The role of antiviral antibodies in resistance to lymphocytic choriomeningitis virus (LCMV) infection was explored. Immune serum and monoclonal antibodies ...
Vol. 66, No. 7
JOURNAL OF VIROLOGY, JUlY 1992, p. 4252-4257 0022-538X/92/074252-06$02.00/0 Copyright ©3 1992, American Society for Microbiology
Mechanisms of Antibody-Mediated Protection against Lymphocytic Choriomeningitis Virus Infection: Mother-to-Baby Transfer of Humoral Protectiont JORY R. BALDRIDGE AND MICHAEL J. BUCHMEIER*
Department of Neuropharmacology, The Scripps Research Institute, La Jolla, California 92037 Received 26 November 1991/Accepted 30 March 1992
The role of antiviral antibodies in resistance to lymphocytic choriomeningitis virus (LCMV) infection was explored. Immune serum and monoclonal antibodies prevented fatal T-cell-mediated immunopathology following acute LCMV infections. In addition, 10- and 14-day-old mice that received maternally derived anti-LCMV antibodies through nursing were protected from an otherwise lethal LCMV challenge. Detailed investigation of the mechanism(s) by which these antiviral antibodies provided protection was carried out by using anti-LCMV monoclonal antibodies. Protection correlated directly with the ability of the antibodies to reduce viral titers in the tissues of conventional (K. E. Wright and M. J. Buchmeier, J. Virol. 65:3001-3006, 1991) and nude mice. However, this reduction was not simply a reflection of virus neutralizing activity, since not all antibodies which neutralized in vitro were protective. A correlation was also found between immunoglobulin isotype and protection: all of the protective antibodies were immunoglobulin G2a (IgG2a), while IgGl antibodies mapping to the same epitopes were not. Protection appeared to be associated with events controlled by the Fc region. Functional F(ab')2 fragments which retained in vitro neutralizing activity were not protective in vivo. Furthermore, this Fc-associated function was not related to complement-mediated cell lysis, since C5-deficient mouse strains were also protected. These results suggest a role for antibody in protection from arenavirus infections and indicate that a distinct immunoglobulin subclass, IgG2a, may be essential for this protection.
though no clear protective advantage has yet been ascribed to the IgG2a immunoglobulins elicited during viral infections, this immunoglobulin subclass efficiently activates the complement system, which may be advantageous to the host
Passive humoral immunotherapy is currently the treatment of choice for the arenavirus infection in humans caused by Junin virus (9) and has been used successfully to treat Lassa fever (24). In nonhuman primates, the combined use of immune plasma and ribavirin is more effective than either therapy alone and thus looks promising for clinical use (19). Despite the demonstrated effectiveness of passive antibody therapy, the mode of action remains poorly understood. Because of the risks inherent in using human convalescentphase sera, monoclonal antibody (MAb) therapy has become an attractive alternative. Defining the protective mechanisms by which antibodies mediate protection is critical so that therapeutic MAbs and vaccine regimens can be appropriately targeted to protective epitopes while avoiding potentially enhancing reactions. In this study, we investigate the requirements for and mechanism of action of humoral protection against lymphocytic choriomeningitis virus
(12, 18). The arenavirus LCMV can trigger three distinct outcomes during infections of mice: (i) an acute asymptomatic infection followed by viral clearance and life-long immunity when immunocompetent adults are inoculated extraneurally, (ii) an acute fatal, T-cell-mediated lymphocytic choriomeningitis (LCM) which develops following intracerebral (i.c.) inoculation of immunocompetent adults, and (iii) a life-long persistent infection following inoculation of neonatal or immunocompromised adult mice. Studies of the humoral response to LCMV infections have demonstrated that antiviral antibodies are produced during both the acute and the persistent infections (4, 26). During acute infections, complementfixing antibodies can be detected as early as 6 days postinfection and reach peak titers in 2 to 3 weeks (4). Neutralizing antibodies appear later and have specificity for the major viral glycoprotein, GP-1 (2, 