Immune responses induced in cattle by vaccination with a recombinant adenovirus expressing Mycobacterial antigen 85A and Mycobacterium bovis BCG.
ABSTRACT Cattle were vaccinated with an adenovirus expressing the mycobacterial antigen 85A (rAd85A), with Mycobacterium bovis BCG followed by rAd85A heterologous boosting, or with rAd85A followed by BCG boosting. BCG/rAd85A resulted in the highest direct gamma interferon responses. Cultured enzyme-linked immunospot assay analysis demonstrated that memory responses were induced by all three protocols but were strongest after BCG/rAd85A and rAd85A/BCG vaccination.
- SourceAvailable from: Mariano Domingo[Show abstract] [Hide abstract]
ABSTRACT: The "One world, one health" initiative emphasizes the need for new strategies to control human and animal tuberculosis (TB) based on their shared interface. A good example would be the development of novel universal vaccines against Mycobacterium tuberculosis complex (MTBC) infection. This study uses the goat model, a natural TB host, to assess the protective effectiveness of a new vaccine candidate in combination with Bacillus Calmette-Guerin (BCG) vaccine. Thirty-three goat kids were divided in three groups: Group 1) vaccinated with BCG (week 0), Group 2) vaccinated with BCG and boosted 8 weeks later with a recombinant adenovirus expressing the MTBC antigens Ag85A, TB10.4, TB9.8 and Acr2 (AdTBF), and Group 3) unvaccinated controls. Later on, an endobronchial challenge with a low dose of M. caprae was performed (week 15). After necropsy (week 28), the pulmonary gross pathology was quantified using high resolution Computed Tomography. Small granulomatous pulmonary lesions (< 0.5 cm diameter) were also evaluated through a comprehensive qualitative histopathological analysis. M. caprae CFU were counted from pulmonary lymph nodes. The AdTBF improved the effects of BCG reducing gross lesion volume and bacterial load, as well as increasing weight gain. The number of Ag85A-specific gamma interferon-producing memory T-cells was identified as a predictor of vaccine efficacy. Specific cellular and humoral responses were measured throughout the 13-week post-challenge period, and correlated with the severity of lesions. Unvaccinated goats exhibited the typical pathological features of active TB in humans and domestic ruminants, while vaccinated goats showed only very small lesions. The data presented in this study indicate that multi-antigenic adenoviral vectored vaccines boosts protection conferred by vaccination with BCG.PLoS ONE 11/2013; 8(11):e81317. · 3.53 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: In this study we have investigated the potential of mycobacterial proteins as candidate subunit vaccines for bovine tuberculosis. In addition, we have explored the use of TLR-ligands as potential adjuvants in cattle. In vitro screening assays with whole blood from Mycobacterium bovis-infected and BCG-vaccinated cattle demonstrated that fusion protein constructs were most commonly recognised, and the ID83 fusion protein was selected for further immunisation studies. Furthermore, glucopyranosyl lipid A (GLA) and resiquimod (R848), agonists for TLR4 and TLR7/8 respectively, stimulated cytokine production (IL-12, TNF-α, MIP-1β and IL-10) in bovine dendritic cell cultures, and these were formulated as novel oil-in-water emulsions (GLA-SE and R848-SE) for immunisation studies. Immunisation with ID83 in a water-in-oil emulsion adjuvant (ISA70) induced both cell mediated and humoral immune responses, as characterised by antigen-specific IFN-γ production, cell proliferation, IgG1 and IgG2 antibody production. In comparison, ID83 immunisation with the novel adjuvants induced weaker (ID83/R848-SE) or no (ID83/GLA-SE) antigen-specific IFN-γ production and cell proliferation. However, both did induce ID83-specific antibody production, which was restricted to IgG1 antibody isotype. Overall, these results provide encouraging preliminary data for the further development of ID83 in vaccine strategies for bovine TB.Vaccine 09/2013; · 3.49 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: There is an urgent need to develop new tuberculosis (TB) vaccines to safely and effectively boost Bacille Calmette-Guérin (BCG)-triggered T cell immunity in humans. AdHu5Ag85A is a recombinant human type 5 adenovirus (AdHu5)-based TB