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Tuberculosis 89 (2009) 389–397

Contents lists avai

Tuberculosis

journal homepage: ht tp: / / int l .e lsevierheal th.com/journals / tube

Evaluation of new vaccines for tuberculosis in the guinea pig model

Ann Williams a, Yper Hall a, Ian M. Orme b,*

a Health Protection Agency, Centre for Emergency Preparedness and Response, Porton Down, Salisbury, Wiltshire, UKb Department of Microbiology, Immunology and Pathology, C331 Microbiology, Colorado State University, Fort Collins Colorado 80523-1682, USA

a r t i c l e i n f o

Article history:Received 10 August 2009Accepted 17 August 2009

Keywords:Guinea pigsVaccinationStatistical limitation

* Corresponding author. Tel.:þ1 970 491 5777; fax:E-mail address: ian.orme@colostate.edu (I.M. Orm

1472-9792/$ – see front matter � 2009 Elsevier Ltd.doi:10.1016/j.tube.2009.08.004

s u m m a r y

The guinea pig is a very useful animal model for evaluating new tuberculosis candidate vaccines. Inaddition to established methods for bacterial load determinations, new technologies are emerging thatallow us to specifically evaluate effects of vaccines on the pathology of the disease process and theexpression by the host of cell mediated immunity. Limitations to the model include housing and relatedcosts, which often contribute to issue with study design and adequate statistical power, and the use oflaboratory strains of Mycobacterium tuberculosis which lack the high virulence and immune evasionproperties of newly emerging clinical isolates.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

Despite the ongoing TB global epidemic, including recent dataindicating that the numbers of multidrug-resistant [MDR] strainsare rising faster than previously recognized, progress to find a newvaccine with which to replace or at least improve the BCG vaccinehas been slow. This situation is gradually changing however, andthere is a growing portfolio of new candidates now being developedand evaluated, with some reaching early stages of clinical trials.1

New vaccines for tuberculosis must first be tested in animalmodels to establish their efficacy and safety.2,3 The most widelyused small animal model is the mouse. Inbred mouse strains areusually used, to minimize variability in responses, the hostresponse and immunopathology is well documented and under-stood, and above all the model is cheap and cost-effective.4,5 Othermodels are also used, and in fact one can argue that the field isundergoing a renaissance, with new information coming fromdiverse animals such as the cotton rat, rabbit, cattle, and non-human primates.

It is generally agreed that following a positive result in the mousemodel further evaluations should be done in the guinea pig model,and this model has been extensively used to test vaccine candi-dates.3,6–13 This model is more expensive and more susceptible to thedisease than the mouse, but has elements of pathology very similarto that seen in humans,8 as discussed extensively below. Moreover,while a lack of immunological reagents was once a serious limitationof this model, the last few years have seen a surge of technicaladvances in flow cytometry, immunohistochemistry, RT-PCR and

þ1 970 491 1815.e).

All rights reserved.

microarray, giving us a much clearer picture of the ability of newvaccines to protect guinea pigs from aerosol exposure to Mycobac-terium tuberculosis.

The guinea pig model has limitations however; the commonlyavailable strain is out-bred and because of its cost [including animalhusbandry under level-III conditions] one cannot do some of theextensive studies possible in the mouse. While an efficacy study inthe mouse is useful as an initial guide, more relevant study designs,such as extending the vaccine to challenge interval or performingprime-boost, studies in the guinea pig are required and these aremore challenging and costly. If anything, this model is still some-what in its infancy, because as discussed in detail in this article weare only now starting to understand whether certain study designsare actually providing the information we need, but equallyimportantly, whether our study design has sufficient statisticalpower. Enabled by a large body of data on novel vaccines of varyingefficacy, the latter is only just starting to be addressed, and hasmajor implications for even more expensive animal models such asthe rabbit and non-human primate, the latter an important end-stage testing model for drugs and vaccines.

2. Studies in the guinea pig

2.1. Course of infection after low dose aerosol exposure

After low dose aerosol exposure, using aerosol generators cali-brated to deliver an inhaled retained dose of 20–30 viable bacteria,infection develops progressively in the lungs for about 3 weeks.During this time, small numbers of bacilli can be detected in thespleen and the draining mediastinal lymph nodes, where they alsoincrease. Pulmonary infection then slows and establishes an

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apparently chronic disease state in which there are [for laboratorystrains] between 105 and 106 viable organisms in the lungs. Thisslowly increases over the next 100–150 days after which time theanimals reach a stage where euthanasia on humane grounds iswarranted.

2.2. Development of pathology

After aerosol exposure, small discrete lesions can be seen, usuallyclose to or within the airway parenchyma.14–17 By day 20 of theinfection these have increased in size [approximately 10-fold] andthe beginnings of central necrosis are already clearly evident. By day30 the ‘‘classical granuloma’’ beloved of textbooks has formed, withan obvious necrotic center, surrounded by viable tissue containinglymphocytes, macrophages, and multinucleate giant cells, and witha fibrotic capsule starting to form around the structure.

