CpG containing oligodeoxynucleotides are potent adjuvants for parenteral vaccination with the fusion (F) protein of respiratory syncytial virus (RSV)

Vaccine 19 (2001) 4874– 4882 www.elsevier.com/locate/vaccine

CpG containing oligodeoxynucleotides are potent adjuvants for parenteral vaccination with the fusion (F) protein of respiratory syncytial virus (RSV) Gerald E. Hancock *, Kristen M. Heers, Jason D. Smith, Catherine A. Scheuer, Alexander R. Ibraghimov 1, Karin S. Pryharski Department of Immunology Research, Wyeth-Lederle Vaccines, 211 Bailey Road, West Henrietta, NY 14586, USA Received 12 January 2001; received in revised form 3 April 2001; accepted 12 April 2001

Abstract The feasibility of using oligodeoxynucleotides (ODN) containing unmethylated CpG motifs as parenteral adjuvants for subunit vaccines against RSV was tested in BALB/c mice. Compared with immunization with natural F protein adsorbed to aluminum hydroxide (F/AlOH) adjuvant alone, coadministration of F/AlOH with CpG ODN resulted in statistically significant increases in serum neutralization titers, an enhanced generation of splenic antigen-dependent killer cell precursors, and accelerated clearance of infectious virus from lungs 4 days after challenge. The statistically significant increases in serum IFNg and anti-F protein IgG2a titers, and significantly diminished pulmonary IL-5 and eosinophilia after challenge indicated that CpG ODN enhanced the ability of F/AlOH to elicit type 1 immune responses. F protein-specific serum IgE titers were also reduced. Further analysis of pulmonary inflammatory cells demonstrated an expansion of CD8+ T cells, relative to the CD4+ T cell compartment. The potency of CpG ODN was not adversely affected in gene knockout mice devoid of the p35 chain of the IL-12 heterodimer. Taken together, the results suggest a novel formulation for naı¨ve recipients of F protein-based subunit vaccines that does not result in a type 2 phenotype. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Eosinophils; Fusion (F) protein; Oligodeoxynucleotides (ODN); Unmethylated CpG; Respiratory syncytial virus (RSV); Type 1 T cells; Subunit vaccines

1. Introduction Respiratory syncytial virus (RSV) is a negative strand RNA virus and a major infectious agent for very young infants, and infants and children with congenital heart disease, bronchopulmonary dysplasia, or cystic fibrosis [1]. RSV causes bronchopneumonia, bronchiolitis, and pneumonia, and RSV infection is associated with asthma in early life [2,3]. RSV is also recognized as a significant agent for respiratory tract disease in aged adults and patients with immunological abnormalities [4]. Towards the development of subunit vaccines for * Corresponding author. Tel.: + 1-716-273-7682; fax: +1-716-2737665. E-mail address: [email protected] (G.E. Hancock). 1 Present Address: Department of Immunology, AstraZeneca R & D Boston, Inc., 35 Gatehouse Drive, Waltham, MA 02451.

seropositive populations, several clinical trials in institutionalized or ambulatory aged human volunteers, normal children greater than 18 months of age, and seropositive children with cystic fibrosis or bronchopulmonary dysplasia have been performed [5]. The results from these studies demonstrated that immunoaffinity or ion exchange chromatography purified natural F protein (adsorbed to alum adjuvant) was safe, and when compared with pre-vaccination, elicited greater than four fold increases in serum neutralizing titers in approximately 50% of the recipients. To increase the number of recipients with augmented serum neutralizing titers improved formulations may be required. Recently, we reported that coformulation of F protein with the attachment (G) glycoprotein significantly augmented systemic neutralizing titers in seropositive chimpanzees [6]. Thus for seropositive recipients, a vaccine composed of F and G proteins adsorbed to alum is

0264-410X/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 4 - 4 1 0 X ( 0 1 ) 0 0 2 2 8 - 6

