113

Immune Responses Stimulated by Percutaneous and Intradermal Bacille

Calmette-Guerin
E. B. Kemp, R. B. Belshe, and D. F. Hoft National Institute ofAllergy and Infectious Diseases Vaccine and
Treatment Evaluation Unit, Division of Infectious Diseases, Department
of Internal Medicine, Saint Louis University Health Sciences Center,
St. Louis, Missouri

Healthy volunteers were randomized to receive percutaneous or intradermal bacille Calmette-

Guerin (BCG) vaccination. Delayed-type hypersensitivity (DTH) to tuberculin, as well as prolifera-
tive and interferon-y responses in peripheral blood mononuclear cells stimulated by whole cell
lysates and culture filtrates of Mycobacterium tuberculosis and Mycobacterium avium-intracellulare,
were compared before and after vaccination. Positive DTH reactions were detected in 83% of
intradermal and 40% of percutaneous BCG recipients 3 months after vaccination (P < .004).
M. tuberculosis-specific proliferation was increased after intradermal BCG (P < .01) but not after
percutaneous BCG compared with pre vaccination responses. In addition, M. tuberculosis-specific
interferon-y production was increased after intradermal BCG compared with both prevaccination
responses (P < .04) and those measured after percutaneous BCG (P < .05). Predominant immune
responses stimulated by BCG were directed against antigens present in mycobacterial whole cell
lysate. These results indicate that the BCG vaccination route can affect both in vivo and in vitro
immune responses.