After that stage the appearance of lesions in the lungs becomesfar less uniform, with different lesions representing a mixture ofprimary and secondary granulomas at different levels of develop-ment becoming apparent16,17 (Figure 1). While some primarylesions are central, others occur close to or on the pleural surface.These grossly visible lesions can be used to estimate the inhaledinfectious dose. Destruction of the draining lymph nodes proceedsrapidly, and lymphatic vessels draining the lung can also becomeblocked.14,18 Large secondary lesions, characterized by their lack ofnecrosis, become evident in the pleural regions, but because oftheir large size and continuing inflammation they begin to signif-icantly consolidate the lung tissues. This latter event, in concertwith the large necrotic primary lesions, results in the compromisedrespiratory function that would, in the absence of a humane endpoint, lead to the death of the animal.

Most of the macrophages in these lesions [both in the mouseand the guinea pig] do not actually harbor bacteria. Most of theseare in foamy macrophages within the viable tissue layers of thegranuloma, and some are in the central necrosis. Although olderdogma saw the latter as the primary site for production of largenumbers of bacteria, there are apparently at least as many ina slowly developing acellular matrix or rim structure outside thecentral area of necrosis, where they exist in an iron-rich region asextracellular biofilms or clusters.19 Indeed, it is quite possible thatthese structures continuously feed individual bacilli into the morehypoxic central necrosis.

Figure 1. Granulomas in the lungs of infected guinea pigs. A large primary lesion [top]with a completely mineralized core is effaced by large secondary lesions below, full oflymphocytes and macrophages, but with no necrosis. Figure courtesy of Dr R. Basaraba.

2.3. Emergence of the cellular response

There are various ways to monitor the immune response to M.tuberculosis infection in guinea pigs, but only recently have we beenable to specifically apply various technologies such as flow cytom-etry, immunohistochemistry, microarray and PCR-based assays tothis model.

Recent data from new flow cytometric approaches has allowedus for the first time to follow the host cellular response in the lungsto the infection. Despite the availability of antibodies to CD4 andCD8 antigens for some time, flow cytometry itself has been difficultfor two reasons. The first is simply the nature of the guinea pig lungitself, which unlike the mouse contains an appreciable amount offatty tissue, which confounds the preparation of single cellsuspensions by enzymatic digestion of the lung tissue. The second,more difficult to solve, has been the general clutter seen in thesesuspensions which confounds flow cytometric gating.

Ordway and her colleagues20 recently solved these problems,allowing clean gating of separate lung cell populations. The tacticused was to replace forward scatter analysis using cell staining withan antibody, MIL4, that binds to guinea pig neutrophils [hetero-phils], the primary cause of the autofluorescence. Hence, by usingSSC/MIL4 two-dimensional gating as the primary step, clean pop-ulations of CD4, CD8, B cells, macrophages, and granulocytes couldthen be seen.

An initial analysis of the course of the cellular response imme-diately revealed some interesting surprises. First, there was a rapidand substantial rise in CD4 cells accumulating in the lungs, butunlike the mouse the CD8 response was far smaller. Secondly, thisCD4 response rapidly fell off after day 30 of the infection. As yet, wedo not know why; in both the mouse and guinea pig modelsa chronic disease state persists for some time, but in the mouse thisis associated with the continued presence of activated CD4 and CD8cell populations.21

3. How can we tell if a vaccine is working in this model?

In the absence of a definitive correlate of protection, a wholerange of read-outs are being considered in order to assess theefficacy of a vaccine.

3.1. Simple measures: body weight and survival

The body weight of the animal at the start of the experiment isan important variable. When provided by breeders, weanedanimals are usually around 300 g in weight. If infected at thisweight range, especially if a relatively high inoculum is used [50–100 bacilli], time to death is far shorter. However, the animal is notfully immunocompetent at this stage, and so vaccine results may bemisleading [both in a positive and negative sense]. Animals shouldbe allowed to put weight on, to at least 450–500 g, and kept ingroups rather than in individual cages to acclimatize and establishpecking order [these animals are highly social].

After infection, feed behavior and hence body weights canfluctuate over the first 2–3 weeks, but this usually settles down andanimals will continue to gain weight, often up to 1000 g or more.But then, as the infection worsens around 10–15 weeks or so, somewill start to show evidence of weight loss. Since weights oftenfluctuate, even in adults, this is not an immediate indication foreuthanasia. Humane endpoints must take into account variousfactors in addition to weight loss [> 10%] including generalbehavior, outward appearance and vocalization. These can beassessed using a Karnofsky scale which once triggered indicateseuthanasia.

