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Antimicrobial Agents and Chemotherapy, January 2001, p. 316-318, Vol. 45, No. 1
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.1.316-318.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Effective Combination Therapy for Invasive Pneumococcal
Pneumonia with Ampicillin and Intravenous Immunoglobulins in a
Mouse Model
Laetitia
De Hennezel,1
Françoise
Ramisse,1
Patrice
Binder,2
Gilles
Marchal,3 and
Jean-Michel
Alonso3,*
Centre d'Etudes du Bouchet, 91710 Vert Le
Petit,1 Direction Centrale du Service de
Santé des Armées, 00459 Armées,2 and Institut Pasteur,
75015 Paris,3 France
Received 22 December 1999/Returned for modification 29 April
2000/Accepted 3 October 2000
 |
ABSTRACT |
Intranasal immunotherapy for Streptococcus pneumoniae
invasive pneumonia with polyvalent immunoglobulins (IVIG) was effective in mice against pneumonia but failed to prevent bacteremia. The combination of subcurative doses of IVIG and of ampicillin was fully
protective. Such an approach, successfully applied in the preantibiotic
era, offers new perspectives for modern therapies.
| |
TEXT |
New therapeutic strategies against
pneumococcal diseases are needed due to the multiple resistance to
antibiotics of certain strains (8, 15). In the
preantibiotic era, antibody-based immunotherapy was effectively used,
when pneumococcal infections were treated by serotherapy or combined
serum plus chemotherapy (6). Polyvalent human
immunoglobulins (IVIG) contain a variety of antimicrobial
immunoglobulin G (IgG) antibodies (3, 9, 14, 18),
including antibodies to Streptococcus pneumoniae (12). Experimental pneumonia in leukopenic mice,
induced by a serotype 14 S. pneumoniae strain which
was avirulent for immunocompetent mice, can be cured by the intranasal
administration of IVIG (23). However, the virulence of
S. pneumoniae depends not only on the immune status of the
host (5, 7, 13) but also on the capsular type of the
strain (4). In this study, we evaluated the efficacy of
IVIG and a combination therapy with IVIG and ampicillin against a
serotype 3 S. pneumoniae strain that is virulent for
immunocompetent mice.
Female, 6-week-old BALB/c mice (Charles River Laboratories, Saint
Aubin-les-Elbeuf, France) were challenged intranasally, as previously
described (23), with S. pneumoniae Pn4241
(2). Inocula were prepared from a 6-h subculture in brain
heart infusion broth (Difco, Detroit, Mich.) at 37°C, reaching
109 CFU/ml and diluted in phosphate-buffered
saline (PBS; Sigma, Saint Quentin-Fallavier, France) to a desired
density according to the A650 and CFU counts on
blood-agar (Biomérieux, Marcy l'Etoile, France). Statistical
analysis of CFU counts in blood and lung homogenates in groups of five
mice were performed by using the Student Fisher t test.
Lethality for mice was scored each day for 15 days. The mean 50%
lethal dose (LD50) of S. pneumoniae Pn4241
for intranasally infected mice was 5 × 103. IVIG
(Tégéline [lot 50060432] from the Laboratoire du
Fractionnement et des Biotechnologies, Les Ulis, France) was used at
the dose of 50 mg/kg throughout the study because this was the highest protective dose tolerated intranasally by the mice. Antibodies to
S. pneumoniae in IVIG, either preabsorbed on S. pneumoniae Pn4241 or on the noncapsulated mutant R6 (ATCC 39937)
or not, were titrated by enzyme-linked immunosorbent assay (ELISA) as described previously (17, 23). Twofold dilutions
(100 to 1 µg/well) in PBS-Tween 20-5% skim milk were added to
microtiter plates (Maxisorp Immunoplates; Nunc, Roskilde, Denmark)
coated with 106 heat-killed bacteria. Rabbit anti-human
IgG-peroxidase conjugate (Immunotech, Marseille, France) was
added and 3,3',5,5'-tetramethylbenzidine (Sigma) was used for
detection. The absorbance (A450) was read with
an ELISA reader (Titertek Multiscan; Bioblock, Illkirch, France).
Cross-standardization of parallel total IgG and S. pneumoniae antibody titration curves was used to determine the
specific antibody titers in each assay (19). Specific
S. pneumoniae Pn4241 antibodies accounted for <1% of the
total IgG, including 60% ± 6% noncapsular antibodies.
We compared the effects of an intranasal or an intravenous
administration of IVIG at 3 h after a challenge with 5 × 104 CFU on bacterial loads in the lungs and the blood.
Intravenous injection of IVIG gave effective bacterial clearance from
the lungs and prevented bacteremia. Intranasal treatment was
transiently effective against pneumonia (P < 0.05),
but had no significant effects on bacteremia (P > 0.1), suggesting a short efficacy of locally delivered antibodies
(Fig. 1). Intranasal immunotherapy administered 24 h before challenge with 5 × 105
CFU was about 100 times more effective against pneumonia than when
given at 3 h after challenge by reducing CFU counts at 48 h
from (1.1 ± 0.8) × 104 to (2.1 ± 0.55) × 102 in the lungs (P < 0.01) and from
(8.9 ± 4.9) × 101 to (1.3 ± 0.16) × 101 in the blood (P < 0.01). Human IgG in
lung or serum samples, collected at 2 h and after 1, 2, 4, and 7 days from intranasally or intravenously treated mice, were titrated by
ELISA, as described above. Standard curves were obtained by mixing 1 mg
of IVIG with 1 ml of lung cell-free homogenate or with mouse serum.