Bacille Calmette-Guerin (BCG), an attenuated strain of My-
cobacterium bovis, is the only vaccine available for the preven-
tion of disease related to mycobacterial infection. Although
originally given orally when used against human tuberculosis
(TB) in 1921, percutaneous and intradermal methods of BCG
administration have generally replaced oral vaccination [1, 2].
Cutaneous vaccination methods have largely supplanted oral
vaccination for various reasons, including a lower dose require-
ment for the induction of positive delayed-type hypersensitivity
(DTH) to intradermal tuberculin purified protein derivative
(PPD), suitability for vaccination of infants, cost, and reacto-
genicity profiles [1, 3, 4]. Percutaneous administration with a
multiple-puncture device is generally associated with lower
complication rates, but intradermal injection using a needle and
syringe is the most precise method of controlling the delivered
dose and therefore may more consistently induce mycobacteria-
specific immunity [1, 2, 4]. In the United States, percutaneous
Received 31 October 1995; revised 9 February 1996.
Presented in part: 35th Interscience Conference on Antimicrobial Agents
and Chemotherapy, San Francisco, September 1995 (abstract 2618).
Informed consent was obtained from all subjects, following the guidelines
of the US Department of Health and Human Services. The protocol was ap-
proved by the Saint Louis University Institutional Review Board and conducted
under a National Institutes of Health --sponsored Investigational New Drug
protocol.
Financial support: National Institutes of Health (AI-45250, funding the Vac-
cine and Treatment Evaluation Unit at Saint Louis University); Organon Tek-
nika, Rockville, MD.
Reprints or correspondence: Dr. D. F. Hoft, Dept. of Internal Medicine,
Division of Infectious Diseases and Immunology, 3635 Vista Ave., FDT-8N,
St. Louis. MO 63110.
The Journal of Infectious Diseases 1996; 174:113-9
© 1996 by The University of Chicago. All rights reserved.
0022-1899/961'7401- 00 1550 1.00
administration is the only route currently licensed for use of
BCG as a TB vaccine.
The relative efficacy of percutaneous and intradermal routes
of BCG vaccination is not known. No conclusive trials have
compared the ability of these different techniques of BCG
administration to protect against mycobacterial disease [1]. A
recent metaanalysis of previous BCG efficacy trials has indi-
cated that BCG can prevent 50% of cases of pulmonary TB
and 70% of TB-related deaths, but no conclusions could be
made concerning the effect of vaccine route on protective im-
munity [5]. Historically, emphasis has been placed on DTH
responses to tuberculin as a measure of immunity. Early clinical
trials focused on determinations of the optimal doses of freshly
cultured BCG that would induce high DTH conversion rates
to tuberculin (>90%) without producing unacceptable adverse
events [3]. Reports of similarly high conversion rates after
vaccination with freeze-dried BCG preparations are docu-
mented, although some investigators have reported less suc-
cessful rates, particularly with percutaneous BCG administra-
tion [3, 6, 7]. However, in both animal and human studies,
tuberculin conversion has not been found to be a strong corre-
late of protective mycobacterial immunity [1, 8-12]. To better
understand the nature of protective immunity induced by BCG
vaccination, including the effects of different vaccine routes,
more detailed investigations of immune responses other than
DTH reactions must be done.
We conducted a comparative trial using commercially avail-
able freeze-dried BCG administered either percutaneously by
multiple puncture technique or intradermally by needle and
syringe. For both methods, we used standard doses of BCG
currently recommended for use in the United States and world-
wide. Our specific aim was to test the hypothesis that different
114 Kemp ct al.
methods of BeG vaccination induce quantitative or qualitative
differences in mycobacterial specific immunity.
Materials and Methods
Human subjects and vaccination. Healthy volunteers between
the ages of 18 and 45 were recruited and considered for enrollment
if they had a negative history for previous infection with Mycobac-
terium tuberculosis, no past exposure to an active TB case, and
no previous positive PPD skin test. In addition, all enrolled subjects
had negative human immunodeficiency virus serology, a negative
reaction to a 5-TU PPD skin test, unremarkable complete blood
cell count, and a normal serum alanine liver transaminase level.
Sixty volunteers were enrolled and randomly assigned to receive
either 1.11 X 108 cfu (0.3 mL) of Tice BCG (Organon Teknika,
Rockville, MD) percutaneously or 2 X 106 cfu (0.1 mL diluted)
of Tice BCG intradermally in the deltoid area. The percutaneous
and intradermal groups were well matched for age (mean ages,
37.5 and 34.1, respectively), sex (12 male/l 8 female vs. 11 male/
19 female), and race (28 white/2 African-American vs. 27 white/
3 African-American).
The percutaneous immunization was done by spreading 0.3 mL
of the vaccine preparation on the surface of intact skin, followed
by superficial perforation of the epidermis through the vaccine
preparation with multiple tiny prongs to a depth of <2-3 mm.
The majority of the vaccine suspension used for percutaneous
vaccination was absorbed by sterile bandaging applied over the
site after vaccination. The intradermal vaccination involved injec-
tion of the entire vaccine dose into the dermis within a sequestered