G.E. Hancock et al. / Vaccine 19 (2001) 4874–4882

tenable. However, there are issues with the development of subunit vaccines for seronegative subjects. This is because exacerbated disease was observed in naı¨ve human infants immunized with a formalin-inactivated whole virus (FI-RSV) vaccine upon subsequent infection [7,8]. The current opinion is that polarized type-2 T cell responses were responsible for enhanced disease, and studies in naı¨ve rodents [9– 12] or non-human primates [13] vaccinated with a facsimile FI-RSV vaccine appear to support this conclusion. Therefore, for RSV-naı¨ve populations it is essential that formulations that induce balanced, not polarized type-2 T cell responses be identified. One category of compounds with significant promise as adjuvants for subunit vaccine antigens are synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG dinucleotides [14]. These compounds were reported to have adjuvant activity in mice and drive immune responses towards a type 1 phenotype [15]. CpG ODN activate lymphocytes to secrete IL-6, IL-10, and immunoglobulin, natural killer cells to secrete IFNg, and monocytes and dendritic cells to secrete IL-12. IL-12 [16] and IFNg [17] play key roles in the generation of type 1 immune responses. It was reported [15] that the optimum ODN for murine studies has a sequence that contains a CpG dinucleotide flanked by two 5% purines and two 3% pyrimidines (5% GACGTT 3%). Therefore, we investigated the impact of CpG (5% GCATGACGTTGAGCT 3%) or control (5% GCATGATGTTGAGCT 3%) ODN on both the functional and quantitative aspects of immune responses generated in BALB/c mice following intramuscular injection with natural F protein. Our results indicate that CpG ODN enhances the ability of F protein to induce type 1 immune responses. Coadministration with F/AlOH augmented the secretion of IFNg into the sera, diminished the generation of F protein-specific IgE titers, increased the generation of complement assisted neutralizing titers, and enhanced antigen dependent killer cell activity. CpG ODN are promising adjuvants for subunit vaccines against RSV.

2. Materials and methods

2.1. Animals Female BALB/c mice (8– 10 weeks of age) and Sprague–Dawley rats (retired breeders) were obtained, respectively, from Charles Rivers Laboratories (Wilmington, ME) and Taconic (Germantown, NY). Female BALB/c mice with a disruption in the gene encoding the p35 chain of IL-12 heterodimer were obtained from Dr R.L Peterson, Department of Molecular Medicine and Pharmacogenomics, Genetics Institute/Wyeth, Andover, MA 01810. All animals were housed in a facility

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accredited by the American Association for Accreditation of Laboratory Animal Care.

2.2. Vaccines/challenges All immunizations with F protein (0.03 or 3.0 mg per dose) were intramuscular (0.1 ml, anterolateral thigh muscle). Natural F protein was purified [18–20] from Vero cells (ATCC No. CCL 81) infected with the A2 strain of RSV. The protein was greater than 95% pure as estimated by SDS-PAGE and antigen capture ELISA. F protein was adsorbed to aluminum hydroxide (AlOH, 100 mg per dose) adjuvant (F/AlOH) and then admixed with ascending doses of CpG (0.8– 100 mg per dose) or control ODN (4 or 100 mg per dose). The sequence of the CpG ODN was 5% GCATGACGTTGAGCT 3% and the control ODN had the sequence 5% GCATGATGTTGAGCT 3% [21]. The ODN were synthesized with a phosphorothioate-modified backbone on an Applied Biosystems 380B DNA Synthesizer (Foster City, CA). QS-21 was obtained from Aquila BioPharmaceuticals, Inc, Worcester, MA. Other control vaccines were prepared with F protein adsorbed to AlOH without ODN, admixed with 20 mg per dose QS-21 (F/QS-21), or prepared in PBS alone (F/PBS). Additional control mice were injected with CpG ODN in PBS alone, immunized by infection with the A2 strain of RSV (2× 106 PFU) clarified from HEp-2 cells (ATCC No. CCL 23), or received an equal amount of mock infected HEp-2 cell antigen control. All intranasal (0.05 ml) challenges were performed 4 weeks after primary immunization under injectable anesthesia (60 mg ketamine per kg and 2.5 mg xylazine per kg, The Butler Co, Dublin OH) and used the A2 strain of RSV (  2× 106 PFU).

2.3. Pulmonary inflammatory response The effect of CpG ODN on the morphology of pulmonary inflammatory cells was assessed following bronchoalveolar lavage (BAL, 18–20) 5 days after challenge. The phenotypic distribution of T cells was ascertained using standard flow cytometric techniques (FACSort, Becton Dickinson, Mountain View, CA). Phycoerythrin (PE) conjugated anti-mouse CD3 (17A2) and CD4 (HSGK1.5), and FITC-conjugated antimouse CD8a (53–6.7) antibodies were used to detect T lymphocyte surface markers on pooled BAL cells from five mice per group [20]. The BAL cells were preincubated with Fc block reagent (anti-CD16/CD32, 2.4G2) prior to staining, and propidium iodide was added to stain dead cells prior to data collection. All flow cytometry reagents were obtained from PharMingen (San Diego, CA). The relative percentage CD3+, CD4+ or CD8+ cells was determined after gating on live lymphocytes. The data are presented as the mean (91

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G.E. Hancock et al. / Vaccine 19 (2001) 4874–4882

S.D.) of four independent studies. The relative percentage of eosinophils was ascertained as described [18–20] after the examination of at least 400 leukocytes on slides stained with Diff-Quik (Dade International, Miami, FL).