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While survival is being monitored, trends in weight gain and/orloss across a group may provide further information regarding thelevel of protection conferred. Weight changes can be monitored atregular intervals during a study without too much additionalhandling and the data generated is highly accurate if calibratedscales are used. By employing summary measures such as area-under-the-curve or mean increase in weight it is possible to makegroup-to-group comparisons. However, while the relevance ofweight loss to disease progression (and lack of vaccine efficacy) isclear, the relevance of weight gain as a predictive measure of long-term vaccine efficacy is less well-defined and requires furtherassessment. Given that weight data is such a simple and reliablemeasure, we are currently investigating several different methodsof analysis in order to establish the predictive value of this read-out.

Time to death is recorded in days and analyzed by Kaplan MeierLog Rank statistics. This is a very stringent test and often providesdisappointing results to experimenters who feel their vaccine isshowing a ‘‘trend’’ towards improved survival. The central problemis that the survival data is binominal and hence the statistical powerof such data is extremely low with the small group sizes that are socommonly used, particularly in guinea pig studies and even more sowith larger animals. In fact, if standard statistical power calculationsare applied to Kaplan Meier survival plots the results reveal that thismethod of comparing vaccine efficacy has some serious limitations.In Figure 2 we have simulated a theoretical result in which thesurvival of two groups of animals [n¼ 8] is compared, one groupvaccinated with BCG and the other with a new subunit candidate, inwhich the subunit vaccine group clearly appears to be living longerthan the BCG controls. However, if the (two-sided) Log Rank test isapplied to these data, the test vaccine is not actually significantlydifferent to the vaccinated group. This of course would be a prom-ising result for a subunit vaccine, but if on the other hand the testgroup was a BCG-prime boost regimen or a modified BCG [actually,more likely to give this type of improvement over BCG alone] itwould unfortunately not provide any substantive rationale for thevaccine developer to move forward with this candidate since it hadachieved no greater protection than conventional BCG. Thus this isnot necessarily because of a failure of the vaccine, but because thestudy was insufficiently powered to allow the observation of animproved efficacy.

Extending this argument, it would be reasonable to assume thatincreasing the group size will increase the statistical power of thetest and hence would allow demonstration of a statisticallysignificant increase in animal survival. However, in the example inFigure 2, where there is an apparently substantial increase in meansurvival time [from about 150 days to 250] an appropriate powercalculation would show that a group size of 74 is actually needed toreach a Probability value of 0.05. Having said that, when there is

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Figure 2. Theoretical survival data demonstrating the limitations of small group sizes.

a much greater difference in survival between the control and testvaccines, much smaller group sizes are needed and there areexamples where group sizes as low as 6 can be used to demonstratesignificant improvement upon BCG 22; however, in general theseare rare. In this regard, survival data that is most commonlyreported is similar to the example shown below in Figure 3 inwhich the test vaccines are only marginally better than the BCGcontrol. Such data obviously suggest a promising ‘‘trend’’, butprohibitively large group sizes would be needed to demonstratea statistically significant difference.

3.2. Counting the bacterial load [CFU] in target organs

The most commonly used guide to determining whethera candidate vaccine has had a protective effect is to homogenize thelungs and other target organs, plate on nutrient agar, and calculatewhether the number of colony forming units is lower in the animalsreceiving the vaccine compared to animals receiving the appro-priate controls. However, while this bacteriological data usuallysupport histologic and/or increased survival data, even this simplemeasurement has to be interpreted with some care, particularlywhen used to discriminate between vaccines at a time pointbeyond 100 days (15 weeks) (Figure 4).

As Figure 4 shows, CFU data is at its most powerful early on inthe course of disease, and for this reason protection data in a short-term study are usually acquired at a time point 4–5 weeks afterchallenge. During this period any active vaccine will allow theaccelerated emergence of protective immunity and hence a slowingof bacterial growth in the lungs, whereas inactive candidates willnot show this. Thereafter however CFU differences can change asthe non-vaccinated animals themselves express some degree ofnatural immunity which is variable between animals.

The example shows clearly that such early CFU data is highlydiscriminative and predictive of survival, and importantly thestatistical power of these data is superior to survival data. Allowingfor a statistical power of 0.8–0.9, and a usual variance of 0.2–0.3 log10

CFU, a reduction in the mean CFU values between saline controls andtest groups of about 0.7 log10 CFU is usually significant, when n¼ 5–6animals are used.2,23–26 Thus, subtle improvements in vaccine effi-cacy can be measured in a relatively short time frame and, impor-tantly, vaccines that offer no protection can be identified at an earlystage. The predictive value of early CFU has been demonstratedpreviously,27 but the large data set shown in Figure 5 illustrates veryclearly that CFU levels observed at much later time points do notpredict survival.