interstitial location. Therefore, the inoculative dose of BCG was
probably much smaller after percutaneous than after intradermal
vaccination, despite the increased inoculum size used for percuta-
neous vaccination.
Volunteers were monitored for adverse reactions by examination
and phone contact. In vivo responses, including reactions at the
vaccination site and DTH response to a 10-TU PPD skin test 2
months after vaccination, were measured in the 59 persons who
completed the study. Additionally, repeat PPO responses to a 10-
TU skin test were measured 3 months after vaccination in 47
of the volunteers. Although the 5-TU PPD skin test has been
standardized for use in evaluating persons for evidence of infection
with M. tuberculosis, the 10-TU PPD skin test has been widely
used to evaluate the immunogenicity of commercially available
BCG vaccine strains. Furthermore, measurements of PPD re-
sponses to the IO-TU test are approved by the US Food and Drug
Administration for potency testing of each manufactured lot of
BCG vaccine.
Antigens. M. tuberculosis (Erdman strain) and Mycobacterium
avium-intracellulare (MAl) were grown to mid -log phase in glyc-
erol-alanine salts broth. For whole cell lysate preparation, myco-
bacterial pellets were heat-killed for 1 h at 80°C, disrupted by
sonication or bead vortex, and passed through 0.2-,um filters before
protein quantification (BCA protein assay kit; Pierce, Rockford,
IL). Culture filtrates, representative of actively secreted mycobac-
terial proteins, were prepared from mid-log culture supernatants
passed through 0.2-pm filters and then concentrated and dialyzed
using an Amicon 10 diafiltrator (WR Grace, Danvers, MA). M.
tuberculosis whole cell lysate and culture filtrate were provided
lID 1996; 174 (July)
by 1. Belisle (Colorado State University; under terms of NIH con-
tract AI-25147). The MAl whole cell lysate and culture filtrate
were prepared at Saint Louis University. Preservative-free PPO
for in vitro studies was provided by Connaught Laboratories
(Swiftwater, PA).
Lymphoproliferative responses. Heparinized blood was col-
lected on the day of vaccination and 2 months after vaccination
from 10 volunteers selected randomly from each group. Cell sus-
pensions of human peripheral blood mononuclear cells (PBMC)
were prepared by Histopaque separation (Sigma, St. Louis, MO)
and diluted in RPMI 1640 supplemented with glutamine, penicillin,
streptomycin, (3-mercaptoethanol, and 10% human AB serum be-
fore adding 10 5 PBMC/l 00 pL of media to individual wells of
96-well flat-bottomed plates. Mycobacterial antigens or media
were added and cells incubated for 7 days at 37°C with 5% CO 2 .
During the last 12- 16 h of incubation, cell cultures were pulsed
with 0.5 ,uCi of eH]thymidine before harvesting cells onto filter
mats (Skatron, Sterling, VA) and counting the incorporated radio-
activity. On the basis of our previous studies that optimized the
dose responses (unpublished data), 2-,ug/mL and 1O-,ug/mL quanti-
ties of mycobacterial protein were selected for stimulating cultures
of PBMC.
Cytokine induction assays. The same volunteers examined for
mycobacteria-specific T cell proliferative responses were studied
for T cell cytokine responses. PBMC were incubated in 24-well
tissue culture plates (4 X 106 cells in 1 mLiwell) in the presence
of medium alone or mycobacteriallysates for 5 days at 37°C with
5% CO 2 , The supernatants were collected and stored at - 70°C.
Interferon (IFN)-y and interleukin (IL)-4 levels were measured
by ELISA. Immulon II microtiter plates (Oynatech Laboratories,
Chantilly, VA) were coated overnight at 4°C with mouse mono-
clonal anti-human IFN-y (MAb 400-45/6; Chemicon, Temecula,
CA) or anti-human IL-4 (1842-01; Genzyme, Cambridge, MA) in
0.1 M carbonate buffer, washed with PBS~ Tween 20 (0.05%),
blocked with PBS with 10% fetal calf serum, and washed again
before experimental culture supernatants were added. After 3 h of
incubation at 3rC, wells were washed again and bound cytokine
was detected with polyclonal rabbit anti-human IFN-y (Endogen,
Cambridge, MA) or polyclonal sheep anti-human IL-4 (Genzyme).
Finally, donkey anti-rabbit IgG (Jackson Immunologicals, West
Grove, PA) or donkey anti-sheep IgG (Genzyme) conjugated to
horseradish peroxidase, followed by ABTS substrate (Kirke-
gaard & Perry, Gaithersburg, MO), were added to complete the
assays. Recombinant human IFN-y (Pharmingen, San Diego) and
human IL-4 (Genzyme) were used as standards to determine cyto-
kine concentration. The specificities of these cytokine-specific anti-
bodies have been evaluated by the manufacturers and confirmed
by us. The sensitivities of the IFN-y and IL-4 assays were I ng/
mL and 50 pg/mL, respectively, in our studies.
Statistical analyses. Nonparametric two-tailed Fisher's exact
tests were used to compare the proportions of volunteers devel-
oping positive PPD responses and ulcer formation. Mean values
of PPO size, duration of ulcer drainage, duration of erythema,
and duration of tenderness at the vaccination site were compared
between groups after vaccination with Mann-Whitney U tests
reporting the two-tailed P values. Proliferative and cytokine re-
sponses were subjected to Wilcoxon matched pairs tests (for com-
parisons before and after vaccination within a group) or Mann-
Whitney U tests (for comparisons between the percutaneous and
JID 1996; 174 (July) Percutaneous vs. Intradermal BeG Immunity
Table 1. Mean duration (weeks) of clinical reactogenicityafter per-
cutaneous and intradermal BCG vaccination.
Route of vaccination