vaccine with demonstrated efficacy in a number of animal species, yet it remains to be translated to human applications. In this phase 1 study, we evaluated the safety and immunogenicity of AdHu5Ag85A in both BCG-naïve and previously BCG-immunized healthy adults. Intramuscular immunization of AdHu5Ag85A was safe and well tolerated in both trial volunteer groups. Moreover, although AdHu5Ag85A was immunogenic in both trial volunteer groups, it much more potently boosted polyfunctional CD4(+) and CD8(+) T cell immunity in previously BCG-vaccinated volunteers. Furthermore, despite prevalent preexisting anti-AdHu5 humoral immunity in most of the trial volunteers, we found little evidence that such preexisting anti-AdHu5 immunity significantly dampened the potency of AdHu5Ag85A vaccine. This study supports further clinical investigations of the AdHu5Ag85A vaccine for human applications. It also suggests that the widely perceived negative effect of preexisting anti-AdHu5 immunity may not be universally applied to all AdHu5-based vaccines against different types of human pathogens.Science translational medicine 10/2013; 5(205):205ra134. · 14.41 Impact Factor
INFECTION AND IMMUNITY, Feb. 2006, p. 1416–1418
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 74, No. 2
Immune Responses Induced in Cattle by Vaccination with a
Recombinant Adenovirus Expressing Mycobacterial
Antigen 85A and Mycobacterium bovis BCG
H. Martin Vordermeier,1* Kris Huygen,2Mahavir Singh,3R. Glyn Hewinson,1
and Zhou Xing4
TB Research Group, Veterinary Laboratories Agency-Weybridge, New Haw, Addlestone, Surrey KT15 3NB, United Kingdom1;
Pasteur Institute of Brussels, Brussels, Belgium2; Lionex Limited, Mascheroder Weg 1, D38124 Braunschweig,
Germany3; and Department of Pathology and Molecular Medicine and Division of Infectious Diseases,
Centre for Gene Therapeutics, McMaster University, Hamilton, Ontario, Canada4
Received 1 July 2005/Returned for modification 23 September 2005/Accepted 26 October 2005
Cattle were vaccinated with an adenovirus expressing the mycobacterial antigen 85A (rAd85A), with Myco-
bacterium bovis BCG followed by rAd85A heterologous boosting, or with rAd85A followed by BCG boosting.
BCG/rAd85A resulted in the highest direct gamma interferon responses. Cultured enzyme-linked immunospot
assay analysis demonstrated that memory responses were induced by all three protocols but were strongest
after BCG/rAd85A and rAd85A/BCG vaccination.
Over the last 2 decades, there has been a steady rise in the
incidence of bovine tuberculosis (bTB) in cattle in Great Brit-
ain (7), and the development of a cattle bTB vaccine is con-
sidered the best option for its control (7). Mycobacterium bovis
bacillus Calmette-Guerin (BCG) is associated with variable
efficacy both in humans and in cattle, and improving its efficacy
is a priority (3, 6). Heterologous prime-boost strategies have
been developed to improve its efficacy (2, 8, 9, 13). In partic-
ular, application of the recombinant modified vaccinia virus
Ankara strain expressing the mycobacterial protective antigen
85A (Ag85A) (Rv3804c) has shown promise in small-animal
models of human tuberculosis (5, 19), and human phase I
clinical trials of BCG vaccination followed by MVA85A boost-
ing are under way (9, 10).
Recently, vaccination with a replication-deficient recombi-
nant adenovirus expressing Ag85A (rAd85A) protected mice
from M. tuberculosis infection (12, 17). To assess this viral
vaccine in cattle, calves were vaccinated with rAd85A in het-
erologous prime-boost scenarios together with BCG to deter-
mine its immunogenicity in this natural bTB target species.
rAd85A was prepared as described previously (17), and groups
of five calves (ca. 6-month-old Holstein females) were vacci-
nated with (i) rAd85A (109PFU/0.5 ml, delivered intramuscu-
larly) at week 0 and BCG Pasteur (Staten Serum Institute,
Copenhagen, Denmark; 106CFU/1 ml, delivered subcutane-
ously) at week 6, (ii) BCG Pasteur at week 0 and rAd85A at
week 6, or (iii) rAd85A at weeks 0 and 3. These boosting
intervals were chosen because rAd85A (12, 17) and BCG (16)
trigger peak responses at different times postvaccination (1 to
2 weeks and 3 to 5 weeks, respectively), and the intention was
not to perform the boost during peak responses, but at a time
when effector immune responses had decreased substantially.
