perspectives on clinical and preclinical testing of …candidate tb vaccines better than would...

CLINICAL MICROBIOLOGY REVIEWS, Oct. 2010, p. 781–794 Vol. 23, No. 40893-8512/10/$12.00 doi:10.1128/CMR.00005-10Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Perspectives on Clinical and Preclinical Testing of NewTuberculosis Vaccines

Arthur M. Dannenberg, Jr.*Center for Tuberculosis Research, and Departments of Environmental Health Sciences, Molecular Microbiology and Immunology,

Epidemiology, and Pathology at the Johns Hopkins Medical Institutions, Baltimore, Maryland

INTRODUCTION .......................................................................................................................................................781WHY SOME CLINICAL TRIALS FAIL TO DETECT BENEFITS FROM BCG VACCINES........................782

High and Low Responders ....................................................................................................................................782High Prevalence of HIV .........................................................................................................................................782High Prevalence of Intestinal Helminths and Poor Nutrition .........................................................................782Environmental Mycobacteria ................................................................................................................................782Absence of Booster PPD Skin Testing .................................................................................................................782Variations in BCG Strains ....................................................................................................................................783Human Genetic Differences ...................................................................................................................................783Conclusion ...............................................................................................................................................................783

IDENTIFYING THE “�4%” GROUP THAT WOULD BE HELPED BY BCG VACCINATION...................783STOPPING THE DEVELOPMENT OF PRIMARY PULMONARY TUBERCLES IS A MAIN REASON

FOR VACCINATION AGAINST TUBERCULOSIS FOR BOTH HUMANS AND LABORATORYANIMALS.............................................................................................................................................................783

TUBERCLE COUNT METHOD ..............................................................................................................................784THE LARGEST DIFFERENCE BETWEEN TWO CANDIDATE VACCINES WILL BE FOUND

IN HOSTS WITH THE STRONGEST IMMUNE RESPONSE...................................................................786EFFECTIVE TB VACCINES MUST PRODUCE APPROPRIATE AMOUNTS OF DELAYED-TYPE

HYPERSENSITIVITY AND CELL-MEDIATED IMMUNITY.....................................................................786Role of DTH and CMI in the Pathogenesis of Tuberculosis............................................................................786Antigens Producing DTH and/or CMI.................................................................................................................786

WHEN VACCINATED FOR TB, LABORATORY ANIMAL SPECIES DEVELOP DIFFERENTAMOUNTS OF DTH AND CMI.......................................................................................................................787

Immune Response to DTH Antigens....................................................................................................................787Immune Response to CMI Antigens ....................................................................................................................787Immune Response in both Mice and Guinea Pigs Together ............................................................................787

COMPARISONS OF TUBERCULOSIS IN HUMANS, RABBITS, MICE, AND GUINEA PIGS..................788TUBERCULOSIS IN NONHUMAN PRIMATES ..................................................................................................789BCG IN NEWBORN INFANTS................................................................................................................................790TB VACCINES HAVE THEIR MAIN EFFECTS EARLY IN DISEASE ............................................................790PROPHYLACTIC IMMUNIZATION AND IMMUNOTHERAPY WITH CRITICAL TB ANTIGENS .........790ACTIVATION OF MACROPHAGES IS A MAJOR GENETIC FACTOR IN RESISTANCE TO

TUBERCULOSIS................................................................................................................................................790DISCUSSION OF VARIOUS COMBINATIONS OF FACTORS THAT SHOULD BE CONSIDERED IN

PRECLINICAL (AND CLINICAL) TESTING OF NEW TUBERCULOSIS VACCINES........................791TB Vaccination and Bacillary Virulence .............................................................................................................791TB Vaccination and Bacillary Titers....................................................................................................................791TB Vaccination and Tubercle Counts ..................................................................................................................791Virulence, Bacillary Titers, and Tubercle Counts..............................................................................................791

CONCLUSIONS .........................................................................................................................................................791ACKNOWLEDGMENTS ...........................................................................................................................................792REFERENCES ............................................................................................................................................................792

INTRODUCTION

New tuberculosis (TB) vaccines (better than the current My-cobacterium bovis BCG vaccines) are greatly needed to controlthis disease, which every year kills 2 to 3 million persons in the

world today. Clinical trials of new vaccines are very expensive andless precise than the testing of these vaccines with laboratoryanimals, so it behooves us to obtain as much information aspossible from laboratory animals before clinical trials are under-taken.

This is not a review of the current immunological literaturethat dissects the various components of the immune process, butit is an analysis of how delayed-type hypersensitivity (DTH) andcell-mediated immunity (CMI) in humans, mice, guinea pigs, rab-

* Mailing address: Johns Hopkins Bloomberg School of PublicHealth, 615 North Wolfe Street, Baltimore, MD 21205. Phone: (410)377-7125. Fax: (410) 955-0105. E-mail: [email protected].

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bits, and monkeys could influence TB vaccine selection. In otherwords, this review focuses on the overall “woods” rather than onthe individual “trees” that comprise the woods. The woods are thepresence and the characteristics of the pulmonary tuberculouslesions themselves. The trees are the many individual factors(genes, transduction factors, cytokines, and microbicidins) thatmay affect the development of such lesions.

This review suggests (i) that in clinical trials the selectionamong new TB vaccines would be more precise if the ratesof healing of the positive-control BCG group were takeninto consideration; (ii) that in preclinical trials the selectionof new TB vaccines would be more precise if tubercle count-ing in rabbits was always included, along with evaluations ofmice and guinea pigs; and (iii) that the evaluations of newvaccines in rabbits would detect differences between twocandidate TB vaccines better than would evaluations in miceand guinea pigs, because the immunity to Mycobacteriumtuberculosis developed by rabbits is much stronger than thatdeveloped by the other two species; i.e., the difference be-tween vaccinated and unvaccinated rabbits would thereforespan a larger range.

The inclusion of tubercle counting in rabbits (a species thatdevelops both good DTH and good CMI) would enable a moreprecise selection of new TB vaccines. In current preclinical TBvaccine evaluations, tubercle counting in rabbits has not beenundertaken before the start of more-expensive clinical trials.However, the use of rabbits in preclinical TB vaccine testingcould reduce the number of inconclusive clinical trials and savemuch time (as well as millions of dollars) in the developmentof better TB vaccines for worldwide use. Vaccine evaluationwith rabbits seems to be even more pertinent than vaccineevaluation with monkeys, because monkeys also have a rela-tively weak immune response to M. tuberculosis and thereforewould respond less well to vaccines.

With today’s emphasis on molecular biology, many funda-mental concepts of TB pathogenesis are often overlooked inthe selection of new TB vaccines. This review calls many ofthese fundamental concepts to our attention.

WHY SOME CLINICAL TRIALS FAIL TO DETECTBENEFITS FROM BCG VACCINES

BCG vaccination usually increases host resistance to infec-tion with virulent tubercle bacilli in almost every commonlaboratory animal. In fact, in laboratory animals, BCG is usu-ally the standard to which new candidate vaccines are com-pared. BCG should also increase resistance in humans. There-fore, why do some clinical trials fail to show any benefit fromBCG vaccination? Below are some possibilities.

High and Low Responders

The high-responding group includes individuals who con-vert their tuberculin skin tests but show no evidence oftuberculosis. This group, which comprises roughly 95% ofhealthy human beings, arrests the disease without vaccina-tion (3, 97). These individuals do not need BCG, becausethey produce a good immune response without it.

