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Drug Discovery Today: Disease Models Vol. 3, No. 1 2006

Editors-in-Chief

Jan Tornell – AstraZeneca, Sweden

Denis Noble – University of Oxford, UK

Infectious diseases

Can animal models predict protectionprovided by meningococcal vaccines?Michelle Finney, Denise Halliwell, Andrew R. Gorringe*Health Protection Agency, Centre for Emergency Preparedness and Response, Porton Down, Salisbury, UK SP4 0JG

Animal infection models are valuable for the develop-

ment and assessment of meningococcal vaccines

because interactions of the organism with the entire

immune system can be assessed. There is no ideal

animal disease model that mimics the course of human

disease but the two most widely used are intraperito-

neal (IP) infection of adult mice or infant rats. Recent

developments using transgenic mice expressing human

CD46 give hope that improved models will be devel-

oped that better predict vaccine efficacy.

*Corresponding author: A.R. Gorringe (andrew.gorringe@hpa.org.uk)URL: http://www.hpa.org.uk/cepr

1740-6757/$ � 2006 Elsevier Ltd. All rights reserved. DOI: 10.1016/j.ddmod.2006.03.002

Section Editors:Enitan Carrol – Malawi-Liverpool-Wellcome Trust ClinicalResearch, Blantyre and Liverpool, Malawi and UKAndrew Riordan – Birmingham Heartlands Hospital,Birmingham, UK

Introduction

In vitro serological assays for the assessment of meningococ-

cal vaccines include ELISA, serum bactericidal antibody

assay (SBA) and opsonophagocytosis [1]. However, it is

only in animal models of infection that the interactions of

the pathogen with whole tissues and both the humoral

and cellular immune systems can be assessed. Animal

disease models for meningococcal disease have a long his-

tory [2,3]. Humans are the only natural hosts for Neisseria

meningitidis owing to the specificity of a range of surface

proteins that interact with the host. These include the

interaction of type IV pili with CD46 and Opa and Opc with

human CEACAM1 (CD66). In addition, the meningococcal

iron uptake systems can obtain iron only from human

iron transport proteins. In humans the majority of infections

lead to asymptomatic nasopharyngeal carriage and it is

only in rare cases that infection progresses to invasive disease

and a further difficulty in modelling meningococcal

disease is the complex pathogenesis of the progression from

nasopharyngeal mucosal carriage to septicaemia and/or

meningitis.

Mouse intraperitoneal challenge model

Intraperitoneal (IP) infection of mice has been used as a

meningococcal disease model since the 1930s. Initially the

infection was enhanced by the co-administration of mucin

but in more recent years iron dextran or human transferrin

has been used as an exogenous iron source. It is clear that this

model does not mimic the natural pathogenesis of menin-

gococcal disease but it does provide a model of bacteraemic

disease that is useful for vaccine assessment. Large numbers

of bacteria can be recovered from the blood of infected

animals and a lethal infection can be established. Disease

progress can be monitored by observing the health of the

mice with humane termination before death and/or by

enumeration of viable meningococci in blood samples

recovered at a set time after challenge. In over 15 years of

experience with this model we have found it to be reprodu-

cible and can be used to compare the virulence of meningo-

coccal strains, including knockout mutants [4]. The model

can be used to assess active protection following immuniza-

tion with a candidate vaccine and, in addition, passive

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Drug Discovery Today: Disease Models | Infectious diseases Vol. 3, No. 1 2006

Table 1. Comparison summary table

Mouse IP challenge models Infant rat IP challenge models Intranasal infection models

Pros Interactions of the pathogen with the

entire immune system are assessed.

Interactions of the pathogen with the

entire immune system are assessed.

Interactions of the pathogen with the

entire immune system are assessed.

Allows assessment of active or

passive protection.

Allows passive protection studies.

Lower inocula required.

Development of bacteraemia and

meningitis can be determined.

Uses the natural route of infection.

Reproducible.

Can differentiate the colonizing

potential of strains.

Can differentiate the virulence

of strains.

Protection can be observed in

the absence of bactericidal

antibody.

A panel of challenge strains

can be used.

Cons Animals respond differently

to humans.

Animals respond differently

to humans.

Animals respond differently

to humans.

The only natural host is humans.

The route of infection is different

from human disease.

The only natural host is humans.

The route of infection is different

from human disease.

Can only be used for passive protection

studies.

Rat passage of challenge strains is

required, some case isolates are

not virulent.

Duration of bacteraemia is short and

mortality is low.

Mice have to be humanized or

treated with various agents to

allow nasal colonization.

Colonization is most

effective in infant animals

which are less suitable for

active immunization studies.

Bacteraemia can follow a lung

infection which is not the human

pathogenesis of the disease.

High challenge doses required.

An exogenous iron source

is required.

Best use

of model

Assessment of protection following

active immunization with

candidate vaccines.

Demonstration of passive protection

with antibodies raised against

candidate vaccines.

Studies on the pathogenesis

of meningococcal infection.

Access to

the models

Literature/collaboration. Literature/collaboration. Literature/collaboration.