Peripheral blood mononuclear cells (PBMC) were prepared
and cultured (14, 15) to establish the numbers of ex vivo
gamma interferon (IFN-?)-secreting cells (spot-forming cells
[SFC]) in enzyme-linked immunospot (ELISPOT) assays (14)
after in vitro stimulation with rAg85A (5 ?g/ml; Lionex,
Braunschweig, Germany) or bovine tuberculin purified protein
derivative (10 ?g/ml; Veterinary Laboratories Agency-Wey-
bridge). The results shown in Fig. 1A demonstrate that all
vaccination protocols resulted in the induction of T cells spe-
cific for Ag85A. Priming vaccination with rAd85A resulted in
rAg85A-specific responses peaking at weeks 1 to 3 for the
rAd85-rAd85A and rAd85A-BCG groups (254 ? 131 and 155
? 50 SFC/106PBMC, respectively). Similar response levels
against rAg85A were observed after BCG priming of the BCG-
rAd85A group. Although BCG-induced IFN-? response peaks
may vary between experiments, the responses of these BCG-
vaccinated calves were not significantly different from the re-
sults of earlier BCG vaccination experiments (16). In addition,
the response kinetic was identical to previous results obtained
after a single BCG vaccination, which resulted in peak re-
sponses between weeks 3 and 5 followed by a decline in re-
sponses towards prevaccination levels between weeks 6 and 8
postvaccination (16). Thus, the responses described up to week
6, i.e., immediately before boosting of the BCG prime group,
were identical to those observed previously following a single
BCG vaccination (BCG-alone group) and were expected to
further decline after week 6 if the cattle were not boosted.
Boosting BCG-primed calves with rAd85A resulted in sta-
tistically significant anamnestic IFN-? responses compared to
preboost peak levels (Fig. 1A) (five of five calves; mean pre-
boost BCG-alone peak value at week 4, 206 ? 67 SFC/106
PBMC; mean postboost peak value, 914 ? 255 SFC/106
PBMC; P ? 0.028). This enhanced ex vivo response peaked at
1 week post-rAd85A infection (week 7) and was significantly
different from those of the other two groups at this time point
(P ? 0.011 for comparison to postboost peak response at week
7 with rAd85A-BCG and at week 4 with rAd85A-rAd85A). Ex
* Corresponding author. Mailing address: TB Research Group,
VLA-Weybridge, New Haw, Addlestone, Surrey KT15 3NB, United
Kingdom. Phone: 44 1932 357 884. Fax: 44 1932 357 260. E-mail:
vivo responses contracted over the following weeks (Fig. 1A).
Boosting the animals in the rAd85A-rAd85A group with
rAd85A at week 3 resulted in enhanced responses compared to
preboost peak levels in two of five animals (mean level, 212 ?
111 SFC/106PBMC) (Fig. 1A), peaking 1 week after the boost.
Boosting rAd85A-primed cattle with BCG resulted in en-
hanced rAg85A responses in three of five animals compared to
the levels at the time of boosting at week 6 (100 ? 39 SFC/106
PBMC at week 6 versus 127 ? 80 SFC/106PBMC after the
boost) (Fig. 1A) and never exceeded the IFN-? levels observed
after rAd85A priming. Responses after ex vivo stimulation
with bovine purified protein derivative were also determined
and confirmed the results obtained with rAg85A (not shown).
One mechanism for the failure of BCG to recall rAd85A-
primed responses, or even to induce stronger responses on its
own, could be due to Ad85A-induced preexisting immunity to
BCG. A similar mechanism has been postulated to explain why
exposure to environmental mycobacteria results in immunity to
cross-reacting antigens that limits BCG multiplication and pro-
tective immunity (1). It is also possible that rAd85A is a weak
immunogen in cows, being poor at priming immune responses
but capable of boosting existing responses.
