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Journal of Virology, August 2007, p. 8571-8578, Vol. 81, No. 16
0022-538X/07/$08.00+0     doi:10.1128/JVI.00160-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Measles Virus-Specific CD4 T-Cell Activity Does Not Correlate with Protection against Lung Infection or Viral Clearance

Karen Pueschel,¹ Annette Tietz,¹ Mary Carsillo,² Michael Steward,⁴ and Stefan Niewiesk^1,2,3*

Institut fuer Virologie und Immunbiologie, University of Wuerzburg, Wuerzburg, Germany,¹ Department of Veterinary Biosciences,² Department for Molecular Virology, Immunology and Medical Genetics, The Ohio State University, Columbus, Ohio,³ Immunology Unit, London School of Hygiene and Tropical Medicine, London, United Kingdom⁴

Received 24 January 2007/ Accepted 25 May 2007

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Acute measles in children can be prevented by immunization with the live attenuated measles vaccine virus. Although immunization is able to induce CD4 and CD8 T cells as well as neutralizing antibodies, only the latter have been correlated with protective immunity. CD8 T cells, however, have been documented to be important in viral clearance in the respiratory tract, whereas CD4 T cells have been shown to be protective in a mouse encephalitis model. In order to investigate the CD4 T-cell response in infection of the respiratory tract, we have defined a T-cell epitope in the hemagglutinin (H) protein for immunization and developed a monoclonal antibody for depletion of CD4 T cells in the cotton rat model. Although the kinetics of CD4 T-cell development correlated with clearance of virus, the depletion of CD4 T cells during the primary infection did not influence viral titers in lung tissue. Immunization with the H epitope induced a CD4 T-cell response but did not protect against infection. Immunization in the presence of maternal antibodies resulted in the development of a CD4 T-cell response which (in the absence of neutralizing antibodies) did not protect against infection. In summary, CD4 T cells do not seem to protect against infection after immunization and do not participate in clearance of virus infection from lung tissue during measles virus infection. We speculate that the major role of CD4 T cells is to control and clear virus infection from other affected organs like the brain.

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Measles virus (MV) uses the respiratory tract of humans as route of entry and spreads to lymphoid organs, where it replicates. It is disseminated via the bloodstream to infect endothelial or epithelial cells of various target organs, resulting typically in clinical symptoms like conjunctivitis and rash. During severe disease, MV infection of the gastrointestinal tract may lead to diarrhea and infection of lung tissue to primary pneumonia. In addition, infection of the brain is documented frequently by abnormalities in electroencephalogram (about 50% of cases) (6, 17), pleocytosis of cerebrospinal fluid (16), or in rare instances acute or chronic forms of encephalitis (for a review, see reference 11). Although the immune system clearly cannot protect against primary MV infection, it controls and clears viral infection (in the absence of secondary infections) within 2 to 3 weeks (for a review, see reference 8). Clinical data from patients with immune deficiencies indicate the relative contribution of T cells and B cells in controlling and clearing infection. In patients with a T-cell defect, chronic MV infection (12) persists whereas patients with a B-cell deficiency are able to control and clear the virus (1, 7). These clinical observations are supported by studies in the rhesus macaque model (19, 20). After depletion of CD8 T cells, the duration of MV replication during primary infection is increased, and the extent and duration of rash are prolonged, indicating the importance of CD8 T cells in control and clearance of virus. In the absence of CD8 T cells, MV-specific B cells are generated earlier than in control animals (20). Depletion of B cells clearly delays the generation of MV-specific antibodies, but this has no effect on the course of infection (19). In contrast to individuals experiencing primary infection with MV, humans vaccinated with the live attenuated vaccine virus are protected against disease. The correlate of protection for vaccinated individuals has been shown to be neutralizing antibodies. The level of neutralizing antibodies correlates with protection against measles in children (2), and passive transfer of neutralizing antibodies is protective both in humans and animal models (11, 30, 32). Another indication of the importance of neutralizing antibodies is the fact that seroconversion after vaccination is inhibited by maternal antibodies, which leaves children susceptible to infection. It has been shown recently that at least some vaccinees develop a CD4 T-cell response after vaccination in the presence of maternal antibodies (5). This raises the question whether the induction of a CD4 T-cell response in the absence of neutralizing antibodies may be protective against infection. To address the question whether the CD4 T-cell response is able to control and clear virus infection and whether MV-specific CD4 T cells protect against infection via the respiratory route, we have used the cotton rat model. MV replicates in lung tissue of cotton rats after intranasal (i.n.) inoculation, and immunization is inhibited in the presence of maternal (or passively transferred human) MV-specific antibodies (30). For this model we have developed a monoclonal antibody specific for the cotton rat CD4 molecule, defined CD4 T-cell epitopes, and have thus been able to determine the role of CD4 T cells during lung infection by MV.