When guinea pigs reach humane end-points there can bea surge in CFU counts [often approaching 108 per lung] and this is

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particularly evident in unprotected animals who exhibit classicalcaseating necrosis and related pathology such as lymphadenopathyrelatively early during the course of the infection [see the salinecontrol group in Figure 5]. Usually, changes in CFU show a consis-tent trend, being lower in animals exhibiting prolonged survival,but the variance of the actual data can be large, up to w2 log10. Inpractical terms this means that vaccine groups with well separatedsurvival curves are more likely to have median CFU values consis-tent with such differences in survival. Vaccines with similarprotective efficacy in terms of survival, however, are unlikely to befurther discriminated on the basis of CFU levels. Moreover, at theend of a given study, this variance means that measured bacterialloads in survivors will not be strongly predictive of the degree orextent of survival past that time point. The same argument can bemade for histopathological data.

These observations lead us to conclude that evaluation ofbacterial load at an early time point is the only instance where CFUdata is likely to provide an informative result. However, this is nottrue when we are evaluating the capacity of a vaccine to improve

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Figure 5. Correlation between survival time and bacterial load in the lung taken fromseveral low dose aerosol challenge studies. Arrows point to survivors sampled at theend of a study.

upon BCG because, at this early time point, it may not be possible topush the immune response any further to reduce the bacterial load.We should perhaps remind ourselves that, even at this early timepoint, BCG is not able to prevent the establishment of pulmonaryinfection and will have only restricted further replication of bacillicompared with unvaccinated animals. If a novel vaccine or vaccine/treatment combination was able to have a more prominent impacton the early events in the lung, this would be very sensitivelymeasured by the 4 week CFU assay.

3.3. Practical implications for vaccine study design

Given BCG’s imperfections, it would be highly beneficial todevelop a new vaccine, such as a recombinant BCG or a BCG over-expressing additional antigens. How then should we design studiesin animal models such as the guinea pig to determine this withsome degree of confidence to move the candidate to very muchmore expensive models such as non-human primates?

In considering the most appropriate methods for evaluatinga vaccine in the guinea pig model one must first consider therequirement to demonstrate significant improvement over BCG, andwhat would be sufficient to merit testing the candidate further. In thecase of simple survival studies, as argued above unless there isa strong rationale to expect this improvement to be substantial, suchas an increase in the mean survival time by 100 days or more, there islittle point nor ethical justification for conducting a survival studybecause it would be almost certainly doomed to failure. On the otherhand, if the objective is to demonstrate that a new candidate is atleast capable of conferring equivalent protection to BCG and that thisprotection is sustained, then guinea pig survival studies can beextremely informative and discriminative. In this regard, globalanalysis of data obtained over several years in which comparativeevaluation of novel vaccines was performed at our two sites revealeda high reproducibility in the performance of BCG relative to unvac-cinated controls, and moreover a simple comparison of survival data(BCG versus saline control groups) obtained from several laborato-ries has revealed a similar consistency, despite variations in chal-lenge strains and methods of aerosol delivery [Figure 6]. Overall thisillustrates that if there is appropriate understanding of the funda-mental characteristics of the model, including a recognition of thestrengths and limitations that the chosen read-out offers, the value ofvaccine efficacy data will only increase.

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A. Williams et al. / Tuberculosis 89 (2009) 389–397 393

3.4. Changes in pathology

Lung pathology data can provide very important and convincingevidence that a vaccine is having a beneficial effect. However, theanalysis has to be done carefully and on multiple sections per groupto avoid any possible subjectivity. The best way to deal with this isto have the pathologist read all the slides blind. These can bereported as from ‘‘best’’ to ‘‘worst’’, then when the code is brokena mean ‘‘rank’’ score can be given to each group. The particular slidefrom each group that is closest to the mean rank score for that testgroup is then used for ‘‘representative photographs’’.

In addition to overall rank, further analysis can be performed onthe degree of lung consolidation, which can be estimated visuallyor by using a commercial imaging program. Additional ranking canbe performed on pertinent elements such as degree of lung fibrosis,lesion mineralization, and extent of necrosis, and new stereologybased methods are now under evaluation to reveal more subtlechanges in the pathology of lesions. These parameters are only nowstarting to be routinely applied, and so their exact usefulnessremains to be seen.

With regards to vaccine evaluation, it should also be noted thatthe time post-infection that pathology is assessed will affect itsusefulness. In a way that mirrors CFU data, histopathology resultsbecome more variable at longer times post-infection and, therefore,correlate less strongly with survival.

3.5. Changes in T cell patterns, flow cytometry,immunohistochemistry

To date, other than very recent studies on BCG, there is very littleinformation on the application of flow cytometry to the guinea pigmodel.20,28 However, that information alone indicates that this willbecome a very powerful tool in monitoring the cellular response tonew vaccines, as it has already become in the mouse model[Figure 7]. We can now track CD4 and CD8 patterns in the lungs,and gating versus CD45 is a reasonable starting point to identifyactivated T cells. Identification of antibodies to CD44 and CD62Lwill help further, as will development of intracellular stainingprotocols for IFNg, as mentioned above.