4). Also following an acute infection, the majority of virus-specific antibodies are of the IgG2a isotype, but mice enduring persistent infections produce predominantly low-affinity IgGl antibodies (36, 37). The complexing of these antiviral antibodies with viral antigens during persistent infections leads to chronic glomerulonephritis (29). The protective capacity of anti-LCMV antibodies was first demonstrated by Rowe (34), but with limited success. More recently, Thomsen and Marker (35) demonstrated that LCMV infections are restricted by passively transferred antibodies, suggesting that preformed antibodies may play a dominant role in resistance to reinfection. Finally, Wright and Buchmeier (39) showed that passively acquired MAbs
(LCMV) infection. Although the protective role of antibody in viral infections is appreciated, it remains poorly understood. Antiviral antibodies are predominantly of the immunoglobulin G2a (IgG2a) isotype (8), while antibodies produced against soluble proteins are predominantly of the IgGI subclass (8, 33). This distinct difference in isotype production most likely relates to differences in cytokines induced in response to the antigen (13). Production of IgG2a antibodies requires T-cell help in the form of gamma interferon, which is generated in large quantities during viral infections (13), while IgGl production is more dependent on interleukin-4 (38). Al* Corresponding author. t Publication 7132-NP from the Department of Neuropharmacol-
ogy, The Scripps Research Institute.
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HUMORAL PROTECTION AGAINST LCMV INFECTION
VOL. 66, 1992
directed against the GP-1 glycoprotein can attenuate fatal T-cell-mediated LCM disease. In this report, we extend those observations, using the model of antibody-mediated attenuation of LCM disease to investigate the mechanisms by which antiviral antibodies provide protection.
MATERLALS AND METHODS Virus. The Armstrong strain 4 (ARM-4) and Armstrong strain 5 (ARM-5) variants of LCMV were used for these studies. These strains differ in their sensitivity to neutralization by MAb 2.11.10 (40) but show no difference in the ability to induce lethal acute LCM disease following inoculation of immunocompetent mice (39). Both viral variants were originally plaque purified from a stock of Armstrong CA-1371. Working stocks of virus were obtained from infected BHK-21 cells (multiplicity of infection of 0.1) 48 h postinfection and stored in 1-ml aliquots at -70°C. Mice. Female BALB/cByJ, SWR/J, and A/J mice of matched age (4 to 6 weeks) were obtained from the rodent breeding colony at The Scripps Research Institute. Athymic BALB/Wehi-nude male and female mice of matched age (4 to 6 weeks) were also obtained from the rodent breeding colony at The Scripps Research Institute. Seven-week-old B10.D2/nSnJ and B10.D2/oSnJ female mice were purchased from Jackson Laboratory (Bar Harbor, Maine). MAb treatment. Mice were passively immunized with anti-LCMV MAbs by intraperitoneal (i.p.) injection of 0.2 ml of crude ascitic fluid on the day before and the day of viral challenge, unless otherwise noted. Generation and characterization of the murine anti-LCMV MAbs have been described in detail previously (3, 30). Viral challenge and immunizations. Mice were challenged by i.c. inoculation of 1,000 PFU (unless otherwise indicated) of virus. The virus was diluted in Dulbecco modified Eagle medium for delivery of 50 pl for adult mice and 10 pul for neonates. Immune mothers were immunized with 105 PFU by i.p. inoculation 30 days or more prior to mating. F(ab')2 fragment preparation. MAb 2.11.10 (IgG2a) was purified from ascitic fluid on protein A columns (Pierce, Rockford, Ill.) (11). The antibody was dialyzed and concentrated in an Amicon filtration cell (Amicon, Beverly, Mass.). MAb at a concentration of 10 mg/ml in 20 mM sodium acetate buffer (pH 4.0) was mixed 2:1 with immobilized pepsin in 20 mM sodium acetate buffer (pH 3). The mixture was incubated overnight at 37°C on a tube rotater. The digestion was stopped by removal of the pepsin, and the undigested MAb was removed by protein A column chromatography. The unbound fraction was collected as the F(ab')2 and purity checked by sodium dodecyl sulfatepolyacrylamide gel electrophoresis analysis. Activity of F(ab')2 was confirmed by an in vitro plaque reduction neutralization assay.