Measuring ex vivo IFN-? responses most likely assesses ef-
fector cells and effector memory T-cell responses, yet recent
studies of viral and parasitic infections in mice and humans
have suggested that central memory responses rather than
effector memory responses correlate with pathogen clearance
and protection (11, 18). However, reagents for labeling central
memory responses in cattle, e.g., bovine CCR7?cells, are
unavailable. Therefore, we developed a cultured ELISPOT
system to investigate long-term central memory responses in
cattle. Although central memory T cells have not been formally
defined for cattle, this assay has been shown to assess such
responses in other species, including humans (4). Cultured
ELISPOT analysis was performed at 14 weeks postpriming,
when no significant ex vivo responses were found compared to
prevaccination levels (Fig. 1A), by stimulating 2 ? 106
PBMC/ml with the rAg85 protein (2 ?g/ml). Recombinant
human interleukin-2 (IL-2) (to 10 U/ml; Sigma Poole, Great
Britain) was added to the cultures on days 5 and 8. On days 10
and 12, half of the supernatant was replaced with IL-2-free
medium. On day 13, 2 ? 104cells/well were added to ELIS-
POT plates and incubated together with rAg85A (5 mg/ml) or
an Ag85A peptide cocktail (peptides 1, 3, 4, 6, 8, 11, 16, 17, 18,
19, 21, 22, and 23 ; 6 mg/ml of each peptide). Large num-
bers of memory cells were found after vaccination with all
three protocols (Fig. 1B). However, rAd85A-rAd85A vaccina-
tion resulted in the weakest cultured ELISPOT responses
(rAg85A stimulation, 10,650 ? 1,270 SFC/106cells) compared
to BCG-rAd85A and rAd85A-BCG (22,720 ? 2,780 and
19,380 ? 3,430 SFC/106cells, respectively), although only the
responses of the BCG-rAd85A group were statistically signif-
icantly larger than those of the rAd85A-rAd85A-vaccinated
calves (P ? 0.05) (Fig. 1B). While the cultured ELISPOT
responses observed for the BCG-rAd85A-vaccinated calves
were predictably the strongest, rAd85A-BCG vaccination also
resulted in recall responses comparable to those for BCG-
rAd85A vaccination (Fig. 1B). Thus, despite the absence of
strong ex vivo IFN-? responses following rAd85A-rAd85A or
rAd85A-BCG vaccination, all three vaccination protocols re-
sulted in strong recall memory responses. In a pilot experi-
ment, BCG-rAg85A-vaccinated calves were also found to be
protected from a virulent M. bovis challenge, and the extent of
protection correlated positively with the level of the cultured
ELISPOT response for three individual calves (data not
shown). This is in agreement with our recent data applying
recombinant fowlpox virus or MVA expressing the same anti-
FIG. 1. IFN-? responses after vaccination. (A) Kinetics of ex vivo,
direct responses after stimulation of PBMC (2 ? 105/well) with
rAg85A (5 ?g/ml). Results are depicted as mean net SFC (SFC with
stimulant minus SFC of medium controls) ? standard errors of the
means (SEM) for five cattle per group. *, P ? 0.05 compared to the
other two groups (one-way analysis of variance followed by the Tukey-
Kramer multiple-comparison postanalysis test). Circles, BCG-rAd85A
(n ? 5); squares, rAd85A-BCG (n ? 5); triangles, rAd85A-rAd85A (n
? 5). Priming vaccination was done at week 0. B1, rAd85A boost of
rAd85A-rAd85A group; B2, BCG or rAd85A boost of BCG-rAd85A
or rAd85A-BCG group, respectively. Positive responses were recorded
when the SFC with stimulant minus the SFC of medium controls was
?50 SFC/106PBMC and more than the prevaccination values.
(B) Cultured ELISPOT responses established 8 weeks after booster
injections. PBMC were stimulated with rAg85A and IL-2 for 13 days,
and IFN-? ELISPOT assays were performed by stimulating cultured
cells with rAg85A (5 ?g/ml) or a peptide cocktail derived from Ag85A
(6 ?g/ml of each peptide). Cultures were performed in the presence of
autologous macrophages as a source of antigen-presenting cells. Me-
dium control values were subtracted, and data are presented as means
of groups of five animals ? SEM. Statistical differences between
groups were evaluated using unpaired, two-tailed t tests. Differences
between the BCG-rAd85A and rAd85A-rAd85A groups did not reach
statistical significance. The horizontal line shows peptide-stimulated
cultured ELISPOT results for PBMC from a group of six unvaccinated
control animals (SFC/106cells, 480 ? 480).
VOL. 74, 2006NOTES1417
gen to cattle vaccination, where the BCG-MVA vaccine com-
bination also proved to be the most immunogenic (16).