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Animals. Cotton rats (inbred strain COTTON/NIco) were obtained from Iffa Credo, France. Female animals from 6 to 10 weeks of age were used. The animals were bought specific-pathogen-free according to the breeder's specification and were maintained in a barrier system. Animals were kept under controlled environmental conditions of 22 ± 1°C, 50% humidity, and a 12-h light cycle.

Cells and viruses. Vero cells (African green monkey) were grown in minimal essential medium with 5% fetal calf serum (FCS), and BJAB and B95a cells were grown in RPMI 1640 medium containing 10% FCS, 1% nonessential amino acids, 1% sodium pyruvate, 2 mmol/liter glutamine, 50 IU penicillin, and 50 µg of streptomycin/liter (referred to as RPMI/10 medium). MV strains used were the MV HU2 strain which was isolated from a child with measles-induced encephalitis after vaccination with Schwarz vaccine (31). MV Edmonston B vaccine/60s (Edm) and MV wild-type Erlangen DEU/90 (WTFb) were used for immunization and infection, respectively, of cotton rats. HU2 and Edm were grown on Vero cells, and titers were then determined; WTFb was grown on BJAB cells, and titers were determined on B95a cells.

Antibodies. For the production of monoclonal antibody CR-CD4, BALB/c mice were immunized with spleen cells of cotton rats which had been stimulated with concanavalin A for 24 h. Fusion of activated spleen cells with the myeloma cell line SP2/0-Ag14 was done with polyethylene glycol according to standard procedures. Spleen cells and myeloma cells were mixed at a 5:1 ratio. The cell pellet was stirred in polyethylene glycol 1500 for 1 min and subsequently diluted in RPMI/10 medium. Fused cells were plated in 96-well plates, and selection medium (RPMI/10 medium containing 100 µM hypoxanthine, 400 µM aminopterin, 16 µM thymidine [final concentration]) was added 1 day later. After 2 weeks supernatant from individual wells was tested for staining of spleen cells by flow cytometry, and positive wells were recloned twice.

Antibodies specific for mouse major histocompatibility complex class II (MHC-II) (antibody 13/4) (9) and human CD46 (13/42) (3) were tested for staining of cotton rat spleen cells by flow cytometry. Antibody 13/4 bound to cotton rat spleen cells to a similar extent as to mouse spleen cells. Antibody 13/42 did not bind to cotton rat spleen cells. Monoclonal antibody HM57 specific for human CD79a (B-cell marker) and rabbit anti-human CD3 epsilon chain antiserum (T-cell marker) (both Dako Corp.) were tested on paraffin-embedded sections of cotton rat spleens and stained the B-cell or T-cell areas, respectively. Hybridomas secreting monoclonal antibodies were grown in RPMI/10 medium. Some supernatants were purified on protein G columns.

Infection, depletion, immunization, serum transfer, and virus titration. For i.n. infection MV was given in phosphate-buffered saline (PBS) to ether-anesthetized cotton rats. The i.n. inoculations of virus were administered in a volume of not more than 100 µl. At 5 days (peptide immunization) or 7 days (CD4 T-cell depletion) after infection, animals were asphyxiated using CO₂, and lungs were removed and weighed. Lung tissue was minced with scissors and dounced with a glass homogenizer. Serial 10-fold dilutions of virus-containing supernatant were assessed for the presence and levels of infectious virus in a 48-well microassay using Vero cells or B95a cells with cytopathic effect as an end point. Plates were scored microscopically for cytopathic effect after 7 days. The amount of virus in inocula was expressed as the quantity of virus that could infect 50% of inoculated tissue culture monolayers (TCID₅₀). The TCID₅₀ was calculated according to Reed and Muench (23).