2.4. Determination of percent cytotoxicity The presence of antigen-dependent killer cell precursors in the spleens of vaccinated mice was determined as earlier described [19] using a standard 4 h 51Cr (Amersham Corp., Arlington Heights, IL) release assay. Briefly, spleen cells from vaccinated mice were cocultured 6 days with syngeneic RSV-infected stimulator cells from naı¨ve mice. There were five responder splenocytes to one stimulator cell. Thereafter, killer cell activity was assessed against syngeneic control targets, and target cells actively infected overnight (multiplicity of infection = 10) with the A2 strain of RSV. Percent specific release was calculated by 100×[(mean cpm experimental)−(mean cpm spontaneous release)]/ [(mean cpm total release)− (mean cpm spontaneous release)].

2.5. Determination of RSV infecti6ity The detection of infectious virus in the lungs after challenge was assessed in a plaque assays as earlier described [18–20]. Briefly, the lungs were removed 4 days after challenge, homogenized, clarified, snap frozen, and stored at − 70 °C until assayed on HEp-2 cell monolayers.

of Evan’s Blue Dye in the skin. For the determination of total levels of IgE, paired monoclonal antibodies against murine IgE (clone R35-72 and biotin-labeled antibody, clone R35-92) and anti-dansyl IgE antibody (clone 27–74) were purchased from PharMingen (San Diego, CA). The amount of IgE was ascertained from a standard curve using a VersaMax ELISA plate reader (405 nm) and SoftMaxPro software (four parameter analysis) from Molecular Devices (Sunnyvale, CA).

2.7. Cytokine determinations IFNg and IL-5 levels were determined as earlier described [18–20] by ELISA. For the quantification of IFN-g in sera, samples were collected pre- and 1, 2, 3, 4, 5, and 7 days after injection of vaccine. IL-5 was quantified in BAL fluids 5 days after challenge. Recombinant (r) murine IFN-g and rIL-5, and monoclonal antibodies (mAb) specific for IFN-g (XMG1.2) or IL-5 (TRFK.5 and TRFK.4) were purchased from PharMingen. The R4-6A2 anti-IFN-g mAb was purified from hybridoma supernatants. The optical density was measured on an ELISA reader (Molecular Devices) at 450 nM with a reference of 550 nM. The amount of cytokine was determined by extrapolating on the linear portion of a standard curve (four parameter) that was generated using rIFNg or rIL-5. The limit of detection of these assays is defined as the amount of recombinant cytokine that gives an OD value equal to three S.D. above the average OD450 of eight wells containing no cytokine.

2.8. Statistical analyses 2.6. Serum antibody determinations The geometric mean serum IgG and neutralizing titers were determined by endpoint ELISA and the plaque reduction neutralization test, respectively, 4 weeks after primary vaccination [18– 20]. The neutralizing titers were determined against the A2 strain of RSV in the presence or absence of 5% (V/V) guinea pig serum (BioWhittaker, Walkersville, MD) as a source of complement (C). The neutralization titers were calculated as the reciprocal of the serum dilution that showed 60% reduction (relative to the virus control) in the number of foci per well. F protein-specific IgE titers were determined as earlier described [20,22] using a rat passive cutaneous anaphylaxis (PCA) model. In brief, rats were sedated (40– 80 mg ketamine per kg and 2.5 – 5 mg xylazine per kg, The Butler Co) and injected intradermally (0.1 ml) with serially diluted (three-fold) pooled sera (5 mice per group). The rats were challenged intravenously (1.0 ml) with a solution containing 2 mg natural F protein and 1% Evans Blue dye in PBS 48 h thereafter. The F protein-specific IgE titer is the reciprocal of the greatest dilution showing no evidence

Significant differences (PB0.05) were determined after log transformation by Tukey–Kramer HSD multiple comparison or Student’s t-test using JMP® statistical software (SAS Institute Inc, Cary, NC). The data are expressed9 1 S.D. All data were confirmed in separate studies with similar results.

3. Results

3.1. Humoral and cell-mediated immune responses after coadministration of F/AlOH with CpG ODN Since CpG dinucleotides were reported [23–29] to augment type 1 immune responses against a variety of subunit vaccine antigens, we investigated the capacity of CpG ODN to functions as adjuvants for subunit vaccines against RSV. Subunit vaccines against RSV are contraindicated for fear of inducing type 2 T cell responses [7–13]. Early on in the investigation the effects of CpG ODN on the immune responses induced by F/PBS alone were compared with those generated by