It was reported recently that intranasal rAd85A delivery
protected mice significantly against M. tuberculosis (17). There-
fore, we intranasally boosted three additional subcutaneously
BCG-vaccinated calves with rAd85A at week 7 (109PFU/4 ml,
2 ml/nostril). BCG vaccination resulted in modest blood-based
ex vivo IFN-? responses to a pool of Ag85A peptides at the
time of intranasal boosting with rAd85A. This response was
boosted by intranasal rAd85A vaccination, peaking at 2 weeks
postboost (Fig. 2A, week 9). Enhanced Ag85A-specific central
memory responses were also demonstrable by cultured ELIS-
POT analysis after intranasal boosting with rAd85A (Fig. 2B).
In conclusion, the results reported here demonstrate that a
heterologous prime-boost vaccination schedule based on BCG
and systemic or mucosal vaccination with an adenovirus ex-
pressing Ag85A induced strong cellular immune responses in
cattle and that its assessment in large-scale protective efficacy
studies is warranted.
This study was funded by the Department for Environment, Food
and Rural Affairs, United Kingdom. K.H. was supported by the Fonds
voor Wetenschappelijk Onderzoek-Vlaanderen.
1. Brandt, L., J. Feino Cunha, A. Weinreich Olsen, B. Chilima, P. Hirsch, R.
Appelberg, and P. Andersen. 2002. Failure of the Mycobacterium bovis BCG
vaccine: some species of environmental mycobacteria block multiplication of
BCG and induction of protective immunity to tuberculosis. Infect. Immun.
2. Feng, C. G., U. Palendira, C. Demangel, J. M. Spratt, A. S. Malin, and W. J.
Britton. 2001. Priming by DNA immunization augments protective efficacy
of Mycobacterium bovis bacillus Calmette-Guerin against tuberculosis. In-
fect. Immun. 69:4174–4176.
3. Fine, P. E. 1989. The BCG story: lessons from the past and implications for
the future. Rev. Infect. Dis. 11(Suppl. 2):S353–S359.
4. Godkin, A. J., H. C. Thomas, and P. J. Openshaw. 2002. Evolution of
epitope-specific memory CD4(?) T cells after clearance of hepatitis C virus.
J. Immunol. 169:2210–2214.
5. Goonetilleke, N. P., H. McShane, C. M. Hannan, R. J. Anderson, R. H.
Brookes, and A. V. Hill. 2003. Enhanced immunogenicity and protective
efficacy against Mycobacterium tuberculosis of bacille Calmette-Guerin vac-
cine using mucosal administration and boosting with a recombinant modified
vaccinia virus Ankara. J. Immunol. 171:1602–1609.
6. Hewinson, R. G., H. M. Vordermeier, and B. M. Buddle. 2003. Use of the
bovine model of tuberculosis for the development of improved vaccines and
diagnostics. Tuberculosis 83:119–130.
7. Krebs, J. R. 1997. Bovine tuberculosis in cattle and badgers. Ministry of
Agriculture, Fisheries and Food Publications, London, United Kingdom.
8. McShane, H., R. Brookes, S. C. Gilbert, and A. V. Hill. 2001. Enhanced
immunogenicity of CD4?T-cell responses and protective efficacy of a DNA-
modified vaccinia virus Ankara prime-boost vaccination regimen for murine
tuberculosis. Infect. Immun. 69:681–686.
9. McShane, H., A. A. Pathan, C. R. Sander, N. P. Goonetilleke, H. A. Fletcher,
and A. V. Hill. 2005. Boosting BCG with MVA85A: the first candidate
subunit vaccine for tuberculosis in clinical trials. Tuberculosis (Edinburgh)
10. McShane, H., A. A. Pathan, C. R. Sander, S. M. Keating, S. C. Gilbert, K.
Huygen, H. A. Fletcher, and A. V. Hill. 2004. Recombinant modified vaccinia
virus Ankara expressing antigen 85A boosts BCG-primed and naturally
acquired antimycobacterial immunity in humans. Nat. Med. 10:1240–1244.
11. Reece, W. H., M. Pinder, P. K. Gothard, P. Milligan, K. Bojang, T. Doherty,
M. Plebanski, P. Akinwunmi, S. Everaere, K. R. Watkins, G. Voss, N.
Tornieporth, A. Alloueche, B. M. Greenwood, K. E. Kester, K. P. McAdam,
J. Cohen, and A. V. Hill. 2004. A CD4(?) T-cell immune response to a
conserved epitope in the circumsporozoite protein correlates with protection
from natural Plasmodium falciparum infection and disease. Nat. Med. 10:
12. Santosuosso, M., X. Zhang, S. McCormick, J. Wang, M. Hitt, and Z. Xing.
2005. Mechanisms of mucosal and parenteral tuberculosis vaccinations: ad-
enoviral-based mucosal immunization preferentially elicits sustained accu-
mulation of immune protective CD4 and CD8 T cells within the airway
lumen. J. Immunol. 174:7986–7994.