To mimic maternal MV-specific antibodies, 1 ml of a human serum (antibody concentration of 16 IU/ml, by enzyme-linked immunosorbent assay [ELISA] using human anti-measles serum [2nd International Standard 1990; 5 IU/ml; National Institute for Biological Standards and Control, Potters Bar, United Kingdom]; antibody titer of 320 by neutralization test and 256 by hemagglutination inhibition assay) was injected intraperitoneally (i.p.). One day later, animals were immunized subcutaneously with 10⁵ PFU of the MV Edm strain. For challenge experiments, animals were infected i.n. with 2 x 10⁵ PFU of MV HU2 strain or 10⁵ TCID₅₀ of WTFb, and 5 days later virus was titrated from lung tissue as described (13).

Peptide immunization was performed by subcutaneous injection of 200 µg of a peptide corresponding to amino acids 553 to 567 on the MV hemagglutinin protein resuspended in TiterMax Gold adjuvant (Sigma). In the same experimental setup, a cotton rat serum that had been obtained from an animal immunized with vesicular stomatitis virus expressing the MV hemagglutinin was used; the neutralization titer (NT) of the serum was 60 (30).

For depletion of CD4 T cells, 0.5 mg of CR-CD4 antibody (purified over protein G columns) was injected i.p. on days 0 and 4 after infection.

Plasmid immunization. For DNA vaccination plasmids pSC-N (nucleocapsid protein), pCG-F1 (fusion protein), or pCG-H5 (hemagglutinin) (29) were used. Plasmid DNA was purified using a plasmid Maxi kit (QIAGEN, Hilden, Germany). For intradermal immunization, 150 µg of plasmid DNA was injected into the lateral flank in two different depots. Injection was controlled by blister formation in the skin. Animals were boosted 3 weeks later with the same dose of plasmid DNA.

Flow cytometry. Spleen, thymus, and lymph node cells were incubated with antibody CR-CD4 and a donkey anti-mouse serum labeled with fluorescein isothiocyanate (Dianova, Germany) which had been preabsorbed with cotton rat serum. Subsequently, cells were analyzed by flow cytometry (FACScan; Becton Dickenson).

Histology. Immunohistochemistry on tissue sections was performed using the Dako Autostainer and a Vectastain ABC kit (Vector Laboratories). Slides were deparaffinized and rehydrated through a series of xylene and graded ethanol baths. Heat-mediated epitope retrieval was performed with Dako antigen retrieval solution and heating at 90°C for 20 min. Endogenous peroxidase activity was quenched by incubation with 3% hydrogen peroxide in Tris-HCl (0.05 M Tris, 0.15 M NaCl, 0.05% Tween 20, pH 7.6) for 5 min. Slides were then washed and incubated with blocking solution (Dako protein block) for 10 min to block interactions between hydrophobic groups. Slides were incubated with the primary antibody at room temperature for 30 min. For detection of T cells, a polyclonal rabbit serum specific for the intracytoplasmic portion of the epsilon chain of the human CD3 (1:100 dilution; Dako Corp.) was used, and for detection of B cells, a mouse monoclonal antibody specific for CD79a on human B cells was used (1:25 dilution; clone HM57; Dako Corp.). Secondary biotinylated anti-immunoglobulin G antibody supplied with the Vectastain kit (anti-mouse and anti-rabbit) was applied for 30 min at room temperature, followed by a 30-min incubation with the peroxidase-labeled biotin-avidin complex. Diaminobenzidine (Dako Corp.) and H₂O₂ were used as chromogens. The slides were counterstained with Mayer's hematoxylin.

Neutralization assay. To assay for neutralizing antibodies twofold serum dilutions were incubated with 50 PFU of the Edm strain for 1 h at 37°C and plated in duplicate onto 10⁴ Vero cells/well (96-well plate). Five days later titers were determined microscopically.

