Induction of immunological memory in baboons primed with DNA vaccine as neonates

Vaccine 19 (2001) 1960– 1967 www.elsevier.com/locate/vaccine

Induction of immunological memory in baboons primed with DNA vaccine as neonates Adrian Bot a,*, Michael Shearer b, Simona Bot a, Mary Avriette a, Adolfo Garcia-Sastre c, Gary White b, Catherine Woods a, Ronald Kennedy b, Constantin Bona c a

Department of Exploratory Biological Research, Alliance Pharmaceutical Corp., 6175 Lusk Boule6ard, San Diego, CA 92121, USA b Department of Microbiology and Immunology, The Uni6ersity of Oklahoma, Box 26901, Oklahoma City, OK 73190, USA c Department of Microbiology, Mount Sinai School of Medicine, 1 Gusta6e L. Le6y Place, New York, NY 10029, USA Received 10 March 2000; received in revised form 31 October 2000; accepted 7 November 2000

Abstract DNA immunization is a potential vaccination strategy for neonates and infants. We tested the ability of a prototype DNA vaccine against influenza virus to prime lasting immunity when administered to newborn non-human primates. Neonatal DNA vaccination triggered virus-specific and neutralizing antibodies of titers and persistence depending on the vaccine dose. Subsequent exposure to influenza virus, revealed significantly increased recall responses in the baboons vaccinated with DNA during the neonatal stage. The humoral and cellular responses were enhanced in the baboons primed with DNA vaccine as neonates. Thus, neonatal DNA vaccination of non-human primates triggered immune memory that persisted beyond infancy. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: DNA vaccination; Neonates; Influenza virus; Primates

1. Introduction During the neonatal stage the immune system is immature, prone to high zone tolerance and the repertoire of memory cells is limited [1,2]. Previous studies showed that the inoculation of newborn mice with DNA vectors expressing various microbial antigens, triggered significant immunity rather than tolerance [3 – 10], probably due to the adjuvant activity of immune stimulatory CpG motifs [11]. The response consisted in the priming of specific CTL [3,7], Th1 and Th2 cells [5,6] as well as specific B cells resulting in detectable circulating antibodies of multiple isotypes [4– 6,8,10]. Subsequent microbial challenge or antigen boost generally resulted in protection associated with enhanced specific immunity [3 – 5,9]. In a reported exception to this rule, a plasmid expressing the Plasmod* Corresponding author. Tel.: +1-858-4105259; fax: + 1-8584105612. E-mail address: [email protected] (A. Bot).

ium circumsporozoite protein was shown to induce CD8+ mediated tolerance when inoculated into newborn mice [12,13]. Besides circumventing the intrinsic poor responsiveness of the neonatal immune system to more conventional vaccines [1,2], DNA immunization was shown to be less susceptible to negative interference by maternal antibodies [14 –16]. More recently, it was shown that although the humoral response triggered by neonatal DNA vaccination was inhibited by large titers of antigen-specific maternal antibodies, the Th and CTL responses were not impaired [17]. These findings were particularly important in view of the failure of early vaccination with vaccines like the conventional measles vaccine, due to the interference by maternal antibodies [18]. A limited number of studies have addressed the question whether DNA vaccines are immunogenic in newborn primates. Thus, it was demonstrated that prototype DNA vaccines against hepatitis B virus, HIV or influenza virus are immunogenic rather than tolerogenic

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A. Bot et al. / Vaccine 19 (2001) 1960–1967

in primates [19 –21]. To our knowledge, there are no studies to address the persistence of immunity or to assess whether immune memory is generated subsequent to neonatal DNA vaccination of primates. Previously, we showed that plasmids expressing HA or NP of type A influenza viruses (H1 subtype) were immunogenic when inoculated into newborn BALB/c mice. Co-administration of such plasmids into mice triggered broad immunity, associated with enhanced protection against infectious challenge [22]. More recently, we showed that this prototype vaccine (pHA+ pNP) was also immunogenic when inoculated in neonatal baboons [21]. In the current study, we assessed the magnitude and persistence of the humoral response triggered by neonatal DNA vaccination of baboons. Secondly, we determined whether there had been an induction of humoral and cellular immune memory lasting beyond infancy. We show that the magnitude and persistence of circulating antibodies in baboons vaccinated as neonates with pHA+pNP is dependent on the dose of vaccine and that immune memory elicited by neonatal DNA vaccination of primates persists beyond infancy.