Relevant

patents

None None None

References [2–6] [2,3,7–9] [2,3,10,11]

protection provided by immune sera administered by IP

injection before or at the time of challenge can be assessed

(Table 1).

An understanding of early life responses to meningococcal

vaccines is a key area which requires investigation to develop

effective vaccines for this age group. Immunization of murine

neonates with meningococcal OMVs at 7 and 14 days after

birth provided significant protection against meningococcal

bacteraemia [5] and the use of various adjuvants to enhance

bactericidal antibody responses has been examined [6].

Infant rat intraperitoneal infection model

An alternative animal disease model that has proved useful

for vaccine assessment involves IP infection of infant rats

(Table 1). This has been used to assess passive protection

provided by animal [7,8] and human [9] immune sera raised

against several antigens. This model has the advantage of not

requiring an exogenous iron source. However, it can only be

used to assess passive protection. Although often considered a

78 www.drugdiscoverytoday.com

single model, this has been performed in two distinct ways.

As described in references [2,3,7,9] relatively high challenge

doses of 107 rat-passaged meningococci are administered and

protection is assessed by viable counts of meningococci in

blood samples obtained either 3 h [2] or 6 h [7,9] post infec-

tion. This short timescale is likely to demonstrate protection

with antibodies that have clear bactericidal activity but other

protective mechanisms might be missed. An alternative pro-

tocol has been used in the Granoff laboratory [8]. Here, a

much smaller dose (103–104) of rat-passaged meningococci is

given by IP injection. Blood samples are then obtained 18 h

after infection and viable counts of meningococci compared

in groups of rat pups that received immune or control sera. In

animals that received control sera the septicaemia progressed

to approximately 105 meningococci/ml of blood.

Intranasal infection models

Several groups have attempted to develop meningococcal

disease models where the infection is initiated by intranasal

Vol. 3, No. 1 2006 Drug Discovery Today: Disease Models | Infectious diseases

(IN) instillation of N. meningitidis (Table 1). Mackinnon et al.

[10] infected 4–6-day-old mice with high doses (1010 CFU) of

N. meningitidis, co-administered with iron dextran or human

transferrin. Progression to bacteraemic disease was observed

with either iron source but it was found that the infection

proceeded from nasal colonization to pneumonia and then

bacteraemia, in contrast to human meningococcal disease,

where pneumonia is uncommon. This model was useful for

the differentiation of the virulence of a panel of meningo-

coccal strains differing only in capsule expression and LOS

immunotype. It was demonstrated that, as in human disease,

capsule expression was the primary virulence determinant

with expression of the L3,7,9 LOS immunotype, a secondary

determinant. However, this model required an iron supple-

ment and high challenge doses and was found to be difficult

to reproduce following a change in the source of the mice

used. Kyungcheol et al. [11] have described an intranasal

infection model of outbred adult mice that included daily

administration of iron dextran. They were able to establish

colonization in the nasopharynx of mice given 107 N. menin-

gitidis and found approximately half of the animals inocu-

lated remained colonized 13 days after the initial inoculation.

The model was used to demonstrate decreased colonization

capabilities of several gene-deletion mutants.

Infection models using humanized mice

The shortcomings of the above disease models have shown

the crucial requirement for humanized mouse models.

Johansson et al. [12] have used ‘humanized’ transgenic mice

expressing human CD46 in potential target tissue. Intraper-

itoneal challenge experiments demonstrated that CD46 mice

were susceptible to meningococcal disease in the absence of

an exogenous iron source, providing an important animal

model for studying pathogenesis as well as for assessment of

meningococcal vaccines. In addition, crossing of the blood–

brain barrier by bacteria occurred in CD46 transgenic mice,

but not in nontransgenic mice. Intranasal challenge of CD46

transgenic mice required piliated bacteria for development of

disease, suggesting that CD46 facilitates pilus-dependent

interactions at the epithelial mucosa. In this study, the mice

had to be pretreated with antibiotics for 2 days to develop

meningococcal disease after intranasal infection, demon-

strating the importance of the normal flora in colonization

resistance. Intranasal challenge with wild-type meningococci

caused 15% mortality in CD46 mice, but none in control

mice. Recently, Johansson et al. [13] have evaluated early

immune responses following IP infection with N. meningiti-

dis. Stimulation of TNF, IL-6 and IL-10 was stronger in CD46

transgenic mice and more clearly resembled human

responses. Also, bacterial clearance in the blood of CD46

mice started at later time points and neutrophil numbers

in blood were lower compared with wild-type mice. Most of

the responses were impaired or absent when LPS-deficient

meningococcal were used, illustrating the importance of LPS

in the early responses to meningococcal infection. Taken

together, these data demonstrate an important role of

CD46 in meningococcal disease and argue that the huma-

nized CD46 mouse model is a useful novel system to evaluate

potential vaccine candidates and therapeutic regimes.

Can animal models predict protection provided by

meningococcal vaccines?