Peptides. Overlapping peptides representing MV proteins were synthesized by the rapid multiple peptide synthesis method (DuPont) using Fmoc chemistry employing 4-[2',4'-dimethyloxyphenyl-F-moc-(amino-methyl)-phenoxy] resin (Novabiochem) (15). Each peptide was 15 amino acids long and overlapped the following peptide by 5 amino acids. Cysteine residues in the sequence were replaced with alanine to prevent dimerization, and tyrosine was added at the C termini of peptides that lacked tyrosine for radiolabeling purposes. Fmoc-protected amino acids were converted to the 1-hydroxybenzotriazole-activated esters by treatment with 1-hydroxybenzotriazole and N,N'-diisopropyl-carbodiimide in N,N'-dimethylformamide (DMF). Subsequent coupling reactions were performed in DMF, and the Fmoc groups were removed with 50% piperidine in DMF, followed by a series of alternate washes in DMF and methanol.

After synthesis, side chain-protecting groups were removed, and the peptides were cleaved from the resin with trifluoroacetic acid in the presence of scavengers. After cleavage, peptides were extracted into diethyl ether and purified by gel filtration using Sephadex G-10 (Pharmacia) and by high-performance liquid chromatography. Peptides were analyzed by fast atomic bombardment mass spectrometry, and all had the expected composition.

Proliferation assay. For proliferation assays 50 µl/well (10 µg/ml) of gradient purified, UV-inactivated MV was coated in 200 mM NaCO₃ buffer (pH 9.6) at 4°C overnight in 96-well plates. After being washed twice with PBS, spleen or lymph node cells were plated in triplicate at 5 x 10⁵ cells/well in a 96-well-plate in RPMI 1640 medium with 10% FCS in wells with or without antigen (medium control). After 40 h, 0.5 µCi of [³H]thymidine per well was added, and 16 to 20 h later cells were harvested onto glass filters and counted with a Betaplate Counter (Wallac, Turku, Finland). The stimulation index (SI) was calculated as the mean of the following ratio: proliferation of MV-stimulated cells/proliferation of cells in medium, where proliferation is measured in counts per minute. MV-stimulated spleen cells reached a maximum of 6,000 cpm, and counts of cells in medium were in the range of 100 to 750 cpm. To control for the specificity of the assay, spleen cells from infected animals were tested against Vero cell antigen (the cell line in which MV was grown), and spleen cells from naive animals were tested against MV. In all controls, proliferation did not exceed an SI of 2, indicating the threshold of the assay. To confirm that UV-inactivated MV stimulates CD4 T cells, proliferation was inhibited with CR-CD4 antibody (similar to previous studies [26]). For antibody inhibition assays, antibody was added to the cells at the time of plating at the indicated concentrations (Fig. 4).

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FIG. 4. Inhibition of MV-specific T-cell proliferation by antibodies against MHC-II and CD4. Spleen cells from MV-immune animals were stimulated with UV-inactivated MV (proliferation in counts per minute) and cultured in the presence or absence of various concentrations of antibody. Antibody 13/4 cross-reacts with cotton rat MHC-II (A), CR-CD4 recognizes cotton rat CD4 (B), and 13/42 recognizes human but not cotton rat CD46 (C). nd, not determined.

For the determination of T-cell epitopes, peptides were added directly at the time of plating at concentrations of 10 to 20 µg/ml.

Statistical analysis. For statistical analysis, viral titers and T-cell proliferation were compared by a two-sided t test.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
To evaluate the function of CD4 T cells in the protection against and clearance of MV infection, the following questions were addressed. Is there a correlation between CD4 T-cell responses and viral titers in lung tissue? Does depletion of CD4 T cells increase viral titers? Are MV-specific CD4 T cells alone able to protect against infection?

CD4 T-cell response correlates with viral clearance. To measure CD4 T-cell responses against MV, spleen cells were stimulated in a standard CD4 T-cell proliferation assay with one modification. UV-inactivated MV antigen was coated onto a 96-well plate overnight, which resulted in higher sensitivity of the assay than adding the antigen to the medium. As shown in Fig. 1, T-cell proliferation above the threshold (SI of 2) was first seen on day 5 after i.n. infection with MV, which corresponds with peak viral titers on days 4 and 5 (13). The T-cell response reached peak proliferation on days 7 and 8, which precedes viral clearance by day 10 (13). After day 8, T-cell responses declined but were detectable until at least day 90 (data not shown). These data demonstrate the close correlation between the generation of a CD4 MV-specific T-cell response and clearance of virus from lung tissue. Similar to previously published results (14), we were able to detect MV-specific antibodies by ELISA on day 8 and neutralizing antibodies on day 12.