Reaction Percutaneous Intradermal
Erythema 1.8 ::I:: 0.2 (14/30) 2.8 ::I:: 0.2 (28/29)
Soreness or tenderness 1.4 ::I:: 0.3 (5/30) 4.0 ::I:: 0.3 (26/29)
4.1 ::I:: 0.4 (24/29)
Uleer drainage 1.2 ::I:: 0.2 (5/30)
NOTE. Data in parentheses are no. of subjects with reaction/no. vacci-
nated.
* Mann-Whitney U tests; two-tailed P values are shown.
intradermal BeG groups after vaccination). Pearson product-mo-
ment tests of correlation between in vivo reactogenicity, in vivo
PPD responses, and in vitro immune responses were done. P <
.05 was considered significant.
Results
Reactogenicity. Percutaneous BCG vaccination (by the
multiple puncture technique) was associated with a lower react-
ogenicity profile compared with intradermal vaccination (table
1). Durations of erythema, soreness or tenderness, and drainage
at the site of vaccination were significantly shorter in the percu-
taneous group. In addition, the incidence of ulcer formation
was much lower in the percutaneous group and resolved more
rapidly. Only 5 of 30 volunteers vaccinated percutaneously
noted drainage, while 24 of 29 intradermally vaccinated volun-
teers developed draining ulcers at the vaccination site (P <
.001 by two-tailed Fisher's exact test), with a typical onset 4-
5 days after vaccination and a mean duration of 4.1 weeks.
Although there were significant differences between groups in
terms of clinical adverse events, none of the volunteers lost
working days or required specific treatment related to vaccina-
tion.
PPD responses. Mean size of induration of the DTH reac-
tion to PPD was significantly larger for the intradermal BCG
group than for the percutaneous BCG group at both 2 and 3
months after vaccination (table 2), although the range of re-
sponses varied from 0 to ~ 30 mm of induration in each group.
In addition, the proportions of volunteers converting to a posi-
tive PPD status, defined as ~ 5 mm of induration 48 h after
intradermal placement of 10 TU of tuberculin PPD, were
greater in the intradermal group (table 2). A positive PPD
response occurred in 76% of intradermal vaccinees (22/29),
while 50% of the 30 percutaneous vaccinees converted to a
positive PPD status at 2 months after vaccination (P = .06).
A repeat PPD test with 10 TU of tuberculin at 3 months was
done in 47 of the volunteers. The positive conversion rate
increased to 83% (19/23) for intradermal vaccinees but de-
creased to 40% 00/25) for percutaneous vaccinees (P < .004).
Lymphoproliferative responses. To assess the relative lev-
els of antigen-specific cellular immune memory induced by
115
percutaneous and intradermal BCG vaccination, we measured
the in vitro lymphoproliferative responses in PBMC stimulated
by different mycobacterial lysates before and after vaccination
(figure I). Postvaccination lymphoproliferative responses to
p* mycobacterial antigens were not significantly increased in the
percutaneous BCG group compared with prevaccination re-
.11
sponses. In contrast, significantly increased lymphoprolifera-
<.003
tive responses after vaccination were detected in the intrader-
<.002
mal BeG group after in vitro stimulation with both doses of
M. tuberculosis whole cell lysate (P < .01, Wilcoxon matched
pairs tests) and the higher M. tuberculosis culture filtrate dose
(P < .03, Wilcoxon matched pairs test). In addition, lympho-
proliferative responses stimulated by 2 j..tg/mL M. tuberculosis
whole cell lysate and 10 j..tg/mL M. tuberculosis culture filtrate
were significantly greater in the intradermal group than in the
percutaneous group on day 56 after vaccination (P < .05,
Mann-Whitney Utests). Lymphoproliferative responses stimu-
lated by MAl antigens were not increased in either group after
BCG vaccination (figure I). Mycobacterial whole cell lysates
stimulated higher absolute responses than mycobacterial cul-
ture filtrates both before and after vaccination. The highest
absolute levels of lymphoproliferative responses, as well as
the most significant up-regulation of M. tuberculosis-specific
responses, were detected when PBMC from intradermally vac-
cinated volunteers were stimulated with whole cell lysates of
M tuberculosis (figure lB).
T cell cytokine responses. To measure immune responses
representative of different subsets of CD4 lymphocytes, we did
cytokine-specific ELISAs with PBMC stimulated in vitro with
mycobacterial lysates. We studied IFN-y and IL-4 responses
before and after vaccination as representative cytokines pro-
duced by CD4 Th 1 and Th2 lymphocytes, respectively. IFN-
y responses were not significantly increased after vaccination
in the percutaneous BCG group after in vitro stimulation with
any of the mycobacteriallysates tested (figure 2A). In addition,
the levels oflFN-y induced in PBMC before and after vaccina-
tion in the intradermal BCG group were similar after in vitro
Table 2. Delayed-typehypersensitivity responses to tuberculin after
percutaneous and intradermal BCG vaccination.
Route of vaccination
Time point,
response Percutaneous Intradermal P
2 months
Induration * 7.0 (l.5) 14.3 (1.9) <.005
Positivity" 15/30 (50) 22/29 (76) .06
3 months
Induration* 7.3 (1.9) 13.8 (1.9) <.05
19/23 (83) <.004
Positivity" 10/25 (40)
* Mean ::I:: SE in mm, 48-72 h after 10 TU of tuberculin. Groups were
compared with Mann-Whitney U tests; two-tailed P values are shown.
t No. positive/total (%); positive response was defined as ~5 mm of indura-
tion 48-72 h after 10 TU of tuberculin. Rates were compared by Fisher's
exact tests; two-tailed P values are shown.
116 Kemp et al. JID 1996; 174 (July)