RESULTS
Passively transferred immune serum and maternal antibodies protected mice from T-cell-mediated LCM. Previous experiments from this laboratory have demonstrated that LCMV-specific MAbs attenuate immunopathologic effects of lethal LCM disease (39). In our initial experiments, we sought to determine whether similar attenuation could be achieved by using a passively transferred anti-LCMV polyclonal antiserum. In the first set of experiments, recipient BALB/c mice were given serum from immune BALB/c donors and then challenged by i.c. inoculation with >1,000
4253
TABLE 1. Attenuation of lethal LCM by passively transferred immune serum Expt
Group"
% Survival"
1
Normal control Immune control Serum recipients
0 100 50
2
Normal control Immune control Serum recipients
0 100 100
"Donor BALB/c mice were immunized with io-s PFU of ARM-4 30 days prior to bleeding. Recipient BALB/c mice were injected i.v. with 0.2 ml of serum from immune donors immediately prior to viral challenge for experiment 1 or i.p. infusions of serum (0.2 ml per mouse) the day before and the day of viral challenge for experiment 2. The immune serum donors were from a group of mice treated identically to the immune control mice. " Mice (four per group) were challenged with 500 PFU (.1,000 LD15) of LCMV ARM-4 by i.c. inoculation.
50% lethal doses (LD50) (500 PFU) of the ARM-4 strain of LCMV. Fifty percent of the mice that received only one infusion of serum (0.2 ml) on the day of challenge survived (Table 1, experiment 1). When a second 0.2-ml dose of immune serum was given to recipients, all of the mice survived the challenge (Table 1, experiment 2). Infection of neonatal mice with LCMV normally results in a life-long persistent infection; however, by 10 days of age, mice challenged with virus die of acute LCM. We took advantage of this age-dependent change in susceptibility to disease to study acquired resistance to LCMV infection in a mother-baby model. Passage of immunoglobulin from mother to baby across the placenta and in the colostrum is well known (16). Therefore, we sought to determine whether protective levels of anti-LCMV antibodies could be obtained in this manner. Ten- and 14-day-old suckling pups were challenged i.c. with 1,000 PFU of LCMV (.2,000 LD50). The pups born to and nursed by immune dams had a 100% survival rate, while all pups born to and nursed by normal mothers died (Table 2). Pups nursed by immune mothers had TABLE 2. Protection from lethal challenge of pups nursing on immune dams Age of pups upon
Pups
challenge'
weanedd
% Survivalr (total no. of
Immune Nonimmune
10 days 10 days
No No
100 (6) 0 (4)
Immune Nonimmune
14 days 14 days
No No
100 (6) 0 (7)
Immune Nonimmune
5 wk 5 wk
Yes Yes
29 (7) 0 (4)
14 days 14 days
No No
73 (15) 0 (12)
Birth mothers'
Nonimmune Immune
Foster
mothersb
Immune Nonimmune
mice)
" The pups were born to either nonimmune or immune dams as indicated. Immune dams were given 105 PFU of LCMV by injection 30 or more days
prior to mating. " Within 24 h of birth, foster-nursed pups were switched to either nonimmune or immune foster mothers as indicated. ' Pups were challenged at the indicated age with 1,000 PFU of LCMV via i.c. inoculation. d Weaning was done at 3 weeks of age. ' The surviving mice exhibited no evidence of LCMV in the brain, liver, or spleen 30 days postchallenge.