The remaining 5% of individuals (the low-respondinggroup) develop clinically active disease and may even die from

it. These individuals evidently produce an insufficient immuneresponse, so an effective TB vaccine could reduce the numberof clinically active tuberculosis cases to 1%. In other words, theTB vaccine would protect about 80% of this group (6, 7, 74,92). Complete protection of every individual may never beachieved.

Note that the 95%, 4%, and 1% of individuals approximatethose found in populations in industrial countries (e.g., theUnited States and Europe). Developing countries with a highpercentage of immunodeficient individuals (e.g., human immu-nodeficiency virus [HIV]) would have a different proportion ineach group (27). The percentages found in industrial countriesare used herein merely to designate each group in a simplemanner.

High Prevalence of HIV

A high prevalence of infection with HIV exists in somedeveloping countries, especially in sub-Saharan Africa. HIVinfection lowers host acquired (adaptive) immunity to the tu-bercle bacillus (11, 27). Therefore, HIV-infected personswould respond less well to BCG vaccination than would per-sons who are not infected with HIV.

BCG vaccination would protect some M. tuberculosis-in-fected/HIV-infected individuals from developing clinically ac-tive disease when the HIV only partly decreased their immuneresponse. In other words, HIV infection would transfer someindividuals from the 95% group (who did not need the vaccine)to the intermediate group (who could be helped by the vac-cine). However, BCG vaccination would have no benefit orcould even be detrimental if HIV greatly lowered the immuneresponse. In this case, HIV would transfer individuals from the4% intermediate group (that would benefit from the vaccine)to the 1% immunodeficient group (that could not be helped bythe vaccine). In the Karonga/Malawi BCG trial, 57% of casesof clinical tuberculosis were directly attributable to HIV infec-tion (27).

High Prevalence of Intestinal Helminths and Poor Nutrition

In developing countries, tuberculosis and intestinal worminfections often occur in the same groups of people (106).Worm infections may cause some debilitation and lower hostresistance to tuberculosis (106). Therefore, worm-infestedpopulations would respond less well to BCG vaccination thanwould noninfested populations (47, 106). Poor nutrition maypossibly have a similar effect (18, 62).

Environmental Mycobacteria

Environmental mycobacteria could have increased immunityin unvaccinated control groups (20, 27, 48), so the beneficialeffects of BCG in the Malawi trial (27) and in other inconclu-sive trials (13, 20, 21, 48) would be hard to detect.

Absence of Booster PPD Skin Testing

Many individuals in every unvaccinated control groupmight show a booster reaction if tested again with tuberculin(purified protein derivative [PPD]) (23, 77, 101). If they did,

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they would already have substantial immunity from a healednatural infection, so BCG vaccination would provide rela-tively little additional benefit.

Variations in BCG Strains

Although the currently available BCG vaccines used in thesetrials were genetically different from the original BCG (13, 21,48, 80, 88), recent studies with guinea pigs indicated that thesedifferences may be small and not critical (56). However, whencurrently available BCG vaccines were evaluated with mice,some major differences were found (58).

Human Genetic Differences

BCG vaccination reduced clinical tuberculosis in NorthAmerican Indians by about 80% (6, 7). This indigenous pop-ulation had probably not been exposed to the tubercle bacillusfor as many centuries as had the populations in Europe andAsia and therefore might have had more individuals in thelow-responding group who could benefit from vaccination (98).In other words, fewer individuals were in the high-respondinggroup, who did not need BCG immunization. Also, the BCGstrains used in these studies may have more closely resembledthose originally developed by Calmette and Guerin.

In contrast, the BCG trial in Chingleput, South India(102), could have been unsuccessful partly because most ofthe individuals who were not infected with HIV were in thehigh-responding group, who required no vaccination. Inother words, the number of individuals in the low-respond-ing group who could be helped by vaccination was small andhard to detect. Also, some of the factors listed above mayhave made the benefits of BCG vaccination unrecognizable.

Conclusion

I am not a statistician, but I believe that even the largenumber of individuals in BCG clinical trials does not compen-sate for every factor listed above. Clinical trials would be muchmore precise if more of these factors were identified and wereaccurately balanced between the control and vaccinated groupsbeforehand. In other words, the many factors listed abovemake it hard to detect those individuals for whom BCG vac-cination actually prevented clinical disease (see Identifying the“�4%” Group That Would Be Helped by BCG Vaccinationbelow).

IDENTIFYING THE “�4%” GROUP THAT WOULD BEHELPED BY BCG VACCINATION

Most clinical trials take relatively few of the above-describedfactors into consideration. If we had a way to eliminate fromthe trial the �95% of individuals who can arrest an earlyprimary pulmonary TB lesion without clinically active diseaseas well as to eliminate from the trial the �1% of individualswho cannot be helped by the vaccine (because of some immu-nodeficiency), then the remaining �4% would undoubtedlyshow benefits from BCG vaccination comparable to thosefound for laboratory animals.

This �4% group was never specifically identified in the

human populations that participated in recorded TB vaccinetrials. However, with rabbits, Lurie et al. reported a way to doso (68), and this method could easily be used for human trials.Lurie found that his inbred resistant rabbits healed dermalBCG lesions faster than did his inbred susceptible rabbits.Therefore, Lurie chose the rabbits that healed their dermalBCG lesions the fastest as breeders for his resistant stock andchose the rabbits that healed their dermal BCG lesions theslowest as breeders for his two susceptible stocks.

Similarly, if standardized, the rate of healing of BCG skinlesions could be used on a sample of the human populationfor whom a clinical trial was planned. BCG lesions withintermediate rates of healing would identify the size of the�4% group that could benefit from the vaccine. If the �4%intermediate group was sufficiently large, the benefits of agood tuberculosis vaccine would be easily recognized, but ifthis �4% intermediate group was small, the candidate vac-cines should probably be evaluated in a more favorablehuman population.

Also, during the main clinical trial, the rates of healing in theBCG-vaccinated group could be determined. (BCG is oftenused as a positive control for new vaccines.) Individuals in the�4% group (who could benefit from vaccination) would againshow intermediate rates of BCG healing. Next, several yearslater, during the statistical analysis of the trial, the amount ofclinically active TB developed in these �4% of vaccinees couldbe compared to the amount in the unvaccinated control group.I predict that these �4% of BCG vaccinees will always show areduced amount of clinically active pulmonary tuberculosis.

Unfortunately, during the main clinical trial, there is no way ofidentifying this �4% group within the nonvaccinated controlgroup (which would have greatly improved the statistics). Never-theless, the identification of the �4% group in only the BCG-vaccinated group could reduce statistical variation enough to re-gain confidence in BCG vaccination for clinical use.

Note that (i) since millions of persons receive intradermalBCG, the delayed healing of their BCG lesions could identifyindividuals who are genetically most susceptible to M. tubercu-losis; (ii) the rate of healing of intradermal BCG lesions innewborns should not be compared with rate of healing ofintradermal BCG lesions in older individuals, because the im-mune system of newborns is still developing (see BCG in New-born Infants below); and (iii) also, if an individual had previ-ously received BCG (or had an inapparent TB infection), thenext dermal BCG lesion would progress more rapidly and healfaster (15).