13. Skinner, M., B. M. Buddle, N. Wedlock, D. Keen, G. W. de Lisle, R. E.
Tascon, J. C. Ferraz, D. B. Lowrie, P. J. Cockle, H. M. Vordermeier, and
R. G. Hewinson. 2003. A DNA prime-BCG boost vaccination strategy in
cattle induces protection against bovine tuberculosis. Infect. Immun. 71:
14. Vordermeier, H. M., M. A. Chambers, P. J. Cockle, A. O. Whelan, J. Sim-
mons, and R. G. Hewinson. 2002. Correlation of ESAT-6-specific gamma
interferon production with pathology in cattle following Mycobacterium bovis
BCG vaccination against experimental bovine tuberculosis. Infect. Immun.
15. Vordermeier, H. M., P. C. Cockle, A. Whelan, S. Rhodes, N. Palmer, D.
Bakker, and R. G. Hewinson. 1999. Development of diagnostic reagents to
differentiate between Mycobacterium bovis BCG vaccination and M. bovis
infection in cattle. Clin. Diagn. Lab. Immunol. 6:675–682.
16. Vordermeier, H. M., S. G. Rhodes, G. Dean, N. Goonetilleke, K. Huygen,
A. V. Hill, R. G. Hewinson, and S. C. Gilbert. 2004. Cellular immune re-
sponses induced in cattle by heterologous prime-boost vaccination using
recombinant viruses and bacille Calmette-Guerin. Immunology 112:461–470.
17. Wang, J., L. Thorson, R. W. Stokes, M. Santosuosso, K. Huygen, A. Zgani-
acz, M. Hitt, and Z. Xing. 2004. Single mucosal, but not parenteral, immu-
nization with recombinant adenoviral-based vaccine provides potent protec-
tion from pulmonary tuberculosis. J. Immunol. 173:6357–6365.
18. Wherry, E. J., V. Teichgraber, T. C. Becker, D. Masopust, S. M. Kaech, R.
Antia, U. H. von Andrian, and R. Ahmed. 2003. Lineage relationship and
protective immunity of memory CD8 T cell subsets. Nat. Immunol. 4:225–
19. Williams, A., G. J. Hatch, S. O. Clark, K. E. Gooch, K. A. Hatch, G. A. Hall,
K. Huygen, T. H. Ottenhoff, K. L. Franken, P. Andersen, T. Mark Doherty,
S. H. Kaufmann, L. Grode, P. Seiler, C. Martin, B. Gicquel, S. T. Cole, P.
Brodin, A. S. Pym, W. Dalemans, J. Cohen, Y. Lobet, N. Goonetilleke, H.
McShane, A. Hill, T. Parish, D. Smith, N. G. Stoker, D. B. Lowrie, G.
Kallenius, S. Svenson, A. Pawlowski, K. Blake, and P. D. Marsh. 2005.
Evaluation of vaccines in the EU TB Vaccine Cluster using a guinea pig
aerosol infection model of tuberculosis. Tuberculosis (Edinburgh) 85:29–38.
Editor: J. L. Flynn
FIG. 2. Intranasal boosting with rAd85A results in increased ex
vivo (A) and cultured ELISPOT (B) responses. (A) PBMC were pre-
pared before the rAd85A boost (week 7, black bar) and at week 9 (gray
bar) of the experiment and stimulated with a pool of immunodominant
Ag85A-derived peptides. Results are expressed as mean SFC/106
PBMC ? SEM (n ? 3). (B) SFC were determined after culture with
rAg85A for the cultured ELISPOT protocol at week 7 (black bar) and
week 10 (gray bar) of the experiment. ELISPOT assays were per-
formed 13 days after culture initiation, using the synthetic peptide
cocktail used for panel A in the absence of additional antigen-present-
ing cells. Results are expressed as mean SFC/106cells ? SEM (n ? 3).
*, P ? 0.05 (unpaired t test).
1418 NOTESINFECT. IMMUN.