2. Materials and methods

2.1. Animals and 6accination Baboons (Papio) were housed and bred according to government regulations, at the Regional Primate Center of the University of Oklahoma Health Sciences Center. The newborn baboons were inoculated with plasmids pHA and pNP [21] expressing the hemagglutinin (HA) of A/32/WSN H1N1 and the nucleoprotein (NP) of A/34/PR/8 H1N1 influenza virus on day 1, 14

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and 28 after birth (Table 1). Different doses of plasmids (40, 200 or 1000 mg/plasmid/dose, corresponding to group A, B and C) and controls (2mg of control plasmid CP-group D or 50 mg of inactivated influenza virus on day 1-group E) were included. The plasmid mixture was dissolved in 0.5 ml of sterile PBS and administered bilaterally, in the quadriceps muscle (0.25 ml/site) by injection. The number of DNA vaccinated baboons in each group was four.

2.2. Viruses and administration of influenza 6irus The WSN virus was grown on Madine Darby bovine kidney carcinoma (MDBK) cells and the strains A/HK/ 68 (H3N2), A/Jap/57 (H2N2), A/PR/8/34 (H1N1) as well as the H1 P50 reassortant virus were grown on 10 day embryonated chicken eggs as previously described [23,24]. The viability and titer of viruses was assessed by multiplication on Madine Darby canine kidney carcinoma (MDCK) cells and standard hemagglutination of chicken red blood cells. For certain assays, we have used sucrose-gradient purified, UV-killed WSN virus. For administration of WSN influenza virus, the vaccinated baboons (age at treatment given in Table 1) were sedated with ketamine and placed in dorsal position. Intratracheal intubation was performed using a soft 8 French by 15 pediatric feeding tube (Becton Dickinson) that was inserted through the glottis into the trachea, using a 6 French intubating stilet. The tracheal tube was taped in place and 1 ml of sterile saline containing 108 TCID50 WSN virus titrated on MDCK cells, was slowly administered using a syringe attached to an 8 French by 15-in. catheter inserted 1 cm past the end of the tracheal tube. An additional volume of 2 ml of sterile PBS was instilled in order to clear the dead volume.

Table 1 Schedule of vaccination and instillation with influenza virus of baboons Group designation /animal number

Vaccine

Dose/inoculation Schedule of vaccination

Age at instillation with live virus

A (1–4) B (1–4) C (1–4)

pHA+pNP pHA+pNP pHA+pNP

40 mg+40 mg 200 mg+200 mg 1 mg+1 mg

d1, 14, 28a d1, 14, 28 d1, 14, 28

NDb ND C1, 18 C2, 17 C3, 15 C4, 14

D (1–4)

CP

2 mg

d1, 14, 28

E (1–3)

UV-inactivated WSN virus

50 mg

d1

D1, D2, D3, D4, ND

a b

Days after birth. Not done.

18 17 15 13

mo mo mo mo mo mo mo mo

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A. Bot et al. / Vaccine 19 (2001) 1960–1967

2.3. Sample har6esting and preparation Blood was harvested from anesthetized baboons by venipuncture of the saphena vein. The separated sera were processed before hemagglutination inhibition (HI) by treatment overnight at 37°C with 100 U/ml of receptor destroying enzyme (RDE-neuraminidase; Sigma, St. Louis, MO). The RDE was removed prior to the assay by 30% treatment at 56°C in the presence of calcium chloride [24]. Nasal washes were harvested under anesthesia, by inserting a pediatric feeding tube 2 – 4 cm into one nostril of animals placed in lateral decubit. Three ml of sterile PBS were instilled in the nostril via an attached syringe and the resulting wash collected in a Petri dish was immediately stored at −70°C.