We have recently shown that T cell subset patterns change inBCG-vaccinated guinea pigs compared to controls after aerosolchallenge.28 The CD4 response peaks faster then declines as theinfection is more rapidly contained. Those than remain expressCD45, a marker we are currently using to measure activated cells. Inaddition, analysis of the influx of macrophages into the lungsprovided data consistent with previous pathology analysis. However,the flow cytometric data indicated that not only was there a muchfaster and larger macrophage response in the vaccinated animals butmost of these cells expressed high levels of Class-II MHC molecules,whereas only a low percentage of the macrophages in the controlsdid so. The latter observation is rather unexpected, and seems tosuggest a very weak Th1 response in the control group, perhapsindicating that despite a big influx of CD4 cells into the lungs of theseanimals very few of these are actually secreting IFNg and thus raisingClass-II expression on the macrophages entering the granulomas.

The hallmark of the guinea pig model is the development ofcentral caseous necrosis in granulomatous lesions. Older hypoth-eses suggest that this is caused by exuberant immunity but we havehypothesized, based on our observations that the beginnings of thedevelopment of necrosis is very early and long before acquiredimmunity has developed,16 that this mechanism has an innatebasis. Exactly what this might be is still unclear, but early influx anddegeneration of granulocytes seems to be a primary reason, and ourflow cytometric data clearly show that influx of these cells is almostcompletely reduced in vaccinated animals in direct parallel with

the absence of necrosis.28 There may be other reasons as well, suchas vascular damage early in the process preventing adequatecarriage of oxygen to the inflamed tissues.17

Where flow cytometry becomes even more useful is when it isdone in parallel with immunohistochemistry. This allows trackingof total lung cell populations coupled with information as to theanatomical locations of these cells. In our studies in the guinea pigto date there are interesting parallels with the mouse model; this isparticularly evident in the distribution of CD8 cells in the lungswhich assume similar peripheral positions in each model.20,29

3.6. Changes in PCR values, application of microarrays

There are now a large panel of cytokines and chemokines thatcan be measured by PCR, driven by work almost exclusively fromMcMurray and his colleagues.30,31 These studies systematically andpainstakingly have described RT-PCR assays for several cytokines[IL-1, IL-4, IL-10, IL-12p40, IFNg, and TNFa] and chemokines [IL-8,MCP-1, TGFb, and RANTES]. There are a small number of antibodyreagents specific for some of these cytokines being developed bythis group and others which should increase the sophistication ofthese studies in the near future.

More recently, the McMurray group has applied laser capturemicrodissection to look directly at cytokine/chemokine expressionin primary and secondary lesions in the guinea pig lung.31 Inprimary lesions the dominant cytokine was shown to be TNFa. Ifthe animal was first vaccinated with BCG, then this profile changedto one containing IL-12, IFNg, as well as TGFb. Interestingly, thislatter profile was seen in the secondary lesions of animals that werenot vaccinated, consistent with the idea that by the time theselesions are seeded by bacilli from the blood or lymphatic vessels,acquired immunity has had time to arise and lesion degenerationand necrosis is thus prevented. [In BCG vaccinated guinea pigs,primary lesions do not become necrotic, and development ofsecondary lesions is minimal].

While microarray analysis has been applied to the mousemodel,32 this is still under development in the guinea pig model.28

A boutique array has been developed for use in the guinea pig,initially consisting of an 81-spot microarray covering 65 guinea piggenes, including cytokines, chemokines, and their receptors/ligands.33 This revealed changes in the differential expression ofmany of these genes between naı̈ve and BCG-vaccinated guineapigs, much of it very consistent with earlier PCR data regardingthese molecules. This study also indicated significant changes inhost ferritin expression, again highly consistent with informationarising from direct histopathologic observations of lesions invaccinated and control animals.18 The size of this array has beenextended as more genes become available following the annotationof the guinea pig genome (www.ensembl.org/Cavia_porcellus/Info/Index).

An important consideration when applying any of the abovemeasures to vaccine evaluation studies is whether the analyses canbe conducted on small volume blood samples (that can be takenfrom live animals) or if whole organ or large volume material isrequired, resulting in the need to euthanize additional animals. In thelatter case, large group sizes will be needed if time-course studies arewarranted with obvious cost implications. In this regard, themolecular methods may be advantageous since it is possible toamplify the nucleic acids in order to generate sufficient material and,at CEPR, we are currently investigating different techniques that willpermit amplification of enough mRNA to be applied to arrays fromsmall volume ear bleeds. If this is successful, extremely powerfulimmune profiling can be conducted over time-course studies wherethe same animals are followed from vaccination through to challengein order to define immune correlates of protection.

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Figure 7. GP flow data. [A] Gating on SSC versus the antibody MIL4 allows clean separation of distinct lung cell populations. [B] Kinetics of CD4 and CD8 T cells in the lungs ofinfected guinea pigs [closed circles] and in animals previously vaccinated with BCG [open circles]. [C] Kinetics of CD45þ activated CD4 and CD8 cells. [D] Kinetics of influx ofgranulocytes; BCG prevents this almost entirely in both the lungs and draining lymph nodes. Figure courtesy of Dr D. Ordway.