Capsular polysaccharide conjugate vaccines

It is clear from research with plain polysaccharide vaccines

and from the experience of the introduction of serogroup C

conjugate vaccines in UK, that the presence of serum bacter-

icidal activity correlates with vaccine efficacy. Animal models

have also been used to demonstrate protection provided by

these vaccines and protection can be demonstrated by active

or passive immunization of adult mice or passive protection

in infant rats. Example data with the adult mouse IP chal-

lenge model are presented in Reddin et al. [14] where it can be

seen that immunization with serogroup C polysaccharide

provided some protection against a lethal challenge with

serogroup C N. meningitidis and complete protection against

a high challenge dose (7 � 108 CFU) was provided by an

experimental conjugate vaccine. It might be that animal

models are more sensitive in the detection of a protective

immune response than the standard bactericidal antibody

assay because it has been shown that 19 of 54 human sera

with bactericidal titres of <1:4 conferred protection in an

infant rat infection model [15].

OMV vaccines

OMV vaccines produced in Norway and Cuba have been

assessed in efficacy trials and shown to provide protection

to older children and adults but not to children under 4 years

[1]. Active and passive protection provided by these vaccines

has been shown in mice and passive protection in infant rats

but the majority of studies have used a challenge strain

homologous to the specific vaccine strain. Bactericidal activ-

ity is induced by OMV vaccines but this appears dependent

on the cross-reactivity of antibodies against the immunodo-

minant serosubtyping antigen PorA. The highest bactericidal

activity is determined using human sera and a homologous

target strain and decreased or abolished activity is seen with

heterologous or PorA negative target strains [1,16]. It is

important that new OMV vaccines are assessed using a panel

of challenge strains that represents the diversity of disease-

causing strains. As demonstrated for polysaccharide

responses, OMV vaccines have provided protection in an

animal model in the absence of a detectable bactericidal titre.

OMVs prepared from N. lactamica did not produce detectable

bactericidal antibodies in mice but provided protection

against diverse meningococcal strains in a mouse IP active

protection model [17], indicating that either the bactericidal

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Drug Discovery Today: Disease Models | Infectious diseases Vol. 3, No. 1 2006

Outstanding issues

� A better understanding of the correlates of protection for human

meningococcal disease.

� The use of a panel of challenge strains representative of the major

disease-causing clones.

� The development of more relevant models including the use of

transgenic mice (e.g. CD46) to more closely mimic human

meningococcal disease.

assay is insufficiently sensitive or other protective mechan-

isms, such as phagocytic killing, are important. Toropainen

et al. [9] have used an infant rat passive protection model to

evaluate human sera for protective immunity provided by an

OMV vaccine. Sera from adult volunteers immunized with

the OMV vaccine reproducibly reduced bacterial counts in

the blood and cerebrospinal fluid. They used a range of

immunoassays to dissect the responses that correlated with

infant rat protection [18] and the only clear correlation was

with IgM antibody specific for the serogroup B capsular

polysaccharide.

Isolated antigens

A range of antigens have been considered as candidates for

inclusion in vaccines to protect against serogroup B menin-

gococcal disease and many have shown protection in mouse

and rat disease models. Many antigens have been selected

because they induce bactericidal antibodies and, where

tested, these generally provide protection in animal models.

In addition to OMVs from N. lactamica, there are isolated

antigens that provide clear protection in mouse or rat models

in the absence of bactericidal antibodies. Two examples are

antibodies against genome-derived antigen (GNA) 2132

which provided passive protection against meningococcal

challenge in infant rats [8] and immunization with transfer-

rin-binding protein A provided protection against IP chal-

lenge in adult mice [19].

Model translation to humans

Animal models have long been used for preclinical assess-

ment of efficacy and safety for vaccine candidates designed

for human use. Results from animal studies cannot, however,

be totally predictive of what will happen in humans as the

protective mechanisms in humans are still not fully under-

stood; there is still the possibility that a successful vaccine

candidate from animal model studies might give unexpected

results when applied to humans. Similarly, lack of a bacter-

icidal response does not necessarily predict disease suscept-

ibility and it has been suggested that other mechanisms, such

as opsonophagocytic killing, might confer protection but this

has yet to be established.

Links

� Health Protection Agency, infections http://www.hpa.org.uk/

infections/topics_az/meningo/menu3.htm

� Who Heath Organization, Health topics, meningitis http://

www.who.int/topics/meningitis/en/

� Meningitis Trust http://www.meningitis-trust.org/

� Neisseria.org, a web resource for the Neisseria community http://

neisseria.org/

� Meningitis Research Foundation http://www.meningitis.org/

80 www.drugdiscoverytoday.com

Conclusion

The development of disease models that have relevance to

natural meningococcal infection and disease presents great

challenges but can have value in assessing protection pro-

vided by candidate vaccines. Animal models for meningo-

coccal disease can provide valuable information for studies of

pathogenesis and vaccine development. However, all the

available models have shortcomings and it is clear that a

greater understanding of meningococcal disease in the ani-

mal and human system is still required and in particular the

correlates of protection for protein-based serogroup B vac-

cines.

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