G.E. Hancock et al. / Vaccine 19 (2001) 4874–4882

F/AlOH. In the experiment depicted in Table 1, naı¨ve BALB/c mice were injected on weeks 0 and 4 with vaccines co-formulated with 1, 5 or 20 mg CpG ODN. About 2 weeks after secondary vaccination humoral and cell-mediated immune responses were assessed. When compared with F/PBS without CpG ODN, immunization with F/PBS plus 1 – 20 mg CpG ODN did not result in statistically significant increases in anti-F protein total or subclass IgG, or neutralizing antibody titers. In contrast, coadministration of CpG ODN with F/AlOH resulted in augmented humoral and cell-mediated immune responses when compared with F/AlOH alone. The addition of 5 or 20 mg CpG ODN to F/AlOH resulted in elevated serum anti-F protein total IgG and IgG2a antibody titers 2 weeks after secondary vaccination (Table 1). Indeed, the IgG2a antibody titers following vaccination with F/AlOH plus 20 mg CpG ODN were heightened 50 fold versus F/AlOH alone and were not statistically disparate from titers generated after secondary vaccination with F/QS-21. Moreover, statistically elevated complement assisted neutralizing antibody titers were achieved. The data further indicated that vaccines composed of F/PBS plus CpG ODN were less able to generate antigen dependent killer cells than F/AlOH plus CpG ODN. Antigen dependent killer cells were not detected in the spleens of mice vaccinated with F/PBS coformulated with 1, 5 or 20 mg CpG ODN (Table 1). On the other hand, after in vitro expansion the splenic immunocytes from mice vaccinated with F/AlOH plus 5

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or 20 mg CpG ODN, respectively, had nearly 20 and 30% cytolysis above background. Cytolysis was approximately 5% above background for the group injected with F/AlOH in PBS alone. Immunization with F/QS21 resulted in antigen specific lysis that was 45% above background. The capacity of 20 mg CpG ODN to augment the ability of F/AlOH to generate cell-mediated immune responses was observed in two of three experiments. Taken together, the data indicated that CpG ODN did not enhance immune responses elicited by F/PBS. We, therefore, focused the investigation on the potential of CpG ODN to change the quantity and quality of immune responses generated by F/AlOH. In a separate series of studies groups of mice were injected with F/AlOH prepared with control ODN to confirm the importance of the CpG motif in adjuvanticity. The results depicted in Table 2 verified that immunization with F/AlOH plus CpG ODN elicited elevated serum IgG2a titers. About 4 weeks after primary immunization with F/AlOH plus 4 or 100 mg CpG ODN serum IgG2a titers were significantly increased 8 and 794 fold, respectively, when compared with titers generated after vaccination with F/AlOH without CpG ODN. Moreover, when contrasted with groups primed with F/AlOH plus 4 or 100 mg control ODN, the respective CpG ODN formulated vaccines elicited IgG2a titers that were 25 and 200 fold greater. Coformulation with CpG ODN also significantly increased serum complement assisted neutralization titers. The titers of mice primed with F/AlOH plus 100 mg CpG

Table 1 The effect of CpG ODN on the capacity of F protein to elicit systemic humoral and cell-mediated immune responses 2 weeks after secondary vaccination Geometric mean serum antibody titers (log10)a Anti-F protein

Neutralizing

Vaccine

CpG ODN (mg)

IgG

IgG1

IgG2a

+C

−C

% Cytolysisb

F/PBS F/PBS F/PBS F/PBS F/AlOH F/AlOH F/AlOH F/AlOH F/QS-21

None 1 5 20 None 1 5 20 None

6.09 0.2 5.49 0.5 5.69 0.1 5.99 0.2 6.29 0.2 6.49 0.3 6.8 9 0.1c 6.89 0.1c 6.8 90.2

5.7 9 0.1 5.0 9 0.3 5.1 90.01 5.4 9 0.3 5.9 9 0.2 6.1 90.3 6.2 9 0.2 6.1 90.4 6.29 0.1

3.7 90.6 4.0 9 0.7 4.690.3 4.6 9 0.7 4.5 9 0.2 5.2 90.4 5.7 9 0.2c 6.2 9 0.2c 6.19 0.2

1.5 90.5 1.3 90.3 1.7 9 0.4 1.4 90.3 2.3 9 0.6 2.7 90.4 2.4 90.3 2.6 90.2c 3.1 90.5

B1.3 B1.3 B1.3 B1.3 1.5 9 0.5 1.7 90.5 2.1 90.2c 2.3 90.1c 2.2 90.3

7.7 −0.6 2.8 −0.4 4.6 3.3 18.5 26.0 44.9

BALB/c mice were vaccinated on weeks 0 and 4 with 3 mg natural F protein adsorbed to AlOH, admixed in QS-21, or PBS alone and the denoted doses CpG ODN. a The geometric mean serum endpoint IgG and neutralizing antibody titers ( 9 1 S.D.) were determined by ELISA and the plaque reduction neutralization test respectively. The neutralizing antibody titers were determined in the presence (+C) or absence (−C) of 5% complement. There were 5 mice per group. b Bulk spleen cells were obtained from 5 mice per group 2 weeks after secondary vaccination, pooled, and stimulated in vitro for 6 days with syngeneic stimulator cells infected with RSV. Thereafter the cells were tested for antigen-dependent killer cell activity against syngeneic control or RSV-infected target cells. The results are depicted minus background cytolysis of control targets at a 33:1 effector to target ratio. c PB0.05 versus F/AlOH in PBS alone.