2.4. Measurement of antibody titers by ELISA An indirect ELISA assay was used to determine levels of virus-specific IgG antibodies in the sera and nasal washes of immunized animals. Plastic microwells of Nunc-Immuno-plates (Nalge Nunc, Tustin, CA) were coated overnight with 10 mg/ml of sucrose-purified WSN virus. As control, wells were coated with 0.1% BSA (Sigma). The wells were blocked with non-mammalian proteins (PBS with 30%-Seablock; Pierce, Rockford, IL) and incubated with various dilutions of samples in PBS-10% Seablock, overnight at 4°C. The assay was developed using polyclonal goat anti-human IgG antibody coupled with alkaline phosphatase (Sigma Immunochemical) followed by addition of pNPP substrate. The anti-IgG antibody was previously screened by direct ELISA for binding to purified monkey IgG (Sigma), human IgG (Sigma), baboon serum and lack of binding to purified human IgA (Dako, Carpinteria, CA) and IgM (Sigma). For the characterization of the IgG subtypes and IgA antibodies, we have used anti-baboon IgG1, IgG2, IgG3, IgG4 and IgA reagents developed inhouse (The University of Oklahoma). The absorption (OD405nm) was read using an automated ELISA reader (ThermoMax, Molecular Devices) equipped with specific software (SoftMax). The ELISA assay for the detection of dsDNA-specific IgG antibodies in the sera of DNA vaccinated baboons, was carried out using a commercial kit (Sigma Diagnostics, catalog no. EIA503-A) with goat-anti human IgG reagent.

2.5. Measurement of antibody titers by hemagglutination inhibition (HI) Various dilutions of RDE-treated sera were incubated in 96-well flexible U-bottom plates with defined amounts of live virus diluted in PBS. The amount of live virus used was determined by hemagglutination in pilot

measurements. After 45% incubation of virus with diluted sera, chicken RBC were added and the hemagglutination was read after 1 h incubation at room temperature. Controls like non-immune baboon serum, as well as wells devoid of virus or serum were run simultaneously. The HI titer was read as the highest dilution of serum that inhibited hemagglutination.

2.6. In 6itro assessment of T cell response Four months after the intratracheal challenge with influenza virus, blood was harvested by venipuncture on heparin and peripheral blood mononuclear cells (PBMC) were separated by centrifugation on Ficoll (Pharmacia Biotech, Uppsala-Sweden) gradient (30 min at 2000 RPM and 20°C). The PBMC were washed twice in HL-1 medium (BioWhittaker, Walkersville-MD) supplemented with L-Glutamine and antibiotics. Finally, the PBMC were resuspended in HL-1 medium with 50 mM mercaptoethanol. Part of the PBMC were incubated in 96-well flat bottom plates at a concentration of 4× 105/150 ml of culture medium, with or without sucrose purified WSN virus. The final concentration of UV-killed or live virus was 4 mg/ml. In parallel, the cells were incubated with medium alone. The experiment was carried out in duplicates. The rest of PBMC was divided into three subsets/ each baboon: one was incubated in HL-1 medium overnight (responder cells, 4× 105 cells/ 150 ml) and the other two (stimulator cells) were infected with Vacc-NP or Vacc-T7 (MOI= 10) overnight, at 37°C and 5% CO2 atmosphere. Vacc-NP and Vacc-T7 are recombinant vaccinia viruses that express the NP of influenza virus H1N1 and the negative control T7 protein. The syngeneic stimulator cells were washed twice and were added to the stimulator cells at a ratio responder/stimulator of 5:1. The experiment was run in duplicates. At 72 h after the incubation, 100 ml of supernatant/ well was harvested for the analysis of IFNg by ELISA (monkey IFNg detection kit; Biosource International, Cammarilo, CA). A similar amount of fresh culture medium supplemented with 7.5 U/ml of human recombinant IL-2 (Boehringer Mannheim) was added to the wells. The data were acquired using an automated ELISA reader (ThermoMax, Molecular Devices).

2.7. Statistical analysis The Wilcoxon rank-sum test using the method of small sample table with exact significance levels [25], was applied to compare the magnitude of secondary responses in baboons vaccinated with DNA as neonates with the controls. This statistical method was preferred to analyze HI titers and ex vivo neutralizing titers due to the nonparametric nature of the data (normal approximation not applicable).