STOPPING THE DEVELOPMENT OF PRIMARYPULMONARY TUBERCLES IS A MAIN REASONFOR VACCINATION AGAINST TUBERCULOSIS

FOR BOTH HUMANS ANDLABORATORY ANIMALS

TB vaccines are usually given to prevent clinically apparentdisease, i.e., to prevent early primary pulmonary tubercles fromprogressing to an X-ray-visible size. Vaccines have little or noeffect on the activation of pulmonary alveolar macrophages(AM), because most AM are nonspecifically activated by ingest-ing a variety of inhaled particles (36, 41) and not by the expandedantigen-specific lymphocyte population produced by the vaccine.

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In rabbits and in humans, most early pulmonary tubercles(caused by M. tuberculosis) are arrested by the host’s immuneresponse. Therefore, vaccine efficacy in rabbits can be mea-sured by a reduction in the number of primary tubercles seen5 weeks after the aerosol inhalation of virulent M. tuberculosis(see Tubercle Count Method below). However, early tuberclesin mice and guinea pigs are not easily arrested. Therefore, westrongly advise that tubercle counting in rabbits be included inall preclinical TB vaccine evaluations.

Effective vaccination of mice and guinea pigs slows or inhibitsbacillary growth, decreases bacillary titers in the lungs (see Com-parisons of Tuberculosis in Humans, Rabbits, Mice, and GuineaPigs below), and prolongs the life of the host. Vaccination wouldalso lower the number of visible primary pulmonary tubercles ifthe mice and guinea pigs were sacrificed at the appropriate time.However, mice and guinea pigs develop relatively poor TB im-munity, because they usually die of the disease. Therefore, thereduction in the number of visible primary tubercles should not benearly as great as the reduction produced by the same vaccine inrabbits (and humans). Also, in mice and guinea pigs, many tinyprimary tubercles that were not seen at the time of sacrifice wouldprobably become visible at a later date.

TUBERCLE COUNT METHOD

Tubercle counting has been performed mainly for rabbits.The number of grossly visible primary pulmonary tubercles(Fig. 1) is counted about 5 weeks after an aerosol exposure tovirulent M. tuberculosis (31, 33, 35, 64, 65, 67, 68, 71). Thenumber of visible primary tubercles is decreased by (i) thenumber of M. tuberculosis bacilli that are immediately de-stroyed by pulmonary alveolar macrophages and (ii) the num-ber of early tubercles that the immune response prevents fromreaching a visible size.

Mice and guinea pigs develop one primary lesion for every3 to 15 inhaled virulent M. tuberculosis bacilli (Table 1) (37,55, 85) and usually die of the disease. In other words, they

FIG. 1. Formalin-fixed lungs of a commercial New Zealand Whiterabbit that inhaled about 33,000 virulent human-type tubercle bacilli(H37Rv) 5 weeks previously. Upon dissection, these lungs contained131 grossly visible primary tubercles with no apparent grossly visiblesecondary tubercles. The “ratio” (i.e., the number of tubercle bacilliestimated to be inhaled divided by the number of grossly visible pri-mary tubercles produced) was 250. In other words, in this rabbit, 250viable H37Rv tubercle bacilli must be inhaled to produce each visibleprimary pulmonary tubercle. Effective BCG (and other effective vac-cines for tuberculosis) should increase this ratio at least 5-fold (35, 68).Small areas of caseous necrosis are visible in many of the tubercles. Onthe left, this photograph shows the ventral surface of the right upperlobe, right middle lobe, and azygous lobe. On the right, the photographshows the ventral surface of the left upper lobe and left lower lobe. Theright lower lobe (RLL) had been removed for culture. This RLLcontained 23 grossly visible tubercles and 1.35 � 105 culturable tuber-cle bacilli. Magnification, �1.04. (Reproduced from reference 31 withpermission of the publisher.)

TABLE 1. Number of inhaled tubercle bacilli required to produce one primary pulmonary tubercle (the “ratio”) andthe amount of multiplication during the logarithmic growth phase in

unvaccinated rabbits, mice, and guinea pigsa

Animal

Average no. ofinhaled virulent

bovine-typebacilli (Ravenel)

required to produce1 pulmonary tubercle

Average no. ofinhaled virulent

human-typebacilli (H37Rv)

required to produce1 pulmonary tubercle

Increase in no. ofvirulent bovine-type

bacilli (Ravenel)before stationary phase

Increase in no. ofvirulent human-type

bacilli (H37Rv)before stationary phase

Lurie’s resistant rabbitsb (includingcommercial rabbits)

3–15 300–3,000 1,000,000�e 1,000�e

Mice (C57BL/6)c 3–15 3–15 10,000� 10,000�f

Guinea pigsd 3–15 3–15 No data 1,000�g

a Adapted from reference 33. Note that this table shows the responses of the most frequently used laboratory animal species to the inhalation of virulent M.tuberculosis and virulent M. bovis. It also shows the importance of tissue-damaging DTH in limiting bacillary titers. Guinea pigs (with strong DTH) develop lowerbacillary titers than do mice (with weak DTH). However, in spite of fewer viable bacilli, the progression of the disease seems to be more rapid in guinea pigsbecause of their relatively poor cell-mediated immunity. Monkeys are very susceptible to M. tuberculosis, and, as with mice and guinea pigs, only 3 to 15 inhaledvirulent tubercle bacilli seem to be required to produce one visible primary pulmonary lesion (see Tuberculosis in Nonhuman Primates).

b Lurie’s resistant rabbits (e.g., strain III) (64, 65, 67, 69). Commercially available New Zealand White rabbits seem to be similar (24, 25, 33, 35, 37, 44, 71).c From reference 81. Bacillary multiplication in other mouse strains may be different from those listed here for C57BL/6 mice (see references 75 and 76).

C57BL/6 is a relatively resistant strain of mouse.d See references 55 and 85.e From Fig. 4.f From Fig. 2.g From Fig. 3.



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are much more susceptible than rabbits and humans. PriorBCG vaccination would reduce the amount of tuberculosisin mice and guinea pigs, because their primary lesions wouldbe smaller, and the disease would progress more slowly (Fig.2 and 3). However, in these hosts relatively few primarylesions would probably be fully arrested by the immuneresponse. The challenge of mice and guinea pigs with M.tuberculosis of reduced virulence would make tuberclecounting much more applicable to these hosts, but suchstudies have yet to be done. Counting of primary pulmonarytubercles in mice may require microscopy (see references 38and 39).

Three facts should be considered in evaluating TB vac-cines by tubercle counting in any laboratory animal. (i) Themore virulent the challenge strain of tubercle bacillus, thesmaller will be the number of inhaled bacilli required togenerate a visible primary tubercle (Table 1) and the smallerwill be the difference in tubercle counts produced by effec-tive and noneffective vaccines. (ii) Currently available rab-bits are not inbred. Therefore, at least 18 rabbits in eachgroup will often be required to get statistically significantresults, i.e., 18 in the nonvaccinated group, 18 in the BCG-vaccinated group, and also 18 in each group receiving a new

vaccine. This number was derived from data in reference 35by the late Helen Abbey of our Department of Biostatistics.(iii) Rabbits, mice, guinea pigs, and monkeys often responddifferently to each antigen in M. tuberculosis. Such speciesdifferences are unavoidable. This is the main reason why TBvaccines should be evaluated in several laboratory animalspecies before clinical trials are begun. Nevertheless, rab-bits, like the majority of human beings, have high native andacquired resistance to infection by virulent M. tuberculosisand would therefore be more likely than other laboratoryspecies to respond to TB antigens in similar ways. Unfortu-nately, except for the studies of Lurie et al. prior to 1964 (33,64, 68, 69) and the few studies by our TB group here at theJohns Hopkins Center for Tuberculosis Research (33, 35,

FIG. 2. Number of viable virulent human-type (H37Rv) tuberclebacilli in the lungs of unvaccinated or BCG-vaccinated C57BL/6mice at each interval following quantitative airborne infection.(C57BL/6 is a relatively resistant strain of mouse.) The vaccinatedmice received 106 viable BCG tubercle bacilli subcutaneously 6weeks before they were challenged by aerosol with H37Rv. Otherstrains of mice may respond somewhat differently (45). Note thatthe initial logarithmic growth phase was followed by a plateau andis similar to that of the guinea pigs represented in Fig. 3 and to thatof the rabbits represented in Fig. 4. Note also that the H37Rvbacillary titers reached higher levels in nonvaccinated mice andguinea pigs than in rabbits. Vaccination of mice lowers mycobacte-rial titers, because an effective immune response occurs faster.However, vaccination does not arrest the disease in mice (or inguinea pigs). (Courtesy of Ian M. Orme, Colorado State University,Fort Collins, CO; reproduced with permission.)