p<O.01
120000 A. Percutaneous B. Intradermal I I

100000

80000
:E
0-
c 60000

40000
I
20000

0
Media MtbWL MtbWL MAIWL MAIWL Media MtbWL MtbWL MAIWL MAIWL
2ug/ml 10ug/ml 2ug/ml 10 ug/ml 2ug/ml 10ug/ml 2ug/ml 10 ug/ml

120000 C. Percutaneous D. Intradermal

100000

80000 -
:E:
p<O.03
~ 60000 II

40000

20000 -

0
Media Mtb CF MtbCF MAICF MAICF Media Mtb CF MtbCF MAICF MAICF
2ug/ml 10ug/ml 2ug/ml 10ug/ml 2ug/ml 10ug/ml 2ug/ml 10ug/ml

10 Day a • Day 561

Figure 1. Mycobacterium-specific human Iymphoproliferative responses are induced by intradermal but not percutaneous BCG vaccination.
A, B, Proliferative responses in peripheral blood mononuclear cells after in vitro stimulation with 2 and 10 ,ug/mL of Mycobacterium tuberculosis
whole cell lysate (Mtb WL) and Mycobacterium avium-intracellulare whole cell lysate (MAl WL); C, D, Proliferative responses stimulated
by 2 and 10 ,ug/mL of M. tuberculosis culture filtrate (Mtb CF) and MAl culture filtrate (MAl CF). Data are means (n = 10 for each group)
± SEs for pre- (day 0) and postvaccination (day 56) responses. Significant differences between pre- and postvaccination responses (Wilcoxon
matched pairs tests) are indicated.