J. VIROL.
BALDRIDGE AND BUCHMEIER
4254
TABLE 3. Protection from LCMV challenge of suckling pups of 2.11.10-treated dams Expt"
Expt"
Viral challenge'
c%c no. of mice) (total Survival
IL 0
1
ARM-4 ARM-5
40 (5) 0 (5)
2
ARM-4 ARM-S
83 (6) 0 (3)
"The mothers were nonimmune when their pups were born but were subsequently infused with 0.2 ml of MAb 2.11.10 ascites every third day (experiment 1) or every second day (experiment 2) from the time of birth until the pups were challenged. " Pups were challenged at 14 days of age with 500 PFU ( 1,0t)0 LD5,,) of LCMV ARM-4 or ARM-5 by i.c. inoculation. The ARM-4 strain of LCMV has the GP-1D epitope as defined by MAb 2.11.10 (4t)), while the ARM-5 strain lacks this epitope.
o 0
-J
FI. oc
anti-LCMV antibody titers equivalent to those of their mothers. Titers for mothers and pups were both 1/655,360, as determined by enzyme-linked immunosorbent assay (ELISA). Maternally derived protection was transient; pups born of and nursed by immune mothers lacked resistance (71% mortality) when challenged at 5 weeks of age after weaning at 3 weeks (Table 2). To determine whether protection was being transferred to the pups through the milk or across the placenta, fosternursing experiments were performed. In these experiments, pups born to immune and nonimmune mothers were switched within 24 h of birth and nursed by foster mothers. Those pups born to nonimmune mothers and nursed by immune dams had a 73% survival rate following i.c. challenge. However, none of the pups born to immune mothers and foster nursed by nonimmune dams survived the i.c. challenge (Table 2). In addition, dams passively immunized (postpartum) by infusion of the protective MAb 2.11.10 provided their suckling pups with antigen-specific protection against lethal LCM disease (Table 3). Pups challenged by i.c. inoculation with the ARM-4 strain of LCMV had a high survival rate (40 to 83%), while pups challenged with a 2.11.10-resistant strain of LCMV, ARM-5, had 100% mortality. The survival rate was proportional to the amount of MAb 2.11.10 given to the mothers. Protection is related to reduced viral titers. Having established that antiviral antibody can effectively protect against LCM disease, we sought to investigate the mechanism(s) of protection. Previous studies had shown that viral titers in the brains of BALB/c mice challenged i.c. with LCMV were reduced if protective MAbs were administered near the time of challenge (39). To ascertain that the reduction in viral burden was the result of antibody-mediated events and was not T-cell dependent, we assayed viral titers in the tissues of nude mice. Athymic nude mice were infused with MAb on days -1 and 0 and then challenged with 1,000 PFU of ARM-4 by i.c. inoculation. On days 2, 6, and 10 after virus challenge, tissues samples were removed and LCMV titers were determined. At 2 days postchallenge, virus could not be detected in mice treated with MAb 2.11.10 (Fig. 1), while control mice treated with an unrelated control MAb (antimouse hepatitis virus; 5B-170) had titers exceeding 106 PFU/g of tissue. The threshold for detection of infectious virus in tissues was approximately 600 PFU/g. By 6 days after challenge, viral titers in 2.11.10-treated mice had risen to detectable levels in the spleens and the brains, although the virus concentrations in these tissues remained approxi-
a-
n v0
-5 O IC.
0
54,
c
C~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
0
IL
IL T-
3O 21
DAY O
DAY 2
DAY 6
DAY
10
DAYS POST INFECTION FIG. 1. LCMV titers during acute infection. Nude mice were infused with 0.2 ml of ascitic fluid containing either MAb 2.11.10 (anti-LCMV; *) or MAb 5B-170 (anti-mouse hepatitis virus; 0) by i.p. injection on days -1 and 0. On day 0, the mice were challenged by i.c. inoculation with 1,000 PFU of ARM-4. The spleens (A), brains (B), and livers (C) of infected mice were removed for assessment of viral titers on days 2, 6, and 10 following challenge. Each point on the graph represents the mean of four samples. The limit of sensitivity (about 2.8 log,0 PFU/g) is indicated by the horizontal lines in each graph.
mately 100-fold less than those of the control mice. By day 10 after challenge, the LCMV titers in 2.11.10-treated mice were similar to those in the control group. This same general pattern was also seen in the livers, although the overall titers were lower in both 2.11.10-treated and control mice (Fig. 1C). We also evaluated the capacity of MAbs to reduce viral
HUMORAL PROTECTION AGAINST LCMV INFECTION
VOL. 66, 1992
TABLE 5. Ability of anti-LCMV MAb to protect C5-deficient mice from lethal LCM disease
-
~~~~~~~~0
BANLIVER FIG.