FIG. 3. Number of viable virulent human-type tubercle bacilli(H37Rv) in the lungs of BCG-vaccinated or control guinea pigs ateach interval following quantitative airborne infection. Note thatthe in vivo bacillary growth curves for guinea pigs resemble thosefound for mice (Fig. 2) and for rabbits (Fig. 4). (Other experimentsshowed that the stationary phase in guinea pigs continues at least 18weeks [2]). BCG-vaccinated guinea pigs are tuberculin positive be-fore challenge. Therefore, the logarithmic growth stage endssooner, and the number of viable tubercle bacilli is markedly re-duced. In the vaccinated host, the disease is less severe, and theanimals live longer. However, they usually die of the disease, be-cause they do not develop effective CMI. The guinea pigs wereimmunized intradermally with live BCG 6 weeks before the aerosolchallenge. Note also that BCG vaccination of guinea pigs lowers thebacillary titers 2 to 3 logs, whereas BCG in mice (Fig. 2) lowersthese titers only 1 log (see also reference 49). BCG makes theguinea pigs strongly tuberculin positive but produces only a weakDTH in mice. This comparison further supports the principle thattissue-damaging DTH is an important immunological host defenseagainst the intracellular multiplication of tubercle bacilli. However,Fig. 2 and 3 are not strictly comparable, because the mice repre-sented in Fig. 2 inhaled more tubercle bacilli than the guinea pigsrepresented in Fig. 3. (Reproduced from reference 95 with permis-sion of the American Thoracic Society. Copyright © AmericanThoracic Society.)



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44, 71), tubercle counting in rabbits has been almost com-pletely neglected.

THE LARGEST DIFFERENCE BETWEEN TWOCANDIDATE VACCINES WILL BE FOUND

IN HOSTS WITH THE STRONGESTIMMUNE RESPONSE

The differences between two TB vaccines may be harderto distinguish in mice and guinea pigs than in rabbits, be-cause mice and guinea pigs do not stop the growth of M.tuberculosis as well as rabbits and humans do: the moreeffective the host’s control of virulent tubercle bacilli, themore effective will be its immune response to vaccination. Inother words, differences between two vaccines will be moreeasily recognized for animals that develop the strongestimmune response, because the difference between the con-trol and the vaccinated animals will span a larger range.

This concept was clearly illustrated by Lurie et al. (68), whoshowed that the number of visible primary pulmonary tuber-cles (produced by M. tuberculosis) in susceptible rabbits wasnot appreciably decreased by prior BCG vaccination, whereasthe number of these primary tubercles in resistant rabbits wasdecreased to 20% of those found in the unvaccinated controls;i.e., the increased immunity produced by BCG in Lurie’s re-sistant rabbits prevented 80% of the developing tubercles fromreaching a visible size.

In other words, Lurie’s susceptible rabbits developedrather poor immunity during infection with virulent M. tu-berculosis and therefore developed relatively little increasein immunity from vaccination. His resistant rabbits devel-oped rather good immunity during infection with virulent M.tuberculosis and therefore developed a substantial increasein immunity from vaccination. Commercially available NewZealand White rabbits resemble Lurie’s resistant rabbits(24, 25, 35, 44, 71).

Similar to Lurie’s susceptible rabbits, mice and guineapigs develop relatively poor overall immunity during infec-tion with virulent M. tuberculosis. Therefore, vaccination ofmice and guinea pigs should have relatively little effect onstopping the eventual progression of primary pulmonarytubercles. In fact, the disease progresses in these susceptiblehosts and would usually be lethal in time (83). However,mice and guinea pigs show a good immune response to someM. tuberculosis antigens and would be useful in selectingthese antigens for TB vaccines (see below).

EFFECTIVE TB VACCINES MUST PRODUCEAPPROPRIATE AMOUNTS OF DELAYED-

TYPE HYPERSENSITIVITY AND CELL-MEDIATED IMMUNITY

Role of DTH and CMI in the Pathogenesis of Tuberculosis

TB is the classic disease with which to study the interplaybetween delayed-type hypersensitivity (DTH) and cell-medi-ated immunity (CMI) (30, 33, 40). DTH and CMI are similarimmunological processes produced by Th1 lymphocytes. How-ever, DTH and CMI inhibit the growth of M. tuberculosis bydifferent mechanisms. In guinea pigs, rabbits, and humans,

DTH kills nonactivated macrophages (that become overloadedwith M. tuberculosis) by producing solid caseous necrosis (inwhich the bacilli do not grow) (30, 33), whereas CMI activatesmacrophages so that ingested M. tuberculosis cells are inhibitedor even killed (30, 33, 70). Macrophages that are activated byCMI before they ingest tubercle bacilli are probably moreeffective than macrophages that are activated by CMI afterthey have ingested tubercle bacilli.

In guinea pigs, in rabbits, and, undoubtedly, in humans,tuberculin-like DTH stops the initial (intracellular) logarith-mic growth of tubercle bacilli in early pulmonary TB lesions bycausing solid caseous necrosis, in which the bacillus does notgrow. This conclusion is derived from correlating bacillarynumbers with the histopathology observed. In the tuberculoushost, tuberculin sensitivity and caseous necrosis always developat the same time (64; also see references 16 and 87). Beforetuberculin sensitivity develops, the bacillus multiplies intracel-lularly without injuring the macrophage in which it resides(64).

Nonactivated macrophages continually enter tuberculous le-sions, until the lesions are fully healed (32). Tubercle bacilligrow easily in these nonactivated macrophages. However,DTH kills these overloaded macrophages and stops furtherbacillary growth, often (as stated above) by causing solid case-ous necrosis, in which bacilli do not grow.

Many macrophages that have been activated by CMI sur-round the caseous center of the lesion. Such activated macro-phages ingest, inhibit, and even kill any free tubercle bacillithat they encounter (including the bacilli released from mac-rophages killed by DTH). Numerous CMI-activated macro-phages are necessary to stop the progression of the lesion.

In brief, within every TB lesion, nonactivated and activatedmacrophages are always present. Therefore, (i) both localDTH and local CMI are needed to arrest the disease, and (ii)good TB vaccines will enhance both DTH and CMI in theproper proportion.