Half of the initial intranasal dose of IgG was cleared from the lungs within 48 h, and no human IgG was detectable in the serum, but half of the intravenous dose was detected in serum after 7 days (data
not shown).
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FIG. 1.
Efficacy of IVIG administered intranasally or
intravenously to mice 3 h after intranasal challenge with 5 × 104 CFU of S. pneumoniae Pn4241. The
bacterial counts in the lungs and blood are the means ± the
standard errors of the mean (vertical bars) for five mice per point
treated with PBS or IVIG intranasally or intravenously.
|
|
We compared the efficacy of combined therapy with that of single
therapy with IVIG or with ampicillin (Sigma) against the ampicillin-susceptible S. pneumoniae strain Pn4241 (MIC of
0.016 mg/liter as determined by E-test [AB-Biodisk, Solna, Sweden]). Subcurative doses of ampicillin (200 µg/kg) and of IVIG (10 mg/kg) were selected from preliminary experiments in which mice challenged with 105 or 106 CFU were treated either with
ampicillin at 0, 100, 200, or 1,000 µg/kg subcutaneously in a volume
of 200 µl at 3 h after infection or intranasally with IVIG at 0, 5, 10, or 50 mg/kg given 24 h before infection because these were the
highest doses inducing >10-fold transient reduction in CFU pulmonary
counts at 24 h, followed by a regrowth at 48 h, thus
mimicking a treatment failure. The efficacy of combined therapy was
compared to that of single therapy with ampicillin given at 3 h after
challenge (in mice treated intranasally with PBS 24 h before) or
with IVIG given 24 h before (and PBS given subcutaneously 3 h
after) the challenge. Controls received 50 µl of PBS intranasally
24 h before the challenge and 200 µl subcutaneously at 3 h
after the challenge. CFU counts at 48 h in the groups given single
treatments were lower but not significantly different from those of the
controls (P > 0.1), whereas in the group given both
treatments the CFU counts in the lungs and blood were below the
threshold of detection (Fig. 2). The
survival data were consistent with these results: 9 of 10 mice given
the combined treatment survived versus 2 and 3 of the 10 mice in the
ampicillin and IVIG single-treatment groups, respectively.
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FIG. 2.
Efficacy of combination therapy with ampicillin and
intranasal IVIG for treating S. pneumoniae Pn4241 invasive
pneumonia. The CFU counts in the lungs and blood are the means ± the standard errors of the mean (vertical bars) for five mice per point
(in two independent challenges with 3.2 × 105 and
6.3 × 105 CFU, respectively) treated with PBS, IVIG,
or ampicillin plus IVIG. Values below the detection threshold for CFU
counts (102 and 101 CFU/ml for the lungs and
the blood, respectively) were counted at the threshold value.
|
|
Recent advances in immunology have led to renewed interest in passive
immunotherapy against infectious agents for which there is no effective
treatment, such as with most viruses and multiple-antibiotic-resistant bacteria (6, 18, 27, 28, 29). Topical immunotherapy is a
promising approach for epithelial infections (27, 29), particularly for pulmonary infections (22, 23, 24). In
this study, intranasal administration of IVIG was effective against pneumonia induced by various lethal pneumococcal inocula of 10 to 1,000 times the LD50 but did not significantly neutralize
bacteremia, which is the major threat in pneumococcal infections
(13, 20), probably due to the short lifetime of IVIG in
the lungs after intranasal administration. Indeed, S. pneumoniae Pn4241 antibody titer in IVIG was low, but an ELISA
does not assess antibacterial efficacy which involves not only antibody
binding to surface epitopes but also complement activation and
opsonophagocytosis (16, 25). Specific S. pneumoniae capsule-type antibody would have been more effective,
but the variety of capsular types of S. pneumoniae (>85)
requires a polyvalent immunotherapeutic approach such as that developed
for vaccines. The development of combinatorial antibody library
technology may be the way forward for polyvalent passive immunotherapy
against pathogens with antigenic diversity (28).
The combination therapy with IVIG and ampicillin which we tested in a
way similar to that applied 60 years ago with immune serum and
sulfapyridine (21) and utilized by other investigators more recently (11) was effective for curing invasive
pneumonia. Combining antibody-based immunotherapy with chemotherapy may
make it possible to achieve effective antibacterial therapy with
standard doses of antibiotics for strains with diminished
susceptibility, thereby reducing the risk of selection of more
resistant variants (8, 15).
| |
ACKNOWLEDGMENTS |
This work was supported by the French Ministry of Defense (grant
25149/ETCA/CEB, Department of Biology).
We thank Patrice Courvalin for fruitful discussions and comments on
this work.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Unite des
Neisseria, CNRM, Institut Pasteur, 25-28 rue du Dr. Roux, 75724 Paris
Cedex 15, France. Phone: 33-1-45-68-83-30. Fax: 33-1-40-61-30-34. E-mail: jmalonso{at}pasteur.fr.
| |
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Antimicrobial Agents and Chemotherapy, January 2001, p. 316-318, Vol. 45, No. 1
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.1.316-318.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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