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FIG. 1. Kinetics of MV-specific CD4 T-cell development in cotton rats during infection. The proliferation of MV-specific CD4 T cells was measured by stimulation of cotton rat spleen cells at different time points after infection with UV-inactivated measles virus. An SI above 2 was considered to be positive. The kinetics of viral titers (dotted line) is depicted schematically and is based on previously published data (13).

Inhibition and depletion of CD4 T cells do not lead to increase of viral titers. In order to demonstrate a causal relationship between the increase in the CD4 T-cell response and the decrease in viral titers, it is necessary to deplete CD4 T cells. For this purpose a CD4-specific monoclonal antibody (CR-CD4) was produced by immunizing BALB/c mice with concanavalin A-stimulated cotton rat spleen cells. This monoclonal antibody precipitated cotton rat CD4 (26). By histological examination CR-CD4 stained T-cell areas (identified by an antiserum against CD3) but not B-cell areas (identified by a monoclonal antibody against CD79a) in cotton rat lymph nodes (Fig. 2). As demonstrated by flow cytometry, cotton rat CD4 was expressed on spleen cells, lymph node cells, and thymus cells in a pattern typical for antibodies specific for mouse or rat CD4 (Fig. 3). In tissue culture, CR-CD4 inhibited proliferation of MV-specific spleen cells in a concentration-dependent manner (Fig. 4). The proliferation inhibition was similar in extent to that seen with an antibody (13/4) specific for mouse MHC-II, which cross-reacts with cotton rat MHC-II. In vivo, the injection of 100 µg of CR-CD4 led to complete depletion of CD4 T cells in the spleen of naive animals within 24 h (Fig. 3). In infected animals, higher amounts of antibody (0.5 mg) had to be injected every 4 days to achieve full depletion (data not shown). To test the role of CD4 T cells during infection, cotton rats were inoculated with 0.5 mg of CR-CD4 twice (on day 0 and day 4 after infection), with 200 µg of MHC-II-specific antibody (13/4) or a combination of both or PBS. To evaluate whether depletion of CD4 T cells and blocking of MHC-II would lead to prolonged virus replication, animals were tested for MV-specific T-cell proliferation in spleen and mediastinal lymph nodes and for viral titers in lung tissue 7 days after infection. In PBS-injected animals, T-cell proliferation in spleens was comparable to the kinetics shown in Fig. 1 (SI of 7.6 ± 5.3), and T-cell proliferation was found in mediastinal lymph nodes (SI of 4.4 ± 2.0). Viral titers were already declining from peak titers, as described previously (2.7 ± 0.5 log 10 TCID₅₀/g of lung tissue). In animals treated with CR-CD4 or a combination of CR-CD4 and 13/4, no T-cell proliferation was detectable in either spleen (Table 1) or the lung draining mediastinal lymph nodes. In the group of animals treated with 13/4, none had MV reactivity in lymph node cells, but two out of five animals demonstrated T-cell proliferation in the spleen (with an SI of 3.8 ± 0.8). Viral titers were not significantly different between groups although cotreatment with CR-CD4 and 13/4 led to increased viral titers. In summary, depletion or inhibition of CD4 T cells did not demonstrate an effect on viral clearance of lung infection.

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FIG. 2. Immunohistochemical staining of the B-cell and T-cell areas of cotton rat lymph nodes. Lymph nodes were stained with antibodies against CD79a, CD3, and CD4. The anti-CD79a antibody clearly stains the B-cell areas (germinal centers; brown, left); the anti-CD3 antiserum stains the T-cell areas (brown, middle) in the white pulp. CR-CD4 does not stain the B-cell area but does stain the T cell-area (black, right). The magnifications for the lymph node are x100 (top row) and x400 (bottom row).

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FIG. 3. Staining cells of lymphoid cells with CR-CD4. Lymph node cells, thymic cells, and spleen cells (bottom left) were stained with the CR-CD4 antibody, and the staining was analyzed by flow cytometry. The thin black line indicates values for cell populations stained with an isotype control and secondary antibody, and the thick gray line indicates values for cells stained with CR-CD4 and secondary antibody. After depletion of animals with CR-CD4, spleen cells were stained with CR-CD4 (bottom right, gray line) and compared to spleen cells from an animal not depleted of CR-CD4 (bottom right, black line).

