Respiratory infection models [96,99–101,108]	18323 and multiple clinical isolates	Oral, intranasal and intratrachial (up to 10^11 CFU); shedding in 3–7weeks	Paroxysmal coughing or classical whooping cough; interference by bronchiseptica infections^†	Not established	No reports of vaccination studies			Mostly used to study transmission and infection
Human	Respiratory infection and immunity	Circulating B. pertussis strains	Respiratory (10^2 CFU); shedding in 8–12 weeks in infants and 4–8 weeks in adults	Classical whooping cough (infants) or moderate respiratory disease (adults)	Both acquired humoral and cellular immune mechanisms are implied, but correlates of protection are unknown; anti-Ptx IgG titer is indicative for recent infection	Efficacy highly dependent on quality of WCV in use and estimated between 36 and 96%; efficacy related to potency in Kendrick test	Efficacy depends on formulation of ACV and is estimated between 59 and 85%. None of the clinical trials yielded a direct correlation between antibody titers and protection	Not yet tested	Correlates or surrogates of protection are not established (in humans nor in animals) due to involvement of multiple immune effector mechanisms against multiple antigens

^†Nonpublished observation of authors or personal comments of other scientist.

ACV: Acellular vaccine; CRM: Coughing rat model; FHA: Filamentous hemagglutinin; ic-MPT: Intracerebral mouse protection test; in-MCM: Intranasal mouse challenge model; OMP: Outer membrane protein; OMV: Outer membrane vesicle; RMCM: Respiratory mouse challenge model; WCV: Whole cell vaccine.

Table 2. Re-evaluation of pertussis animal models in various phases of vaccine research and development.

Discovery phase
Basic animal models	Infection model to study pathogenicity of Bordetella pertussis strain variants and component of challenge model	Covariates • Animal species: monkey, rat or mouse† and age of animal • B. pertussis strain variants: old and recent clinical isolates • Route of infection: intranasal, aerosol, intratracheal or intrabronchial • Dose of infection: lethal or nonlethal dose that induces disease features End points • Disease features: lung colonization, lung pathology, coughing, leukocytosis and/or weight retardation, mortality • Time points of sampling to monitor course and severity of infection
	Vaccination model to screen immunogenicity of vaccine preparations and component of challenge model	Covariates • Animal species: mouse, rat, guinea pig, rabbit and/or monkey† • Vaccine preparations (laboratory scale) derived from B. pertussis strain variants • Route of immunization: intramuscular, subcutaneous, intraperitoneal, intranasal or alternative • Schedule of immunizations: dose (0.3 human dose/kg) and number of and time intervals between vaccinations End points • Antigen-specific antibody, T- and B-cell responses • Level of immunogenicity
	Challenge model combining vaccination and infection model to screen for protectiveness of vaccine leads	Covariates • Animal species: monkey, rat or mouse† and age of animal • Vaccination: route depending on animal species and vaccine type; serial dilution of vaccine dose and schedule to be optimized • Infection: route depending on animal species; dose of challenge inoculum and time interval between vaccination and challenge to be optimized End points • Survival • Single or multiple disease features (see basic infection model)
Host immune responses	Innate immunity to (components of) B. pertussis strain variant(s)	Basic model • Infection or vaccination model optimized for B. pertussis strain variants, source and time of host specimen sampling End points • (Early) gene signatures of lymphoid, lung and other tissues • Proteome signatures (e.g., chemokines, cytokines, receptor signaling or marker expression) • Cellular signatures (shifts in immune cell subsets) • Immunomodulation (activation or suppression of particular immune functions)
	Adaptive immunity to (components of) B. pertussis strain variant(s)	Basic model • Infection or vaccination model optimized for B. pertussis strain variants, source and time of host specimen sampling End points • Specificity, level and functionality of antibody responses • Specificity, magnitude and differentiation of B-cell responses (e.g., memory B cells vs plasma cells) • Magnitude, breadth and differentiation of T-cell effector and memory immune responses
Preclinical phase
Selection of vaccine candidates	Cross protection of vaccine candidates against B. pertussis strain variants	Basic model • Challenge model optimized for type of vaccine candidate and B. pertussis strain variants for infection, time interval between vaccination and infection End points • Level of protection against one or multiple disease features • Level of cross protectiveness against B. pertussis strain variants
Scientific substantiation of mechanisms of protection	Characterization of immunity after vaccination	Basic model • At least two vaccination models (animal species) adjusted to the type of preclinical (up-scalable) vaccine candidate and its delivery vehicle, with short- and long-term follow-up of immune responses End points • Features of antigen-specific host (innate and) adaptive immune responses and effector mechanisms • Duration of immune responses (or waning of immune responses)
	Optimization of vaccine formulation for long-term duration of protection against B. pertussis strain variants	Basic model • Challenge model reflecting waning of vaccine-induced immunity using variable vaccine formulations (i.e., adjuvants, solvents, preservatives or stabilizers) End points • Type and concentration of adjuvant to sustain protection • Identification, quality and quantity of innate and adaptive immune parameters that correlate with short- and/or long-term protection
Postmarketing surveillance
Monitoring vaccine failure	Periodical control of effectiveness of licensed pertussis vaccines against circulating B. pertussis strains	Basic model • Challenge model suitable for use of licensed pertussis vaccine, for use of emerging B. pertussis strain(s) as challenge inoculum, and to sense (rapid) loss of vaccine-induced protection End points • Monitoring of short- and long-term vaccine-induced protection against one or multiple disease features • Duration of survival of B. pertussis strain(s) in the immune host

Covariates and end points: choices depend on the aim of the study and take into account B. pertussis strain or allelic variants.

^†Depending on availability of immunoreagents, knowledge of and experience with the model, and/or ethical considerations.

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