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3.7. Other techniques

Further assays have been successfully applied to the guinea pigmodel that potentially could be applied to testing new vaccines.Advances are now being made in magnetic resonance imaging, andin positron emission tomography [PET]. The guinea pig lung is largeenough to be visualized by MRI, and three-dimensional images canbe constructed by stacking individual images of slices of tissue.34 Infact, because slices of tissue can be used that are far thicker thanmicroscope slides, resolution of lesions appears to be far moresensitive. The MRI data also confirm the classical data, by showingthat BCG does not reduce the number of infectious lesions, butprevents them from increasing in size.34 New MRI software whichcaptures images in between breaths taken by living animals couldallow real-time evaluations of lung pathologic changes against timeof the infection, thus reducing the total number of animals thatwould have to be needed for a given study.

Another approach being assessed is Flame Atomic AbsorptionSpectroscopy. Simply, the lung is reduced to ash then processed forspectroscopy to detect molecular ions including iron, calcium andphosphorus. Because these are central elements of the necroticprocess in the lung lesions, they are modulated by vaccination, asrecently demonstrated for BCG.18

Finally, two potentially simple methods are to measure theanimal body temperature, which rises in unprotected animals, andthe degree of saturation of peripheral arterial oxygen, which islower in control animals compared to vaccinated, and may reflectthe oxidative stress the former are experiencing.17 These methods

could be useful to supplement other read-outs but are not suffi-ciently robust nor validated as yet to be used exclusively as indi-cators of vaccine efficacy.

4. BCG prime-boosting models; what should we try tomeasure?

Most children in the world are vaccinated as neonates with theBCG vaccine. It is cheap, is effective against TB in children, but thereis now a general consensus that it does not work in adults. Initially,it was considered that BCG could be replaced, but that now seemsless likely and a bigger emphasis is now being placed on newvaccines that could be used either to directly boost immunity that iswaning in BCG vaccinated individuals, or to replace this immunity.Because we still not understand the basis of these events, i.e.whether memory immunity is even induced by BCG [see below] orwhether memory immunity established by neonatal vaccinationcan subsequently be expanded by a new candidate, what we expectprime-boost regimens to actually achieve has yet to be clearlyarticulated. That aside, evidence that BCG can be boosted in animalmodels is now clearly established.2,35

In such regimens guinea pigs are vaccinated with BCG thenrested a finite time [10 weeks or more], then vaccinated with theboost candidate. After another six weeks or so [note the shortvaccine to challenge interval] the animals are challenged. One canthen evaluate CFU at an early time point, but as mentioned above,a boosting effect is not always evident compared to the BCG-onlycontrol. Because of this, a second tactic is to follow long term

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survival to see if boosted animals can live longer than animals justreceiving BCG alone; if so, this can provide convincing information.

Even if the group size is modest, e.g. w10 animals, these have tobe kept under, stringent, clean, level-III biosafety conditions, whichis extremely expensive. Because of the latter, group sizes are ofteneven smaller. As discussed above this impacts on statistical power,to the point that it may provide meaningless or misleading data.Moreover, as animals die [trigger Karnofsky scale and are eutha-nized] there is the temptation to harvest organs for CFU evaluationsand pathology assessments. In addition to the important limita-tions discussed above, a further problem here is obvious; dataarising from this approach are obtained from animals that aredying. One way around this is to perform sequential analysis byharvesting animals at certain times during the period after 4 weeksand up until a time point where either deaths or prohibitively largevariation (see Figure 4) negates the discriminative power of theassays. Notwithstanding the considerable costs of such studies, thismay yield limited information if CFU data are used alone, and hencethis evaluation would have to be combined with flow cytometricand immunopathology analysis. This would be prohibitivelyexpensive, especially if the newer technologies described above areapplied, and this of course explains why no-one has seriouslyattempted to do this. Sadly, this is short-sighted, given the fargreater expense that would be involved if an inadequate vaccinereached the non-human primate testing point or even worse, wentstraight to clinical trials.

One must note that people do not get tuberculosis a few weeksafter prime-boosting, and a more realistic method would be toconsiderably extend the vaccine boost to challenge interval; afterall, we want people to be boosted for decades. But again, this wouldbe prohibitively expensive, even for one or two of the very bestvaccine candidates.

Given these issues there is a requirement for more robustmethod of testing BCG-boost regimes in the guinea pig. Modelingthe partial protective efficacy of BCG observed in humans may beone way of evaluating boost candidates with improved sensitivity.

5. Existing and newly emerging potential barriers tosuccessful testing of vaccines

Much of what we know about the immune response to tuber-culosis has been provided by the mouse model. We have nowhowever reached a stage in the development of the guinea pigmodel where we are beginning to be able to specifically measureCD4 and CD8 responses, including activated and IFNg-secretingpopulations. While such information may prove invaluable giventhe high relevance of the model, the mouse model continues toprovide new information that tells us that the immune response totuberculosis is far more complicated than we thought, and whichwe must try to assimilate in the evaluation of new candidates.