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TABLE 1. Depletion of CD4 T cells does not influence viral clearance from lung tissuea

Immunization with CD4 T-cell epitope from the hemagglutinin protein does not lead to protection against infection. Although CD4 T cells apparently have no effect on clearance of virus from lungs during primary infection, they might protect against MV infection after immunization. In order to be able to specifically stimulate CD4 T cells, epitopes were determined for MV-specific cotton rat T cells. Spleen cells from cotton rats that had been immunized with plasmids expressing the hemagglutinin protein, the fusion protein, or the nucleocapsid protein were stimulated with peptides 15 amino acids long and overlapping by 5 amino acids (15). For peptides to be recognized as epitopes, the threshold was set to three times the background. For the hemagglutinin only one peptide corresponding to amino acids 553 to 567 on the hemagglutinin protein stimulated T-cell proliferation; on the fusion protein amino acids 348 to 372 were recognized, and on the nucleocapsid protein, amino acids 261 to 285 and 348 to 372 were recognized (Fig. 5). We have shown previously that immunization with a plasmid expressing the nucleocapsid protein of MV induces a strong CD4 T-cell response and nonneutralizing antibody response but does not induce protection against infection (29). On the other hand, neutralizing antibodies against the hemagglutinin protein are protective against infection. We chose to stimulate the hemagglutinin-specific T-cell response because these T cells might not only have a direct antiviral effect but also provide intramolecular B-cell help. Immunization with peptide (H553/567) induced an MV-specific T-cell response in cotton rats, which peaked 10 days after immunization and was abrogated by injection of CR-CD4 (data not shown). In order to test the effect of T-cell immunization with this peptide, cotton rats were immunized and infected with MV 10 days later. Some groups were also inoculated with the equivalent of 1 ml of a hemagglutinin-specific neutralizing antiserum (NT of 60) which leads to undetectable levels of neutralizing antibody in blood (NT of <10). Five days after infection with MV, virus was titrated from lung tissue homogenates, and T-cell proliferation measured from spleen cells. In animals immunized with hemagglutinin peptide (either with or without coinjection of antiserum), all but 1 (9 out of 10) demonstrated a T-cell response (Table 2). In animals without peptide immunization (either with or without coinjection of antiserum) only 5 (out of 10) had a detectable T-cell response. In addition, the SI of the T-cell response was higher in animals immunized with the hemagglutinin peptide (SI of 4.5 compared to SI of 2.4). Independent of the T-cell response, viral titers in lung tissue were not significantly different from nonimmunized animals or animals inoculated with serum (around 4 log 10 TCID₅₀/g of lung tissue). The only exception was the group of animals which had been immunized with hemagglutinin peptide and inoculated with neutralizing antiserum. This treatment led to a small but statistically significant reduction (P > 0.03) in viral titers. These data indicate that a hemagglutinin-specific T-cell response does not protect against infection alone but may be able to reduce viral titers in conjunction with even low antibody titers.

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FIG. 5. Determination of CD4 T-cell epitopes on MV hemagglutinin by peptide scanning. Cotton rats were immunized intradermally with plasmid pSC-N which expresses the nucleocapsid protein, with pCG-F1 which expresses the fusion protein, or pCG-H5 which expresses the MV hemagglutinin, as described previously (29). Spleen cells were stimulated with peptides 15 amino acid long that overlapped by 5 amino acids. The threshold for positive proliferation was set as three times the lowest counts (broken line). The one-peptide or two-peptide combinations which stimulated T-cell proliferation are signified by an asterisk. For the hemagglutinin (H) only one peptide corresponding to amino acids 553 to 567 on the hemagglutinin protein stimulated T-cell proliferation; on the fusion (F) protein amino acids 348 to 372 were recognized, and on the nucleocapsid (N) protein amino acids 261 to 285 and 348 to 372 were recognized.