stimulation with M. tuberculosis culture filtrate or MAl whole
cell lysate (figure 2B). In contrast, the levels oflFN-y induced
by M. tuberculosis whole cell lysate were significantly greater
in PBMC from intradermal vaccinees 2 months after vaccina-
tion compared with baseline responses (P < .04, Wilcoxon
matched pairs test). Furthermore, the levels of IFN-y stimu-
lated by M tuberculosis whole cell lysate were significantly
greater in PBMC from intradermal BCG recipients than in
PBMC from percutaneous BCG recipients on day 56 after vac-
cination (P < .05, Mann-Whitney U test). IL-4 responses were
not detected in the culture supernatants of PBMC harvested
from either vaccine group after stimulation with any of the
mycobacterial lysates tested (data not shown).
In vivo and in vitro correlations. When we used Pearson
product-moment correlation analyses with the data obtained
from all 20 volunteers studied for in vivo and in vitro responses,
significant correlations between reactogenicity, in vivo DTH
responses, and in vitro immune responses were detected. The
size (millimeters of induration) of the DTH response to PPD
measured 3 months after vaccination correlated with the dura-
tion of ulcer drainage (R = .502; P = .04). The duration of
soreness or tenderness at the vaccination site was correlated
with both the up-regulation of in vitro proliferative responses
after vaccination (R = .510; P = .022) and the up-regulation
of in vitro IFN-y production after vaccination (R = .606; P =
.005) stimulated by the optimal dose (2 I-Lg/mL) of M. tubercu-
losis whole cell lysate. Furthermore, in vivo responses to PPD
(millimeters of induration) at 2 months after vaccination were
correlated with both in vitro proliferative responses (R = .600;
P = .005) and in vitro IFN-y responses (R = .569; P = .009)
stimulated by the optimal dose (2 I-Lg/mL) of M. tuberculosis
whole cell lysate 2 months after vaccination.
JID 1996; 174 (July) Percutaneous
A. Percutaneous
20
-
Figure 2. Mycobacterium - specific
interferon (I FN )-1' responses are in-
duced by intradermal but not per-
-
E
.s 15
0)
-
cutaneous BCG vaccination. Mtb, <t-
Z
Mvcobacterium tuberculosis; MAl, ~
Mvcobacterium avium-intracellulare; "0 10
Q)
WL, whole cell lysate; CF, culture
~
filtrate. Data are means (n = 10 for o
Q)
each group) ± SEs for pre- (day 0) en 5
and postvaccination (day 56) re-
sponses. Significant difference be-
tween pre- and postvaccination re-
sponses (Wilcoxon matched pairs
tests) is indicated. Media
Discussion
Cellular immune responses are believed to be the most im-
portant protective responses that develop naturally during pri-
mary infection with M. tuberculosis. It is also reasonable to
believe that cellular immunity is important after BCG vaccina-
tion in protecting humans against active TB, and work in animal
models confirms this hypothesi s [13, 14]. The detai led mecha-
nisms responsible for the induction of this protective immunity
are not known [1, 8], and there are no known clinical laboratory
markers that can be used to accurately predict the protective
efficacy of BCG vaccine strains. Analyses of the detailed im-
mune subsets stimulated by BCG vaccination will be important
for the rational design of more effective TB vaccines and for
the most appropriate use of BCG as a vector for the construction
of vaccines directed against other infectious pathogens.
The importance of cellular immunity in control of M. tuber-
culosis infection has led to the practice of monitoring the levels
of OTH responses to PPO stimulated by BCG vaccination as
a presumed measurement of vaccine efficacy. However, OTH
responses measure only a subset of host cellular immune re-
sponses, and OTH responses to PPO do not closely correlate
with BCG-induced protective immunity [1, 8]. In general,
BCG-vaccinated animals that become PPO-positive have been
observed to be better protected against virulent M. tuberculosis
challenges than similarly vaccinated animals that do not de-
velop detectable OTH to PPO [8, 9]. However, strong OTH
responses have been stimulated in some trials of BCG vaccina-
tion without the induction of protective immunity [10]. Con-
versely, PPO-negative animals after BCG vaccination have
been found to be protected from mortality after virulent M.
tuberculosis challenge despite their negative OTH status [8]. In
humans, although it has been reported that there is a reciprocal
relationship between the extent of TB disease and the OTH
VS. Intradermal BeG Immunity 117
B. Intradermal
p<O.04
,------,
MtbWL MtbCF MAIWL Media MtbWL MtbCF MAIWL
2ug/ml 2ug/ml 2ug/ml 2ug/ml 2ug/ml 2ug/ml
I D Day 0 • Day 56 1
reaction, large field trials have failed to show that PPO reactiv-
ity after BCG vaccination correlates with protection against
TB [I, 8, 12].
Recent evidence has shown that two populations of C04 T
helper lymphocytes that produce distinct profiles of cytokines
after antigenic stimulation can be differentially stimulated and
can have opposing effects on resistance and susceptibility [15-
17]. Th 1 cells, which produce IFN-y and IL-2 after antigenic
stimulation, are increased in animals resistant to infection with
various intracellular parasites. Th2 cells secrete IL-4, IL-5, TL-
6, and IL-IO after antigenic stimulation and are preferentially
expanded in mice with increased susceptibility to intracellular
parasite infections. Furthermore, IFN-y can increase the killing
activity of murine macrophages for mycobacteria, although it
has been difficult to demonstrate such killing activity in human
macrophages [14, 18, 19]. Acquired immunity to TB appears
to be more complex than exclusively Th I or Th2 cytokine