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Table 2 The humoral immune responses of BALB/c mice 4 weeks after primary immunization with F/AlOH coformulated with CpG ODN Serum antibody titersa Anti-F Protein (Log10)

Neutralizing (Log10)

Vaccine

ODN (mg)

IgG

IgG1

IgG2a

IgE

Total IgE (ng/ml)

+C

−C

F/AlOH F/AlOH F/AlOH F/AlOH F/AlOH

None Control (4.0) Control (100.0) CpG (4.0) CpG (100.0)

5.99 0.3 5.29 0.2 6.49 0.3 6.5 9 0.6 7.39 0.6b

5.3 90.2 6.3 90.9 5.9 90.4 6.9 91.2 6.79 0.2b

3.9 90.2 3.4 90.4 4.5 9 1.0 4.8 90.5b,c 6.8 90.3b,c

2.1 1.7 1.7 2.1 1.2

3.6 9 0.2 3.3 9 0.2 3.2 90.5 3.4 90.2 3.4 90.4

2.3 90.4 1.9 9 0.4 2.2 90.6 2.7 90.1 3.79 0.2b,c

B1.3 1.6 9 0.4 1.4 9 0.1 1.4 90.2 1.99 0.6

a BALB/c mice were primed with 3 mg natural F protein adsorbed to AlOH, F/AlOH admixed with 4 or 100 mg of CpG or control ODN, or F/PBS alone. The geometric mean serum IgG and neutralizing titers were determined by ELISA and the plaque reduction neutralization test respectively. The neutralizing titers were ascertained in the presence (+C) or absence (−C) of 5% sera as a source of complement. Total and F protein specific IgE titers were determined by ELISA and a PCA reaction, respectively. Pooled sera were used to ascertain F protein specific IgE titers. There were 5 mice per group. b PB0.05 versus F/AlOH formulated without ODN. c PB0.05 versus F/AlOH formulated with the respective 4 or 100 mg control ODN.

were 32 and 25 fold greater when compared with those primed with F/AlOH plus 100 mg control, or no ODN, respectively. Statistically significant increases in neutralization titers were not observed when F/AlOH was coadministered with 4 mg CpG ODN.

3.2. The addition of CpG ODN to F/AlOH facilitates accelerated clearance of RSV from the lungs We next investigated if the heightened immune responses resulted in an enhanced capacity to inhibit virus replication in the pulmonary tissues after challenge. To accomplish this, vaccines were formulated with a suboptimal amount (0.03 mg per dose) of F protein adsorbed to AlOH. The data indicated that CpG ODN enhanced the capacity of F/AlOH to elicit immune responses that were more efficacious (Table 3). When compared with vaccination without CpG ODN, priming with F (0.03 mg)/AlOH + 0.8 or 4 mg CpG ODN resulted in significant reductions in infectious virus in the lungs 4 days after challenge. Complete clearance of infectious virus was observed when F (0.03 mg)/AlOH was coadministered with 20 or 100 mg CpG ODN. Infectious virus was also not detected in the lungs of mice immunized by experimental infection after challenge.

3.3. CpG ODN enhance the capacity of F/AlOH to generate type 1 immune responses The enhanced cell-mediated immune responses and statistically significant increases in IgG2a and complement assisted neutralizing titers suggested that CpG ODN increased the generation of type 1 immune responses. To test this hypothesis sera were analyzed for the effects of CpG ODN on the generation of IgE

antibodies (Table 2) and IFNg secretion (Fig. 1). In addition, the phenotype of T cells infiltrating the lungs after challenge was examined (Table 4). Upon initial analysis, IgE did not appear to be diminished 4 weeks after primary vaccination with F/AlOH plus CpG ODN. Statistically significant differences between the groups in total IgE were not observed. However, when the sera were compared in the rat PCA assay, a ninefold reduction in F protein specific IgE titers (from 2.1 log10 to 1.2 log10) was observed (Table 2). In contrast, F protein specific IgE titers of groups primed with F/AlOH + 4 or 100 mg of control ODN were 1.7 log10 and only three fold less than that generated after vaccination with F/AlOH without ODN. Table 3 The effect of CpG ODN on the capacity of F/AlOH to generate protective immune responses in BALB/c mice Vaccine

CpG ODN (mg)