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TABLE 2. Peptide immunization does not influence viral clearance from lung tissuea

CD4 T-cell responses developed during immunization in the presence of maternal antibodies do not protect against infection. MV immunization of children in the presence of maternal antibodies inhibits seroconversion. Recently, it has been reported that in spite of the lack of antibody production, T-cell proliferation can be detected (5). In order to investigate the generation of CD4 T cells after immunization in the presence of maternal antibodies, we used an established regimen of substituting maternal antibodies with passively transferred human MV-specific antibodies (30). Four groups of cotton rats were inoculated with either PBS or the equivalent of 1 ml of antiserum with an NT of 320, 160, or 80. One day later, this transfer resulted in NTs in serum of cotton rats of 60, 30, and 15, respectively. (These levels of antibody would be enough to protect cotton rats against i.n. infection with MV [28]). At this point, cotton rats were immunized i.p. with 10⁵ PFU of MV vaccine virus. After 9 weeks when the passively transferred antibodies had been metabolized, animals were challenged with MV i.n., and T-cell proliferation, neutralizing antibodies, and virus titers in lung tissue were measured. Immunization in the presence of high levels of maternal antibodies (NT of 320 and NT of 160) reduced (although did not abolish) the T-cell response whereas low levels had no effect (Fig. 6). Similarly, the total MV-specific antibody response as measured by ELISA was inversely correlated to the amount of passively transferred antibody present at the time of immunization. In contrast, the neutralizing antibody response was blocked completely after immunization in the presence of NTs of 320 and 160, and only a low-level NT of 12.5 was generated after immunization in the presence of an NT of 80. The lack of NT antibody generation correlated with a lack of protection after viral challenge (after immunization in the presence of NTs of 320 and 160). Generation of a low NT response resulted in partial protection (after immunization in the presence of an NT of 80) (Fig. 6). These data demonstrated that the generation of neutralizing antibodies is much more sensitive than the generation of T-cell responses to the presence of passively transferred antibody at the time of immunization. They also showed that the CD4 T-cell response generated after immunization in the presence of human MV neutralizing antibodies was not able to protect against infection in the absence of neutralizing antibodies.

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FIG. 6. Immunization in the presence of maternal antibodies. One day after the transfer of an equivalent of 1 ml of antibody with a neutralization titer of 320, 160, 80, or 0 of human MV-specific antibodies, groups of four cotton rats were immunized i.p. with 105 PFU of MV (Edm strain). Seven weeks later, serum samples were taken, and animals were challenged i.n. with MV (HU2 strain). Five days later, T-cell proliferation was measured from spleen cells (C), and virus titers were determined from lung tissues (D). Serum samples were analyzed by ELISA (A) and neutralization assay (B). Immunization in the presence of NTs of 320 and 160 did not result in neutralizing antibody titers above the threshold level of 10. The asterisk indicates significant differences compared to animals immunized in the absence of MV-specific antibody (0) for ELISA titers (at least P < 0.002) and NTs (at least P < 0.001); for virus titers, the asterisk indicates significant differences compared to nonimmunized animals (P < 0.002).