Antigens Producing DTH and/or CMI

The main difference between antigens eliciting DTH andantigens eliciting CMI is the concentration at which they pro-duce these in vivo effects. In tuberculous lesions, tuberculin-like DTH antigens kill nonactivated macrophages within whichtubercle bacilli have multiplied extensively at very low localconcentrations, because the local concentration of the tuber-culin-like products soon reaches tissue-damaging levels (30,33). CMI antigens evidently activate macrophages to inhibit M.tuberculosis growth at higher concentrations (30, 33).

For skin testing of people, 1 tuberculin unit (1 TU) of PPD(first strength) or 5 TU (intermediate strength) is frequentlyused. One TU contains 0.00002 mg of PPD in the 0.1 ml usedfor the intradermal injection. If the second strength of PPD(250 TU, i.e., 0.005 mg) is injected intradermally in a personwho is known to be strongly tuberculin positive, caseous ne-crosis will develop at the site of the tuberculin injection. Inother words, the concentration of tuberculin is still very lowwhen it can stop the intracellular multiplication of the bacillusby killing bacillus-laden macrophages.

In a host with a good immune response, tuberculin-likeDTH antigens may be toxic when the host becomes hypersen-



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sitive to them, but CMI antigens are usually nontoxic. Never-theless, CMI antigens in very large concentrations would prob-ably produce necrosis, and tuberculin-like DTH antigens invery small concentrations do, in fact, activate macrophageswithout necrosis (5).

A list of DTH- and CMI-producing antigens has never beenmade. Many of those so far identified are reviewed in refer-ences 3, 4, 52, 61, 64, 79, and 89. In general, the proteins,peptides, and carbohydrates in tuberculin seem to produceDTH, and other proteins complexed with carbohydrates andlipids seem to produce CMI (reviewed in reference 33).

Note that to date, no laboratory has analyzed the known TBantigens for the amount of DTH and the amount of CMI thateach antigen produces. For DTH, such an investigation wouldinvolve determining the minimal concentration of each antigenthat elicits a positive (antigen-specific) skin test in tuberculousrabbits, guinea pigs, or even humans and, possibly, the minimalconcentration of each antigen required in vitro to kill macro-phages that contain live tubercle bacilli (see reference 61). ForCMI, such an investigation would involve determining the con-centration of each antigen that activates macrophages suffi-ciently to inhibit the intracellular growth of virulent tuberclebacilli. Unfortunately, the concentrations found in vitro may ormay not match those found locally in vivo.

WHEN VACCINATED FOR TB, LABORATORY ANIMALSPECIES DEVELOP DIFFERENT

AMOUNTS OF DTH AND CMI

Immune Response to DTH Antigens

Mice develop little DTH to tuberculin-like antigens.Guinea pigs and rabbits develop considerable DTH. Hu-

mans are more sensitive to tuberculin than any laboratoryanimal species (Table 2) (37, 42). In other words, tubercu-lin-like DTH antigens produce caseous necrosis in humansat very low concentrations and in guinea pigs and rabbits atsomewhat higher concentrations, but caseous necrosis usu-ally does not occur in mice. For this reason, I propose thatmice would be a poor species with which to recognize tu-berculin-like DTH antigens in new vaccines, and guinea pigsand rabbits would be rather good species.

Immune Response to CMI Antigens

CMI antigens (see Effective TB Vaccines Must ProduceAppropriate Amounts of Delayed-Type Hypersensitivity andCell-Mediated Immunity above) that are as specific as tuber-culin is for DTH remain to be identified. CMI antigens shouldbe studied (i) for their ability to activate macrophages and (ii)for their ability to cause caseous necrosis. I propose that theimmune response in mice (Fig. 2) is mainly a CMI response,because mice show little (or no) caseous necrosis and showonly a weak reaction to tuberculin. Also, I propose that theimmune response in guinea pigs (Fig. 3) is mainly a DTHresponse, because guinea pigs show considerable caseous ne-crosis and show a rather strong reaction to tuberculin. For thisreason, I propose that mice would be a good species with whichto recognize CMI antigens in new vaccines and that guinea pigswould be a rather poor species to do so.

Immune Response in both Mice and Guinea Pigs Together

The evaluation of new vaccines with both mice and in guineapigs may compensate for the proposed deficiencies in bothspecies. However, the inclusion of rabbits (a species for which

TABLE 2. Characteristics of tuberculosis in humans and in laboratory animalsa

Species

TB characteristicf

Tuberculin-typeallergy (DTH)

Caseous necrosis(due to DTH)b CMI Cavity

formation

Isolated human populationsc ��

Rhesus monkeysd ��

Guinea pigs ��

Modern humansImmunocompetent �� Immunosuppressed � ��

Rabbitse

Resistant �� Susceptible �� 0

Mice � �/� �� 0

a Reproduced from reference 37 and based on data from a table by Francis (50). References 29 and 100 reproduce the entire Francis table (which contains manyother animal species, including elephants).

b Tissue-damaging DTH is the cause of caseous necrosis. Tubercle bacilli are dormant in solid caseum, and many do not survive there. In arrested human and rabbittuberculous lesions, solid caseum is often encapsulated by fibrous tissue.

c Isolated human populations refer to those who had been exposed to M. tuberculosis only during the last several centuries, such as the Senegalese troops broughtfrom Africa to Europe during the first World War (14, 28; see reference 98).

d Cynomolgus monkeys are more resistant to tuberculosis than rhesus monkeys and may even arrest the disease (see the text).e The characteristics of tuberculosis in Lurie’s inbred resistant and susceptible rabbits are reviewed in references 64 and 66.f The assignment of � to �� is an estimate for a given species as a whole. Good CMI and DTH are both needed to arrest the disease. Guinea pigs have good

DTH but relatively poor CMI, and mice have good CMI but relatively poor DTH. Both species show progressive disease leading to their death. Most humans and rabbits(infected with virulent human-type tubercle bacilli) have adequate amounts of DTH and CMI to arrest the disease.



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DTH and CMI usually work together to arrest the disease)would make such evaluations more complete.

Since different antigens are recognized to different degreesby each laboratory animal species, vaccine evaluation with allcommon laboratory species—mice, guinea pigs, and rabbits(and even monkeys)—should provide the most informationbefore expensive clinical trials are begun.

Note that after M. tuberculosis is inhaled, the strong DTHdeveloped by humans probably stops the logarithmic growth ofthe bacillus sooner than does the DTH in rabbits or guineapigs. In fact, in most tuberculin-positive humans, arrested TBprimary lesions are so small that they cannot be identified by Xray during life (99).

COMPARISONS OF TUBERCULOSIS IN HUMANS,RABBITS, MICE, AND GUINEA PIGS

In rabbits, numerous inhaled M. tuberculosis cells are de-stroyed by pulmonary alveolar macrophages (Fig. 4) (30, 33,

37, 64, 69), but in mice and guinea pigs, relatively few inhaledM. tuberculosis cells are destroyed by alveolar macrophages(Table 1) (33, 37, 55). In humans, many tubercle bacilli seem tobe destroyed soon after their inhalation by alveolar macro-phages, because family members of tuberculous patients caninhale tubercle bacilli for many months and even years and stillremain tuberculin negative (see reference 98).

In rabbits, the innate and acquired immune responses arehighly effective in preventing tiny pulmonary tubercles fromreaching a visible size. Rabbits must inhale an average of 300to 3,000 M. tuberculosis cells to produce one visible primarytubercle (Table 1) (33, 64, 69). Humans must usually inhale anaverage of 20 to 200 bacilli to do so (estimated by the lateRichard L. Riley). However, in mice and guinea pigs, themajority of inhaled M. tuberculosis bacilli produce visible tu-bercles (Table 1) (33, 55, 78, 85).