GMT RSV (log10)a

F/AlOH F/AlOH F/AlOH F/AlOH F/AlOH PBS/AlOH RSV

0.0 0.8 4.0 20.0 100.0 100.0 None

4.2 90.9 2.7 90.9c,d 2.3 9 0.6c B2.090.1c B1.99 0.1 4.8 9 0.2b B1.89 0.1c

BALB/c mice were primed with 0.03 mg F protein adsorbed to AlOH plus the indicated ascending doses of CpG ODN. Control mice were injected with PBS/AlOH plus 100 mg CpG ODN or experimentally infected with the A2 strain of RSV. a The mice were challenged with the A2 strain of RSV 4 weeks after primary vaccination and the geometric mean titer (GMT) RSV was determined 4 days thereafter. There were 10 mice per group. b PB0.05 versus GMT RSV from all groups except F/AlOH without ODN. c PB0.05 versus GMT RSV from F/AlOH without ODN. d PB0.05 versus GMT RSV from F/AlOH plus 100 mg CpG ODN.

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Table 5 The absence of eosinophilia and elevated IL-5 in the lungs upon challenge of IL-12 knockout mice primed with F/AlOH plus CpG ODN

Fig. 1. The temporal secretion of IFNg into the sera of BALB/c mice primed with F/AlOH plus CpG ODN. Mice were primed with 3 mg F protein adsorbed to AlOH (F/Al) or admixed in PBS alone (F/PBS). CpG ODN were added to the vaccines at 100 mg per dose (F/Al/CpG or F/PBS/CpG). At the indicated time points thereafter IFNg was determined in the sera. Each data point represents the mean of five mice ( 9 1 S.D.). Additional control mice were injected with CpG ODN (100 mg per dose) in PBS alone (PBS/CpG) or F/QS-21. Statistical differences (P B0.05) were determined after log transformation.

For the experiments depicted in Fig. 1 and Tables 4 and 5, the 100 mg-dose of CpG ODN was used. This dose resulted in the greatest enhancement in neutralization titers. Coadministration of F/AlOH with 100 mg CpG ODN increased the secretion of IFNg into sera (Fig. 1). Peak amounts of IFNg were observed 2 days after injection in the sera of all recipients of vaccines containing CpG ODN. In comparison, peak amounts of IFNg occurred 1 day after injection of F/QS-21 and were approximately three fold less than that of mice primed with CpG ODN. The data indicated that the quantity and duration of IFNg in the sera were not dependent on the presence of F protein or AlOH. The amounts of IFNg in the sera of mice administered

IL-12

Vaccine

% EOSa

IL-5(OD450)

IFNg (OD450)

IL-12 −/−

F/AlOH

239 10b

IL-12 −/−

F/AlOH/CpG 1 9 0.7

IL-12 +/+

F/AlOH

IL-12 +/+

F/AlOH/CpG 1 9 1

2.293 90.801b 0.056 90.032 0.616 90.155b 0.115 90.129

0.356 90.405 1.283 9 1.032 3.207 90.197 2.778 90.863

15 96b

Normal (IL-12 +/+) or IL-12 knockout (IL-12−/−) BALB/c mice were primed with F protein (3 mg/dose) adsorbed to AlOH (100 mg/dose), or F/AlOH plus 100 mg CpG ODN. All mice were challenged with the A2 strain of RSV 4 weeks after immunization. a The relative mean percentage eosinophils (EOS), and IL-5 and IFNg ( 91 S.D.) were determined in the BAL fluids 5 days thereafter. There were 5 mice per group b PB0.05 versus respective IL-12 +/+ or IL-12 −/− BALB/c mice primed with F/AlOH/CpG.

F/AlOH + 100 mg CpG ODN were not significantly different from that of mice immunized with F/PBS plus CpG ODN, or CpG ODN in PBS. The characterization of the pulmonary inflammatory infiltrates for T cell phenotype after challenge suggested that coadministration of CpG ODN resulted in an expansion of CD8+ T cells relative to the CD4+ T cell compartment (Table 4). The lungs of mice primed with F/AlOH, or F/PBS + 100 mg CpG ODN had 14 95 and 169 6% CD8+ T cells 5 days after challenge. This was, respectively, 2.3 and 2.7 times greater than that observed in the lungs of mice primed with F/AlOH or F/PBS without CpG ODN. In comparison, priming with F/QS-21 resulted in a CD8+ T cell compartment that was relatively three fold greater when compared

Table 4 The phenotype of pulmonary T cells following challenge of mice primed with F/AlOH plus CpG ODN Vaccine