As an example, there is now firm evidence for the existence oftwo additional new T cell subsets within the mouse CD4 response.The first is a CD4 subset that produces members of the cytokine IL-17 family. This cytokine is produced earlier during the inflamma-tory response [by gd T cells as well as CD4 cells36] and its expressionis influenced by prior vaccination.37 Because of its central role incontrolling cell influx by inducing secretion of key chemokinesallowing accelerated expression of immunity and also its influencein the expansion of memory T cells, the ability of certain vaccines[especially live recombinant BCG vaccines or live attenuatedmutants of M. tuberculosis] to selectively induce this subset couldbe an important factor. However, as yet, virtually no studies havetried to measure the emergence of this subset in this particularcontext.

The second subset comprises CD4 regulatory T cells. In fact, thisis probably not a single subset but a relatively heterogeneouspopulation of cells designed to keep florid immunity under check[some CD8 cells can also work in this manner]. Within the CD4subset, these cells express the intracellular Foxp3 marker, someexpress high levels of CD25, and some secrete IL-10. The laboratorystrains H37Rv and Erdman [in our hands] induce only low levels ofthese cells, but highly virulent and [perhaps more importantly]highly inflammatory clinical isolates strongly induce regulatory Tcells; an excellent example is the W-Beijing strain HN878.38 Wecurrently speculate that this happens to try to dampen theinflammatory response in the lungs induced by such infections, butas a consequence this response competes with the protectiveresponse being mediated by CD4 TH1 cells. This of course has allsorts of implications for whether a vaccine might work in the caseof infections in which regulatory T cells are strongly induced, andindeed we have preliminary information that certain vaccines canthemselves potentially induce this subset. In terms of the guineapig model, this subset cannot be directly measured as yet but theCSU can now at least detect Foxp3 by RT-PCR.

In addition, the CD8 response is clearly more complicated thanfirst realized. The importance of CD8 cells in the mouse is longestablished,39–41 but our data discussed above show that this subsetaccumulates in relatively low numbers in the guinea pig granulo-matous response, where [as in the mouse] they assume a peripheralposition. What the mouse has told us, in the context of chronic viralinfections, is that CD8 cells gradually become unresponsive overtime, in some sort of state of exhaustion, and indeed this couldexplain our own observations regarding the kinetics of infection inchronically infected CD8 gene knockout mice.41 This can be fol-lowed by looking at expression of certain markers, principally PD-1.Levels of this marker rise in mice infected with M. tuberculosis[S. Irwin, I.Orme, unpublished] and remain high, but are reversed ifthe mouse is treated with drugs. We do not know if this happens inguinea pigs, because again there are no reagents, but the observa-tion is important. Improved CD8 responses are a major target for atleast two types of vaccine candidates, pro-apoptotic mutants42 anda leading rBCG candidate,43 so whether these can prevent CD8exhaustion or themselves promote it remains unknown. We wouldpoint out that while the pro-apoptotic DsecA2 mutant stronglyprotects in the guinea pig model,42 these were short-term protec-tion assays, whereas the CD8 exhaustion mechanism takes longerto gradually appear.

Above all, perhaps, we need to develop reagents that can allowus to specifically monitor memory immunity in vaccinated guineapigs. Having said that, information from the mouse model is itselffar from clear, with at least two cell transfer studies givingsurprising results in terms of the cell phenotype capable of trans-ferring immunity.44,45 In acute models [mostly viral or in responseto defined antigens in TCR transgenic mice] there appears to bea logical progression from activated/effector T cells to memoryT cells. In the latter case, there is now good evidence for twoseparate lineages. The first, effector memory cells, remainpredominantly in the blood and peripheral tissues, where theyprobably act as a first line of defense. A second, central memory Tcells, can be found in the spleen and other lymphoid tissues.

Recent studies [Orme, submitted] seem to suggest that BCGvaccination of mice generates significant numbers of effectormemory T cells [both CD4 and CD8] and these reside in thelungs, lymph nodes, and other lymphoid tissues. In contrast, thegeneration of central memory cells is minimal, with only thespleen [and to a lesser extent the bone marrow] containing anapparent reservoir of these cells. In this regard, it has beensuggested that in chronic infection memory per se may notexist,46 and in the context of BCG [which itself is only slowly

Figure 8. Severe pathology seen in the lungs [left] and cavitation of draining lymph nodes [right] of guinea pigs infected with a recent outbreak-associated clinical isolate. The CSUgroup now has multiple examples of isolates causing this degree of pathology, representing a range of W-Beijing, non-Beijing, and MDR strains. Photo courtesy of Dr G. Palanisamy.