Rabbits and most humans will eventually stop the progres-sion of visible primary pulmonary tubercles produced by viru-lent M. tuberculosis (33, 64), but mice and guinea pigs usually

FIG. 4. Number of viable human- or bovine-type tubercle bacilli in the lungs of Lurie’s natively resistant and susceptible inbred rabbitsat each interval following quantitative airborne infection (1, 33, 64). Commercially available New Zealand White rabbits resemble Lurie’sresistant strain of rabbits (24, 25, 33, 35, 37, 44, 71). This graph makes several points: (i) higher bacillary titers occur with the bovine typethan with the human type, because the bovine type is much more virulent for rabbits; (ii) higher bacillary titers occur with Lurie’s susceptiblerabbits, because they have less native and acquired immunity; (iii) many human-type tubercle bacilli (but not many of the more-virulentbovine type) are destroyed soon after they are inhaled by the resident pulmonary alveolar macrophages (AM); (iv) the AM of the resistantrabbits destroy more human-type tubercle bacilli than the AM of the susceptible rabbits; (v) the multiplications of the highly virulentbovine-type and the less-virulent human-type bacilli in both susceptible and resistant rabbits are the same during the logarithmic growthphase (i.e., both types of bacilli grow equally well in nonactivated macrophages); and (vi) acquired (adaptive) immunity has less effect onreducing bacillary numbers when the infecting strain is more virulent. Human-type tubercle bacilli are more virulent for mice and guineapigs than they are for rabbits. Therefore, TB vaccines would be less beneficial for mice and guinea pigs than would TB vaccines for rabbits.Also, TB vaccines in mice and guinea pigs would have less ability to detect differences in two or more proposed vaccines than would TBvaccines (tested by tubercle counting) in rabbits. This graph shows the increase in the number of viable bacilli relative to the initial numberdeposited in the pulmonary alveoli, which is the number zero in the graph. (The inhaled dose of the human type was roughly 100 times theinhaled dose of the bovine type because of differences in their virulences.) Additional rabbits would be needed to obtain the standard errorsfor each time point. However, these results are consistent with the tubercle count data on these same rabbit races (1, 64, 69). The numberof human- and bovine-type tubercle bacilli in the lungs of the resistant rabbits failed to decrease during the period illustrated, becauseliquefaction and cavity formation occurred (with the extracellular multiplication of the bacilli) (64). Liquefaction usually did not occur inthe susceptible rabbits (64), probably because their macrophages develop lower levels of hydrolytic enzymes (see reference 34). (Reproducedfrom reference 1 with permission of the American Thoracic Society. Copyright © American Thoracic Society.)



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cannot do so (75). Therefore, most primary tubercles in miceand guinea pigs progress, cause metastatic lesions, and even-tually kill the host (83).

In rabbits, guinea pigs, and humans, the solid caseuminhibits and even kills some tubercle bacilli due to toxic fattyacids and low pH (33, 54, 63, 84). However, in mice, over-loaded macrophages apparently undergo apoptosis, usuallywithout forming necrosis. In mice, the vasculature in thetubercles usually remains patent (33, 37), whereas in rabbits,guinea pigs, and humans, thrombosis of the tubercle’s cen-tral vasculature is a major cause of the caseous necrosis (seereference 26).

With time, cavitary tuberculosis usually occurs in rabbitsinfected with virulent M. bovis and occasionally occurs in rab-bits infected with M. tuberculosis (24, 25, 33, 34, 64). Cavitiesfrequently occur in tuberculous adult humans beings. How-ever, cavities do not occur in mice and only rarely occur inguinea pigs (96). Many of these species differences are de-scribed in references 12, 37, and 49.

Note that since a typical caseum does not form in mice,extracellular tubercle bacilli are not inhibited but remain readyto divide. These bacilli are soon ingested by viable macro-phages, and if these macrophages have not been activated, thebacilli will again start intracellular growth. Actively multiplyingtubercle bacilli released from killed mouse macrophages intononnecrotic tissues probably grow better when ingested bynearby nonactivated macrophages than do relatively dormantbacilli escaping from the solid caseous tissue found in otherhosts. Therefore, the inability of mice to form typical caseousnecrosis seems to be a factor in explaining why mice usually dieof progressing pulmonary granulomas in spite of their ability todevelop appreciable CMI. (References 104 and 105 discuss therole of CD8� T cells in mouse tuberculosis.)

TUBERCULOSIS IN NONHUMAN PRIMATES

Monkeys are genetically more similar to humans than toother animal laboratory species, but their response to inhaledM. tuberculosis is more like that of “isolated” human popula-tions (Table 2) (14, 37, 50, 53), such as the Senegalese troopswho were brought from Africa to Europe during the firstWorld War (14, 64). These troops developed the susceptible(hematogenously spread) childhood type of tuberculosis ratherthan the chronic cavitary (bronchial spread) type found in mostadults today. In other words, such “isolated” human beings didnot have the resistance to M. tuberculosis that most humanbeings developed after living with the disease for numerouscenturies (see references 28 and 98).

Two strains of nonhuman primates are being used to eval-uate TB vaccines: rhesus macaques (Macaca mulatta) (43a, 49,53, 59, 60) and cynomolgus macaques (Macaca fascicularis)(17, 49, 59, 103). Quantitative airborne infection of youngrhesus monkeys showed that a progressing primary tuberclewas produced by the majority of inhaled viable bacillary unitsof virulent M. tuberculosis that reached the alveolar spaces (10,86). Cynomolgus monkeys are natively more resistant thanrhesus monkeys. In fact, some cynomolgus monkeys infectedwith a low M. tuberculosis dose become tuberculin positive withno other evidence of the disease (17, 103). However, bothmonkey strains seem to be more susceptible to M. tuberculosis

than the majority of today’s human population (Table 2). Also,in both monkey strains, the type of tuberculosis produced var-ies considerably.

Younger monkeys in both groups (2 to 5 kg in weight) tendto develop a more rapid childhood type of TB, and oldermonkeys (over 7 kg in weight) tend to develop a more chronic,slowly progressive type (51). In other words, the immune sys-tem of monkeys (like that of humans) takes several years tomature. Young monkeys were used for studies reported inreferences 53 and 103, and older, mature monkeys were usedfor studies reported in references 17 and 59. Liquefaction andcavity formation were occasionally observed for older monkeysafter an intratracheal challenge with a rather high dose of M.tuberculosis (59) and after an aerosol challenge with a lowerdose (9).

Quantitative airborne infection of rhesus monkeys with 12 to49 units of M. tuberculosis was sufficient to produce multiplegrossly visible pulmonary tubercles (9, 86). Therefore, rhesusmonkeys are as susceptible as guinea pigs to this disease.

Quantitative airborne infection of cynomolgus monkeysremains to be reported. Scientists at the Tulane NationalPrimate Research Center in Covington, LA, are exposingvaccinated cynomolgus monkeys (along with controls) toaerosols of M. tuberculosis. If they count the number ofprimary tubercles developed, they could calculate the “ra-tio” (31, 33, 64, 68), i.e., the number of inhaled M. tubercu-losis cells required to produce one visible primary tubercle(Table 1). Ratios of 10 to 50 define a susceptible animal thatwould develop rather poor immunity from a vaccine. Ratiosof 500 to 1,200 would define a resistant animal that (simi-larly to commercial rabbits) would develop good immunityfrom a vaccine. We truly need such information to decidewhether two vaccines compared by using monkeys wouldproduce as decisive a result as the same vaccines comparedby using rabbits. Such “ratios” cannot be obtained if mon-keys are challenged intratracheally (59, 103) or broncho-scopically (17, 60), because these procedures deposit numer-ous tubercle bacilli into one locale.