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The immune system is not able to protect against clinical signs after primary MV infection but eventually controls and clears viral infection. In patients with specific immune deficiencies, primary infection can be controlled and eventually cleared in the absence of a B-cell response (1, 7) but not in the absence of a functional T-cell response (12). Studies in rhesus macaques also demonstrate that B cells do not protect against primary infection and do not have a role in control and clearance of the virus (19). CD8 T cells, on the other hand, although not able to protect against infection, play a role in the control and clearance of infection (20). In this study, we have addressed the role of CD4 T cells during lung infection. Although during primary infection the kinetics of CD4 T-cell generation is inversely correlated with viral titers, depletion of CD4 T cells has no effect on virus replication. In contrast to these findings, it has been demonstrated in the mouse-MV encephalitis model that in resistant mouse strains the primary CD4 T-cell response is sufficient to protect the brain against MV infection (24). In susceptible and intermediately susceptible mouse strains, the generation of a CD4 T-cell response is not efficient enough to contain virus replication. In these mouse strains, CD8 T cells can protect against brain infection if they receive help from CD4 T cells or are already antigen specific ("primed") (33). In the brain, the antiviral mechanism of CD4 and CD8 T cells was shown to be the secretion of interferon gamma (4, 18). Although cotton rat MV-specific CD4 T cells secrete interferon gamma after in vitro stimulation with MV antigen (data not shown), currently no data are available to elucidate the role of interferon gamma in lung infection. In other experimental respiratory infections like influenza A virus (25) and respiratory syncytial virus infections (10), it has been demonstrated that protection against lung infection is achieved by virus-specific immunoglobulin G. The same is true for MV-infected cotton rats and has also been demonstrated in the rhesus macaque model and humans (11, 30, 32). After immunization with the live attenuated measles vaccine virus, T-cell and antibody responses are induced. However, only the level of neutralizing antibodies has been correlated with protection (in humans and experimental models). This might explain why induction of a hemagglutinin-specific CD4 T-cell response alone does not protect against lung infection. If this immunization is combined with passive transfer of a low (nonprotective) dose of neutralizing antibodies, a low level of protection is observed. This is in agreement with other studies where induction of T-cell responses alone by immunization with the nucleocapsid protein (expressed in various vector systems) was not protective against infection via the respiratory route (29, 34). Only when the vaccine vectors used expressed either the fusion protein or the hemagglutinin protein were neutralizing antibodies generated and protection achieved (21). In aggregate, these studies suggest that neutralizing antibodies present in the circulation protect against infection via the respiratory route. In contrast, studies in the mouse encephalitis model suggest that CD4 T cells might have a role in protection of other organs including the skin, the gastrointestinal tract, and the brain. It seems that control of (residual) virus infection and eventually resolution of infection in these target organs are important roles of both CD4 and CD8 T cells.

The relative contribution of the various parts of the immune system in protection, control, and clearance of MV is important in devising a strategy for vaccination in the presence of maternal antibodies. It has been a long-standing problem that MV vaccination is inhibited even by nonprotective titers of maternal antibodies (11). Until these maternal antibodies are fully metabolized, children are susceptible to infection with the wild-type virus and do not generate neutralizing antibodies after vaccination. However, the generation of CD4 T-cell responses has been observed in at least some individuals immunized in the presence of maternal antibodies (5). The present study in cotton rats confirms that the neutralizing antibody response is suppressed by maternal antibodies (even at low titers), whereas a CD4 T-cell response is always detectable but unable to protect against challenge with MV. This helps to explain disparate results for immunization in the presence of maternal antibodies with different vector systems. Our studies suggest that the immune response generated depends on the level of MV-specific antibody present at the time of immunization (high levels, no response; low levels, low response). Usually, CD4 T-cell responses and MV-specific antibodies measured by ELISA are detectable (indicating immunization success) whereas neutralizing antibodies and lung protection are not achieved (indicating immunization failure). Based on the T-cell responses induced in the presence of maternal antibodies and the increased clearance due to CD8 T cells, it has been suggested that a boost-prime strategy which induces only MV-specific T-cell responses followed by vaccination with the standard live attenuated vaccine might be advantageous. Based on vaccination studies in cotton rats and rhesus macaques, one would predict that T-cell vaccination alone would not protect against infection as such but would enhance control and clearance of infection (e.g., in the intestine and brain, where infection otherwise might lead to complications like diarrhea and encephalitis, respectively). In addition, there is some evidence that partial immunity might help to reduce immune suppression by MV (22), which is thought to be the major cause of severe measles cases with secondary infections.

 
* Corresponding author. Mailing address: Department of Veterinary Biosciences, The Ohio State University, 1925 Coffey Road, Columbus, OH 43210. Phone: (614) 688-4257. Fax: (614) 292-6473. E-mail: niewiesk.1{at}osu.edu

Published ahead of print on 6 June 2007.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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Journal of Virology, August 2007, p. 8571-8578, Vol. 81, No. 16
0022-538X/07/$08.00+0     doi:10.1128/JVI.00160-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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• Pasetti, M. F., Ramirez, K., Resendiz-Albor, A., Ulmer, J., Barry, E. M., Levine, M. M. (2009). Sindbis Virus-Based Measles DNA Vaccines Protect Cotton Rats against Respiratory Measles: Relevance of Antibodies, Mucosal and Systemic Antibody-Secreting Cells, Memory B Cells, and Th1-Type Cytokines as Correlates of Immunity. J. Virol. 83: 2789-2794 [Abstract] [Full Text]  

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