A. Williams et al. / Tuberculosis 89 (2009) 389–397396

cleared] there may only be partial memory generation47 with theBCG, for whatever reason, incapable of driving emerging effectormemory T cells fully through to a central memory population. Infact, this could be the central reason why BCG is not particularlyeffective; it may be capable of generating an initial effectormemory T cell response in young children, but this is graduallylost over the next 10–15 years [by T cell senescence, attrition, orperhaps because of exposure to environmental mycobacteria] andthere is no central memory immunity to provide an adequatesecond line of defense when the individual is exposed at thatlater time to tuberculosis infection.

This is a fundamental issue. Above all, it is hard to verifywhether a new vaccine candidate generates a memory T cell pop-ulation that will protect the individual for many decades, when wehave no validated way of measuring this in the first place. This iscentrally important in the context of BCG prime-boosting strate-gies, where it is reasonable to believe that the boost is expanding orat least prolonging the lifespan of such T cells. Another possibility toconsider is that highly immunogenic recombinant BCG vaccinecandidates might actually exacerbate matters by further preferen-tially expanding effector memory T cells; one way to address thismight be to design prime-boost strategies in which the boosttargets the expansion of central memory cells.

BCG results in the accelerated containment of the infection invaccinated guinea pigs but does not sterilize. Some of the remain-ing bacilli are in small lymphocytic lesions but are often hard tofind. A similar reduction is seen in drug-treated guinea pigs, buthere residual bacteria can be found in an extracellular state in anacellular rim on the surface of primary lesions, in what appears tobe a biofilm.19 While there is no evidence to date that vaccines forcethe same survival of small numbers of bacilli in a similar way, if sothis has confounding implications for vaccine design, especially inthe context of candidates that may be designed in the near future toprevent reactivation of latent disease.

Finally, virtually all vaccines are tested against the laboratorystrains H37Rv and Erdman. These are certainly virulent, and causenecrosis and death in guinea pigs, but they pale in comparison withcertain recent outbreak strains.48 In some cases, there is extremelyrapid development of severe pathology in lung lesions, with asso-ciated lymph node cavitation [Figure 8]. Given the recent veryimportant and troubling observation43 that BCG is poorly protectiveagainst a Beijing isolate in the mouse model [although anotherstudy49 now contests this], it seems imperative to determine if newvaccine candidates can protect against these ‘‘real’’ M. tuberculosisstrains. To date, this question has never been addressed partly dueto a lack of consensus on which of the clinical isolates should beused in the animal models to be representative of the strains

causing disease in humans, and because the reported ‘virulence’ ofthe clinical strains varies depending on the animal model.

6. Conclusions

The guinea pig model is a highly reproducible and sensitivemeans of evaluating new candidate vaccines for tuberculosis. Therecent emergence of new technologies that can be applied to themodel, the development of new reagents and assays, and a newappreciation of the pathogenesis of the disease process in thisanimal and its similarity to this process in humans, makes ita powerful tool for new vaccine selection. In this regard it canprovide vaccine developers with important data to progress theircandidates to early stage human trials and offers a means ofdiscriminating the most promising candidates for further testing.As novel vaccines advance to clinical efficacy trials, the data willstart to reveal which of the read-outs in this animal model bestpredict the capacity of these vaccines to protect humans.

We now have the opportunity to take this realistic and relativelycost-effective animal model to the next level, but we also have to bemindful of three issues. The first is study design issues includingimportant statistical elements that are often ignored, and which ofcourse will be relevant to similar studies in larger animal models.The second is that we have to take into consideration the continuedimprovement of our knowledge of the host immune response andhow this might influence vaccine efficacy, both in positive ways[strong development of long lived memory and capacity to push theeffector memory to central memory transition] and negative ways[depression of TH1 immunity by regulatory T cells for example].Thirdly, all our efforts to develop new vaccines may be for naught ifthey are incapable of protecting against highly virulent, rapidlypathogenic, clinical isolates.

Finally, it is imperative that there is dialogue and close inter-action between those with expertise in this and other models withthose who wish to demonstrate efficacy of their candidate vaccines.

Acknowledgements

We thank our colleagues at HPA, particularly Mr Simon Clarkand Mr Graham Hatch for guinea pig modeling and aerobiologyexpertise, and Prof Philip Marsh for scientific leadership of the teamand critique of the text. We thank our colleagues at CSU, includingDrs Diane Ordway, Randall Basaraba, Anne Lenaerts, MarcelaHenao-Tamayo, Gopinath Palanisamy, and Crystal Shanley. We arevery grateful to Prof David McMurray for sharing information oncytokine assays.

A. Williams et al. / Tuberculosis 89 (2009) 389–397 397

Funding: This work was supported by European Union grants5 (QKL2-CT1999-01093) and 6 (LSHP-CT-2004-503367 and LSHP-CT-2003-503240), and by the Department of Health, UK, and byNIH Grant AI070456.

Competing interest: None declared.

Ethical approval: Not required.

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