In nonhuman primates, tubercle counts would best be made5 to 10 weeks after challenge with virulent M. tuberculosis byaerosol. At such times, the primary lesions could be easilydistinguished from metastatic lesions, because the primary le-sions are much larger. To compare two or more TB vaccines innonhuman primates, further studies are needed on the effect ofage and on the effect of very low inhaled doses of M. tubercu-losis. However, all monkeys appear to be much more suscep-tible to M. tuberculosis than are human beings and thereforewould not be an adequate substitute for rabbits.

Primate facilities that expose monkeys to M. tuberculosis byaerosol could easily be used to infect rabbits. The TulaneNational Primate Research Center uses USAMRIID’s head-only aerosol exposure apparatus (46), which works well forcommercial New Zealand White rabbits (24, 33, 35, 44, 71).Comparisons of the efficacy of new TB vaccines in rabbits bytubercle counting should be more pertinent to the majority ofmodern-day human beings than comparisons of them in mon-keys and should also be less costly.

In brief, human beings and rabbits prevent most primarypulmonary tubercles caused by M. tuberculosis from developinginto clinically active disease, whereas monkeys (being much



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more susceptible) do not do so. The good overall immuneresponse of human beings is best modeled by tubercle countingin rabbits, where the protective effects of a good vaccine areeasily recognized by the reduction in the number of visibleprimary tubercles produced by an aerosol of M. tuberculosis.Good vaccines would show less benefit in monkeys, mice, andguinea pigs, because they are species that develop a less effec-tive immune response to M. tuberculosis than that which rabbitsand humans develop (see above).

BCG IN NEWBORN INFANTS

The vaccination of newborn infants is somewhat differentfrom the vaccination of more mature individuals. In clinicaltrials, newborn infants have usually benefited from BCG im-munization (8, 19, 20, 22, 48, 91). Since the cell-mediatedimmune system of newborns is relatively underdeveloped, liveBCG would multiply more extensively in them than in olderindividuals. The larger number of BCG bacilli would persistuntil these newborns become more immunocompetent. At thattime, these young vaccinees would be more effectively immu-nized than older vaccinees, because these youngsters had re-ceived a greater antigenic stimulus. On the other hand, be-cause of their underdeveloped immune system, newbornswould respond less well than older individuals to nonviablevaccines, unless those antigens persisted until the newbornswere more immunocompetent.

TB VACCINES HAVE THEIR MAIN EFFECTSEARLY IN DISEASE

Prophylactic vaccination expands antigen-specific T-lympho-cyte populations producing DTH and CMI and also expandsantigen-specific B-cell populations producing antibodies.These expanded antigen-specific lymphocyte populations rap-idly enter developing TB lesions (94). The logarithmic growthof inhaled virulent bacilli is stopped sooner, bacillary titers arereduced, and the host lives longer.

However, in both vaccinated and unvaccinated hosts, thevirulent tubercle bacilli in the challenge infection will soonsupply large quantities of antigens that will probably exceedthe quantity supplied by the vaccine. These M. tuberculosisantigens may, in time, cause the acquired (adaptive) immunitydeveloped in unvaccinated hosts to approximate that devel-oped in vaccinated hosts. Therefore, the main effect of pro-phylactic TB vaccination would be the rapid immune responsesoon after M. tuberculosis enters the host and not after theinfection has induced its own immunity, but see ProphylacticImmunization and Immunotherapy with Critical TB Antigensbelow.

PROPHYLACTIC IMMUNIZATION ANDIMMUNOTHERAPY WITH CRITICAL

TB ANTIGENS

Vaccines containing critical antigens may, however, increasehost resistance above that produced during an active TB in-fection. Vaccines containing critical antigens would expand thecorresponding Th1 lymphocyte population, and this Th1 pop-ulation would be increased even further by an active TB infec-

tion, even though the infecting virulent strain contained onlysmall amounts of such critical antigens.

Immunotherapy with critical antigens in patients who al-ready have active tuberculosis could have a similar beneficialeffect. Unfortunately, the use of critical antigens for both pro-phylactic immunization and immunotherapy is still in develop-mental stages (reviewed in references 3, 4, 43, 43a, 57, 89,and 90).

The effects of primary vaccination with live attenuated tu-bercle bacilli (often BCG) followed by a booster vaccinationwith critical antigens months or years later are currently beingevaluated (3, 43, 73, 79, 82, 89). This two-step vaccinationregimen is most promising, because it combines the multipleantigens of intact viable tubercle bacilli with the critical anti-gens that are found to have the greatest effect on host resis-tance.

Modified vaccinia virus Ankara expressing immunodomi-nant secreted antigen 85A (MVA85A) is already in clinicaltrials as a booster for persons who have had a positive tuber-culin skin test from BCG or a naturally acquired (arrested orlatent) TB infection (43, 72, 73, 93). It is too soon to knowwhether individuals receiving an MVA85A booster vaccinationwill develop less clinical tuberculosis than BCG-vaccinated in-dividuals who did not receive the booster.

Early-secreted antigenic target 6-kDa protein (ESAT-6),culture filtrate protein 10 (CFP-10), recombinant fusion pro-tein Mtb72F, and others (see references 3, 4, and 46) might bepromising critical antigens to boost the host’s immune re-sponse (89). To date, however, ESAT-6 and CFP-10 have beenused mainly with human peripheral blood mononuclear cells to(i) diagnose latent and active TB and (ii) assess the immuneresponse to new TB vaccines (43, 72, 73, 93). (BCG does notcontain these two antigens.)

With the genome of M. tuberculosis now known, many pos-sible critical antigens should soon become available for testing(discussed in reference 82). Some of these M. tuberculosis an-tigens may be more effective (or critical) than others in con-trolling the growth of the tubercle bacillus. The best TB vac-cine would enhance those critical antigens the most. Theaddition of pulmonary tubercle counting in rabbits to the cur-rent methods of antigen selection for TB vaccines should makesuch selections more precise.

ACTIVATION OF MACROPHAGES IS A MAJORGENETIC FACTOR IN RESISTANCE

TO TUBERCULOSIS

Pulmonary alveolar macrophages (AM) are nonspecificallyactivated by ingesting inhaled particles. The macrophages intuberculous lesions are specifically activated by immune pro-cesses. In Lurie’s inbred susceptible rabbits, both nonspecifi-cally activated macrophages (i.e., pulmonary alveolar macro-phages) and immunologically activated macrophages (intuberculous lesions) cannot destroy virulent human-type tuber-cle bacilli as well as can the macrophages in his inbred resistantrabbits (Fig. 4) (64, 69). Therefore, his susceptible rabbitsapparently have a defective macrophage-activating system.Whether such a defect occurs in other susceptible animals (oreven in humans [98]) remains to be determined. In humans,defects in the immune system, such as that found in HIV/



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AIDS, are a major cause of the increased susceptibility totuberculosis (11, 27).