response profiles [20, 21], but it is reasonable to hypothesize
that the induction of IFN-y responses in Th 1 cells is one im-
portant component of the protective immune responses induced
by BCG against mycobacteria. Our results demonstrating that
intradermal BCG vaccination induced a significantly increased
Th I-like cytokine response to M. tuberculosis whole cell lysate
antigens compared with percutaneous BCG vaccination suggest
that intradermal BCG vaccination may induce higher levels of
protective immunity.
Cellular immune responses induced by whole cell lysates of
mycobacteria were the most consistent marker of a vaccine
effect. Both T cell proliferative and cytokine responses induced
by whole celllysates were significantly increased after intrader-
mal BCG vaccination. In contrast, only T cell proliferative
responses induced by a single dose of M. tuberculosis culture
filtrate were significantly increased after intradermal BCG vac-
cination. Recent work on mycobacterial immunity has focused
118 Kemp et al.
on antigens secreted by viable mycobacteria, using the proteins
present in the culture filtrate or supernatant of mycobacterial
cultures during an early logarithmic phase of growth as a source
for these antigens [18, 22-28]. Major components of these
secreted mycobacterial proteins have been shown to induce
immune responses that protect animals from virulent M. tuber-
culosis challenge and are being developed as candidate subunit
vaccines [27,28]. Our results demonstrating that BCG vaccina-
tion induced only limited immunity directed against mycobac-
terial culture filtrate antigens suggest that BCG does not induce
optimal immune responses against these important antigens.
This may explain why BeG is only partially protective against
tuberculosis. On the other hand, a recent metaanalysis con-
cluded that BCG significantly reduces the risk of pulmonary
TB by 50% and decreases TB-related deaths by 71% [5]. M.
tuberculosis whole cell lysates may contain non secreted anti-
gens not found in M. tuberculosis culture filtrates that are in-
volved in mycobacterium-specific protective immunity.
In addition to the need to identify the subsets of cellular
immunity and specific mycobacterial antigens important in the
protective immunity induced by BCG vaccination, it will be
important to consider the effects of different routes of BCG
administration. Conclusive efficacy trials focusing on the role
of vaccine route in humans have not been published. The induc-
tion of increased PPD reactions by intradermal BCG vaccina-
tion compared with percutaneous BCG vaccination has been
documented [6, 7]. To our knowledge, our study is the first to
report that in addition to increased DTH responses, both human
lymphoproliferative and IFN-y responses are greater in magni-
tude after intradermal than after percutaneous BCG vaccina-
tion. This was in spite of the use of 40-50 times more colony-
forming units of the same lot of Tice BCG per volunteer for
the percutaneous vaccination protocol than was used for the
intradermal vaccination protocol. These results suggest that
intradermal BCG vaccination may represent a more efficient
method of inducing mycobacteria-specific cell-mediated im-
mune responses.
The percutaneous dose of Tice BCG used in our trial is
currently licensed by the US Food and Drug Administration.
The intradermal dose used in our trial corresponds to a standard
dose recommended by the World Health Organization for intra-
dermal BCG vaccination. These doses for percutaneous and
intradermal BCG vaccination were optimized in previous clini-
cal trials based upon the highest doses that induced maximal
PPD conversion rates with minimal reactogenicity. Although
the amount of BCG required for percutaneous vaccination is
~ 50 times greater than the amount required for intradermal
vaccination, the "effective" or "inoculative" dose is probably
much smaller for percutaneous than for intradermal BCG vacci-
nation. The vast majority of the percutaneous vaccine prepara-
tion never penetrates the stratum corneum and therefore cannot
infect suitable host cells, replicate, or be involved in immune
induction. Therefore, it is unlikely that the higher dose used
for percutaneous vaccination could have induced high-dose
.TID 1996; 174 (July)
tolerance as a potential cause for the lower levels of cell-
mediated immune responses measured in our experiments.
Until more is known about how the specific subsets of immu-
nity are related to protection, it is difficult to recommend one
route of BCG vaccination over another. Our study supports
others in noting a relatively high incidence of ulcer formation
with drainage that may persist for several weeks after intrader-
mal vaccination [29], although our volunteers considered these
adverse reactions to be mild to moderate in severity and easily
tolerated. The percutaneous route of BCG vaccination was as-
sociated with a much lower incidence of ulceration and drain-
age but was also less effective in the induction of immune
responses. In addition, we detected significant correlations be-
tween reactogenicity, in vivo DTH responses, and in vitro im-
mune responses in our volunteers. These results suggest that a
higher level of in vivo vaccine replication is necessary to induce
optimal immune responses, and it may be difficult to separate
clinical reactogenicity from the stimulation of protective immu-
nity by live BCG vaccination.
Acknowledgments
We thank Carolyn DeRousse and .Jan Tennant for excellent
nursing assistance and Tammy Grant for help with manuscript
preparation.
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