2.
Virus challenge'
treatment"
A/J
Yes
ARM-4 ARM-4 ARM-5 ARM-5
+ +
SWR/J
Yes
ARM-4 ARM-4 ARM-5 ARM-5
+ +
B1O.D2/oSnJ
Yes
ARM-4 ARM-4
+
0 (4) 100 (4)
B1O.D2/nSnJ
No
ARM-4 ARM-4
+
0 (4) 100 (4)
IC. 0
SPLEEN
IEUS
2.11.10
0 100 0 0
(4) (4) (4) (4)
0 (12) 83 (12) 0 (12) 0 (12)
Viral titers during persistent LCMV infection. To estab-
lish persistent infections, nude mice were inoculated i.c. with 500 PFU of ARM-4 35 days prior to MAb treatment. Mice received no
MAb (U) or ascitic fluid containing MAb 2.11.10
(ESl)
% Survival (total no. of mice)
C5 deficient
Mouse strain
IL
IL
4255
(g)
or MAb 36.1
by i.p. injection. Twenty-four hours after MAb infusion, viral
titers were determined in each tissue. Each bar represents the mean of three samples except that for the normal control, which repre-
sents titers from one mouse.
titers of persistently infected nude mice. We found that 24 h
"Mice were challenged i.c. with 1,000 PFU of either ARM-4 or ARM-5 on day 0. The ARM-5 strain lacks the GP-1D epitope that is recognized by 2.11.10 (40). " Mice were infused with 0.2 ml of 2.11.10 MAb ascitic fluid on days -1 and 0 by i.p. injection.
after MAb infusion, viral titers in mice treated with protective MAb 2.11.10 were decreased by 100- to 1,000-fold (Fig. 2) compared with the titers of mice treated with the MAb 36.1 (nonprotective, anti-LCMV; Table 4). This reduction in
viral titers occurred in the spleen, sera, and livers of mice infused with group of
of
mice
MAb
2.11.10. Titers in
the
brains
the MAbs to
of either
not reduced, likely reflecting the cross the blood-brain barrier (15).
were
Isotype and epitope specificity
inability
of the in vivo protection was
restricted to MAb of the IgG2a subclass. MAbs specific for the peripheral glycoprotein GP-1 protected mice from lethal but MAbs against
LCM,
or the nucleocapsid
GP-2
the
transmembrane
glycoprotein
protein did not (39). We set up
experiments to further investigate the mechanisms by which the anti-GP-1 MAbs mediated protection. Specifically, MAbs of the IgG1 and IgG2a isotypes were compared by in vitro neutralization assays, ELISA, and in vivo protection
assays. All of the MAbs which protected mice were of the
while neither of the IgG1 was protective even though MAb 36.1
IgG2a isotype, 18.8)
in vitro (Table
TABLE
4.
4). The
Comparison
assay,
MAb"
protective
MAbs (36.1 and neutralizes virus
capacity of the anti-LCMV
of anti-GP-1 MAb by neutralization
ELISA, and passive protection assay
Epitope"
Isotype
Neutrali-
Nzeutriaot
i
ELISA ieL
~~~~~%
Survival
(total
no.
m ic e
of
)
197.2.1 258.2.11 36.1
GP-1A GP-1A GP-1A
IgG2a IgG2a IgGl
3,548 2,512 >25,000
218,700 218,700 656,100
75 (8) 88 (8)
67.2 18.8
GP-1C GP-1C
IgG2a IgGl
4,266