DISCUSSION OF VARIOUS COMBINATIONS OFFACTORS THAT SHOULD BE CONSIDERED INPRECLINICAL (AND CLINICAL) TESTING OF

NEW TUBERCULOSIS VACCINES

TB Vaccination and Bacillary Virulence

Vaccination will have less effect on arresting the disease inhosts where M. tuberculosis is of high virulence. The amount ofacquired (adaptive) host resistance is superimposed and deter-mined by the amount of innate (genetic) host resistance (64).Therefore, the disease produced in mice, guinea pigs, andmonkeys (which have rather low resistance to M. tuberculosis)would be helped less by vaccination than would the diseaseproduced in rabbits and humans (which have high resistance toM. tuberculosis). The differences between two candidate vac-cines would be more apparent in rabbits and humans, becausethey would develop considerable acquired (adaptive) resis-tance to all good TB vaccines.

TB Vaccination and Bacillary Titers

Good TB vaccines would lower the bacillary titers in thelungs of all common laboratory species. These vaccines wouldproduce good DTH in guinea pigs, good CMI in mice, andboth good DTH and good CMI in rabbits and humans. Thegood DTH response of guinea pigs seems to lower the pulmo-nary bacillary titers more effectively than the good CMI re-sponse of mice (compare Fig. 3 with Fig. 2). However, becauseof their poor CMI, guinea pigs seem to die sooner than domice.

TB Vaccination and Tubercle Counts

Good TB vaccines lower primary pulmonary tubercle countsby producing both DTH and CMI. In immunized rabbits, mostprimary pulmonary tubercles that are not seen at 5 weeks afteraerosol infection remain arrested. In mice and guinea pigs,many nonvisible pulmonary tubercles at 5 weeks may becomevisible at 10 or 20 weeks, but such studies remain to be per-formed. These species differences reflect the susceptibility ofthe host (Table 2). However, tubercle counting in all labora-tory animals (including monkeys) could measure vaccine effi-cacy if the animal is necropsied at the right time.

Virulence, Bacillary Titers, and Tubercle Counts

In general, the greater the bacillary virulence, the greaterwill be the bacillary titer in the host (Fig. 4) and the fewer thenumber of inhaled bacilli will be required to generate onevisible primary pulmonary tubercle (Table 1).

CONCLUSIONS

This review is an effort to improve the selection of new TBvaccines by providing certain perspectives on the immunizationof humans, mice, guinea pigs, rabbits, and monkeys that have

not usually been considered in TB vaccine selection. Briefly,these perspectives are as follows.

(i) In human trials, BCG vaccination has not been consis-tently beneficial. However, in laboratory animals, BCG hasconsistently increased host resistance to challenge with M. tu-berculosis. We propose that the rate of healing of BCG lesions(used as a control for new vaccines in clinical trials) will iden-tify the 95% of humans who arrest infection with M. tubercu-losis without the need for vaccination. In the remaining 5% ofindividuals, the benefits of BCG vaccination should be easierto recognize and should be more consistent with those found inlaboratory animals.

(ii) The arrest of early pulmonary tubercles by the immuneprocess before they become clinically apparent is the verypurpose of TB vaccination. Early tubercles in mice and guineapigs are not as easily arrested, but most early pulmonary tu-bercles caused by M. tuberculosis in rabbits and humans arearrested.

(iii) Because of the expense, tubercle counting in rabbits hasnot been undertaken before starting much more expensiveclinical trials. However, tubercle counting in rabbits could se-lect the most effective new TB vaccines more precisely than anyother procedure. Therefore, tubercle counting could savemuch time and could save millions of dollars in getting betterTB vaccines into clinical use.

(iv) In mice and guinea pigs, differences between two TBvaccines are harder to distinguish, because the ability of theseanimals to stop the growth of M. tuberculosis is much lesseffective than the ability of rabbits and humans to do so. Themore effective the immune response to virulent tubercle bacilli,the more effective will be the immune response to vaccination.In other words, differences between two vaccines may be moreeasily recognized by pulmonary tubercle counts in animals thatdevelop a good immune response, because the difference be-tween the control and the vaccinated animals spans a widerrange.

(v) Both cell-mediated immunity (CMI) and delayed-typehypersensitivity (DTH) must be produced in a host to arrestthe progress of tuberculosis. CMI and DTH are similar immu-nological processes involving Th1 lymphocytes. However, CMIand DTH inhibit the growth of M. tuberculosis by differentmechanisms. CMI activates macrophages so that they inhibitthe growth of the M. tuberculosis cells that they ingest. DTHkills nonactivated macrophages that become overloaded withM. tuberculosis by producing solid caseous necrosis in whichthe bacillus does not grow. (Nonactivated macrophages arepresent in every active tuberculous lesion and may ingest tu-bercle bacilli.)

(vi) DTH and CMI are produced by different M. tubercu-losis antigens, and new vaccines must contain these antigensin the proper amounts. Mice (infected with M. tuberculosis)have weak tuberculin sensitivity (DTH) and apparently goodCMI, and they usually die of the disease. Guinea pigs (in-fected with M. tuberculosis) have good tuberculin sensitivity(DTH) and apparently weak CMI, and they also usually dieof the disease. However, most humans and rabbits (infectedwith M. tuberculosis) usually survive the disease. Therefore,we concluded that mice do not respond well to DTH-pro-ducing antigens and that guinea pigs apparently do notrespond well to CMI-producing antigens. However, humans



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and rabbits (species that usually arrest the disease producedby M. tuberculosis) evidently respond well to both DTH- andCMI-producing antigens.

(vii) The antigens recognized by mice and those recog-nized by guinea pigs together may (or may not) be the sameas the antigens recognized by rabbits. Also, the antigensrecognized by rabbits may (or may not) be the same as theantigens recognized by humans. Such differences and simi-larities remain to be investigated. Therefore, we urge inves-tigators to always include rabbits along with mice, guineapigs, and, perhaps, monkeys in the preclinical testing of newTB vaccines in order to make preclinical studies more com-plete.

(viii) Vaccines containing critical antigens (possiblyESAT-6 or CFP-10) might increase the immunity of the hostto a greater extent than antigens produced by a natural M.tuberculosis infection. Such critical antigens would increasethe host’s ability to neutralize key components of M. tuber-culosis. However, only some critical antigens have so farbeen identified. When identified, these critical antigenscould then be used for TB prophylaxis and/or TB immuno-therapy.

ACKNOWLEDGMENTS

I am indebted to Paul J. Converse for many helpful suggestionsconcerning this report and to William R. Bishai and Ying Zhang forreviewing it. They are members of our Johns Hopkins Center forTuberculosis Research.

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Arthur M. Dannenberg, Jr., spent his 60-year research career unraveling the intrica-cies of tuberculosis in the rabbit model. Hereceived his M.D. from Harvard MedicalSchool in 1947 and Ph.D. in Microbiologyfrom the University of Pennsylvania in 1952under Professor Max B. Lurie, who was theforemost experimental pathologist of tuber-culosis at that time. Dr. Lurie outlined theresearch career that Dr. Dannenberg pur-sued for the rest of his life. Dr. Dannenbergwas a Postdoctoral Fellow in biochemistry at the University of Utahunder Emil L. Smith (1952 to 1954), a Lt. Cdr. in infectious disease atU.S. Naval Medical Research Unit 1 at the University of California(1954 to 1956), an Assistant Professor in the experimental pathology oftuberculosis at the University of Pennsylvania (1956 to 1964), and anAssociate Professor, and then a full Professor, at the Johns HopkinsBloomberg School of Public Health, where he continued his studies(1964 to present).



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