CpG ODN

% Lymphocytes

% CD3

% CD4

% CD8

CD4/CD8

F/AlOH F/AlOH F/PBS F/PBS F/QS-21 PBS

100 mg None 100 mg None None 100 mg

599 6 369 9a 48 99 3592a 57 9 3b 37 98a

3699 24 9 11 24 9 9 14 9 2a 32 9 7b 13 9 5a

23 9 6 14 94c 12 95a 10 9 2a 19 9 2b 8 92a

14 9 5 6 92a 16 9 6d 6 9 1a 18 9 5b 8 93a

1.6 2.3 0.8 1.7 1.1 1.0

BALB/c mice were primed with F/AlOH co-formulated with CpG ODN. Control mice were immunized with F/AlOH alone, F protein admixed with CpG ODN, F protein admixed with QS-21, or CpG ODN in PBS alone. All mice were challenged with the A2 strain of RSV 4 weeks thereafter. The data are the mean percent lymphocytes (Lym.), CD3+, CD4+, or CD8+ T cells 5 days after challenge. Statistical differences (PB0.05) were determined by Student’s t-test after log transformation. CD4/CD8 denotes the ratio of CD4 to CD8 T cells. a PB0.05 versus F/AlOH plus CpG ODN and F/QS-21. b P\0.05 versus F/AlOH plus CpG ODN. c PB0.05 versus F/AlOH plus CpG ODN. d PB0.05 versus F/AlOH without CpG ODN.

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with immunization with F/AlOH or F/PBS without CpG ODN. The resultant CD4/CD8 T cell ratios in the lungs of mice primed with F/AlOH plus CpG ODN, F/PBS + CpG ODN, or F/QS-21 5 days after challenge were 1.6, 0.8, and 1.1, respectively. In comparison, the ratios of T cells in the lungs of mice primed with F/AlOH or F/PBS without CpG ODN was 2.3 and 1.7, respectively after challenge. Adsorption of F protein to alum did not expand the CD8+ T cell compartment. The percent of CD8+ T cells observed in the lungs of mice primed with F/AlOH was identical to that of mice primed with F/PBS after challenge. The effects of CpG ODN on the generation of type 2 immune responses was further explored in other experiments by examining the lungs of mice for IL-5-dependent eosinophilia [19,30] after challenge. In addition, the role that IL-12, a potent inducer of type 1 T cells [16] could have on CpG ODN adjuvanticity was investigated using IL-12 knockout mice. In the first series of studies, pulmonary eosinophilia (Fig. 2A) and IL-5 (Fig. 2B) were significantly reduced 5 days after challenge when F/AlOH was co-formulated with 20 or 100 mg CpG ODN and compared with F/AlOH alone. The reduction in type 2 phenotype appeared to be dependent on dose, as the 0.8 and 4 mg CpG ODN did not result in statistically significant reductions in IL-5 or eosinophilia. As earlier reported [18– 20] pulmonary eosinophilia upon challenge of mice vaccinated with F/QS-21 was not observed (Fig. 2A). The potency of CpG ODN did not appear to be absolutely reliant on the presence of IL-12 heterodimer (Table 5). Significant reductions in IL-5 and eosinophilia were still observed in IL-12 knockout mice vaccinated with F/AlOH plus CpG ODN.

murine studies [15]. To evaluate the effects of CpG ODN on the generation of distinct T cell subsets, antibody isotypes [32] and IFNg [17] were determined in sera following vaccination. In addition, the lungs of vaccinated mice were examined after challenge for accelerated clearance of infectious virus, the phenotype of infiltrating T cells, and IL-5-dependent eosinophilia [19,30]. The effect of CpG ODN on the generation of antigen dependent killer cell precursors was also monitored. When viewed in sum, the data strongly suggest that CpG ODN increase the capacity of F/AlOH to induce more balanced immune responses in naı¨ve BALB/c mice. Coadministration of CpG ODN with F/AlOH led to significant increases in IFNg, IgG2a, and complement assisted neutralizing titers in the sera. F protein-specific IgE titers were also diminished. In

4. Discussion In several murine models, CpG ODN have been reported to augment type 1 immune responses against a variety of subunit vaccine antigens. These include hepatitis B [23], HIV [24], influenza [25], leishmania [26], pneumococcal polysaccharide conjugated to protein carrier [27], and cancer [28] antigens. CpG ODN have also been shown to diminish schistosome egg-induced pathology mediated by type 2 dominated immune responses [29] and lessen airway inflammation in a murine model of asthma [31]. Thus it was logical to test whether CpG ODN could boost immunogenicity and reduce the type 2 immune responses generated in naı¨ve mice injected with natural F protein-based vaccines adsorbed to AlOH. In the studies presented herein, an ODN with the sequence 5% GCATGACGTTGAGCT 3% was evaluated. This sequence is flanked by two 5% purines and two 3% pyrimidines and contains the 5% GACGTT 3% sequence reported to be optimal for

Fig. 2. The pulmonary inflammatory response upon challenge of BALB/c mice primed with F/AlOH co-formulated with CpG ODN. Naı¨ve BALB/c mice were primed with 3 mg F protein adsorbed to AlOH (F/Al) adjuvant +five fold ascending doses (0 – 100 mg) of CpG ODN. For comparison, additional mice were immunized with F protein admixed with CpG ODN without AlOH (F/PBS/CpG ODN), or F/QS-21. All mice were challenged with the A2 strain of RSV 4 weeks later. Eosinophilia (A) and IL-5 (B) were assessed in the lungs 5 days after challenge. The results are the means of 5 mice per group 9 1 S.D. The pulmonary tissues of mice primed with F/ AlOH+20 or 100 mg CpG ODN had significantly less (PB 0.05) eosinophilia and IL-5 when compared with mice primed with F/ AlOH alone.

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addition, pulmonary eosinophilia and IL-5 were significantly reduced, and splenic antigen dependent killer cell precursors were increased, when CpG ODN was added to the vaccines. The greatest potential for subunit vaccines appears to be when CpG ODN is added to F protein adsorbed to alum adjuvant. The resultant cell-mediated immune responses were greater in magnitude than those generated by F/PBS +CpG ODN. In addition, vaccination with F/AlOH +CpG ODN generated enhanced humoral immune responses when compared with those elicited by F/PBS +CpG ODN. Thus the data are similar to those reported using HIV gp160 protein [24], leishmania [26], and hepatitis B [23] antigens. However, it remains unclear what role alum may have on CpG ODN adjuvanticity. The presence of alum was not associated with an increased persistence of IFNg in the sera. Indeed, IFNg was significantly increased in the sera following injection of CpG ODN in PBS alone, without F protein or alum. Moreover, in data not presented herein CpG ODN binding to alum did not appear to be a pre-requisite for enhanced immunogenicity. When co-formulated with 3 mg F protein, approximately 20 of 100 mg CpG ODN bound to 100 mg AlOH. In comparison, binding of CpG ODN to 100 mg aluminum phosphate (AlPO) was negligible. In a direct side by side comparison the humoral immune responses elicited after vaccination of BALB/c mice with 3 mg F protein adsorbed to 100 mg of either AlOH or AlPO, +100 mg CpG ODN were identical. Thus the data imply that CpG ODN not only have adjuvant properties, but also may have the power to modify developing immune responses facilitated by other adjuvants. The results presented herein do not fully address the mechanism(s) of how CpG ODN transform the capacity of F/AlOH to elicit more balanced immune responses. The primary effects of CpG ODN appear to be increased IFNg production and generation of CD8+ T cells. Thus, the data are consistent with several reports [15,24,36–39] which demonstrate that the adjuvant activities of CpG ODN are likely associated with the actions of IL-12 and IFNg and further support the notion [12,33,34] that IFNg-secreting CD8+ T cells regulate the pulmonary eosinophilia associated with RSV infection. However, the role of IL-12 in CpG ODN adjuvanticity must be qualified by the observation that coadministration of rIL-12 with F/AlOH [20] or a facsimile FI-RSV vaccine [35] preferentially expanded CD4+ T cells. In addition, coformulation with rIL-12 did not ameliorate pulmonary eosinophilia [20]. Moreover, the data presented herein and those of others [29,31] clearly demonstrated that CpG ODN diminished type 2-dominated immune responses in the absence of endogenous IL-12. Thus, CpG ODN may regulate immune responses to F/AlOH via several mechanisms.

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To increase immunogenicity and elicit more balanced T cell responses it is likely that future subunit vaccines will require novel adjuvants and/or immune response modifiers. Currently, the only adjuvants approved for humans are the aluminum-based salts and a squalene containing oil/water emulsion [14]. The data presented herein demonstrate for the first time the potential of CpG ODN for subunit vaccines against RSV. Coadministration of F/AlOH with CpG ODN generated immune responses in naı¨ve mice that were more balanced and not polarized towards a type 2 phenotype. Elevated serum F protein specific IgE titers, and eosinophilia and IL-5 in the lungs, hallmarks of the type 2 phenotype in BALB/c mice and reminiscent of prior human experience with FI-RSV vaccine [40,41], were significantly diminished to background levels. Nonetheless, to achieve similar results in human populations it is likely that sequences other than the CpG ODN used in the presented studies will be required. The suggestion is that ODN containing more than one CpG motif may prove to be more promising for human recipients [15]. Once identified, the data indicate that CpG ODN should be considered as adjuvants in subunit vaccines for naı¨ve populations against RSV.

Acknowledgements The authors are grateful to R. Hazelo, K. McGuire, G. Maine, and K. Mason for their technical assistance. In addition, the authors thank Dr J.H. Eldridge and Dr P.W. Tebbey for constructive reviews of the manuscript.

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