A. Bot et al. / Vaccine 19 (2001) 1960–1967

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been vaccinated with DNA as infants, by ELISA and HI (Fig. 1). Persistence of circulating virus-specific IgG antibodies beyond 6 months, depended on the dose of vaccine. Thus, whereas the virus-specific antibody titers in the baboons immunized with intermediate (group B) or low (group A) doses of DNA vaccine (200 or 40 mg of each plasmid/dose) decayed within months, the antibody titers in the baboons immunized with the highest dose of DNA vaccine (1 mg/plasmid/dose; group C) were sustained through more than 6 months. The titer of virus-neutralizing antibodies, a subset of the virusspecific antibodies detectable by ELISA, was measured by hemagglutination-inhibition. Only the baboons immunized with the highest dose of DNA vaccine showed detectable titers of HI antibodies, that represent virusneutralizing antibodies (Fig. 1). By the age of 1 year and a half, three out of four baboons from group C still displayed reduced HI titers against homologous WSN virus (Table 2). No antibody titers, as determined by ELISA or HI, were triggered by inoculation of dosematched control plasmid (group D) or single injection at day 1 after birth, of UV-inactivated influenza virus (group E) (Fig. 1). A potential concern associated with DNA vaccination is whether antibodies may be induced against ds-DNA, since such antibodies have been implicated in the pathogenesis of systemic lupus erythematosus (SLE). The measurement of anti-ds-DNA IgG antibodies in the serum of DNA vaccinated baboons showed titers that were not significantly increased over the background (B 150 IU/ml), irrespective of the dose of vaccination and time-point of measurement (between 0 and 6 months after birth). Thus, naked DNA vaccination of newborn non-human primates triggered antibody responses specific for the expressed antigen rather than plasmid vector.

Fig. 1. Magnitude and kinetics of antibody titers in sera of baboons vaccinated as neonates with plasmids expressing influenza virus antigens. (A) WSN-specific IgG antibodies were measured by indirect ELISA and endpoint titers were estimated as corresponding to the highest dilution associated with a signal equal or above 3 × background. Results are shown as means 9SE of log2 endpoint titers in various groups (hd, high dose; md, medium dose; ld, low dose). (B) WSN-specific HI antibodies were measured by standard inhibition of hemagglutination. The results are represented as means 9SE of endpoint titers, corresponding to each group.

3. Results

3.1. Generation and kinetics of 6irus specific antibodies by neonatal DNA 6accination of baboons We assessed the long-term kinetics of influenza virusspecific antibodies in the sera of the baboons that had

Table 2 Titer of hemagglutination-inhibiting antibodies in the sera of baboons immunized as neonates with pHA+pNP (group C) or control plasmid (group D) and subsequently instilled with 108 TCID50 of WSN influenza virus; results are representative for two independent measurements Baboon number

D1 D2 D3 D4 Geometric mean (group D) C1 C2 C3 C4 Geometric mean (group C) a b

Day 0a

Day 14

Day 134

WSN

PR8

P50

JAP(H2)

HK(H3)

WSN

PR8

P50

JAP(H2)

HK(H3)

0b 0 0 0 0 0 80 160 160 92

0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0

5120 2560 5120 2560 3620 10 240 40 960 10 240 5120 12 177

80 80 160 160 113 640 640 160 160 320

80 0 80 160 80 320 160 80 80 135

0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0

HI titers at various intervals after the challenge (time of challenge is ‘Day 0’). Titers below 80 were expressed as ‘0’.

WSN 2560 640 2560 1280 1522 640 2560 1280 1280 1280

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Table 3 Virus specific IgG antibodies in the sera and nasal wash of baboons inoculated as neonates with DNA vaccine (group C) or control plasmid (group D), before or after the instillation of live WSN virus Baboons

C1 C2 C3 C4 Geometric mean D1 D2 D3 D4 Geometric mean

Serum

Nasal lavage

Day 0a

Day 14

Day 0

200b B200 200 B200 – B200 B200 B200 B200 –

51 200 51 200 25 600 25 600 43 054 12 800 6400 12 800 12 800 10 763

B10 B10 B10 B10 – B10 B10 B10 B10 –

Day 12 80 40 20 10 28 B10 B10 B10 B10 –

a

Time of sample harvest relative to the day of live virus instillation. b End-point titers calculated as the last dilution corresponding to an OD \background against BSA+2SD.

3.2. Memory response to influenza 6irus, of baboons immunized as newborns with DNA 6accine In order to test the induction of immune memory to viral epitopes expressed by the DNA vaccine, WSN virus was administered to the baboons primed with the highest dose of DNA vaccine (group C) as well as to controls (group D). The virus was administered via the respiratory tract (108 TCID50/baboon, titrated on MDCK cells and corresponding to 106 mouse infectious doses) around the age of 1 year and a half. The presence and titer of antibodies in the sera of baboons before and subsequent to viral exposure, were assessed by ELISA (Table 3). The virus-specific antibody response was not mirrored by detection of infectious virus and viral antigen in the respiratory tract of baboons, or by clinical signs reminiscent of influenza (data not shown). The results suggest that despite a lack of productive infection, the administration of WSN virus to baboons resulted in significant humoral responses that allowed the assessment of immune memory. Before the boost, only modest reactivity was noted, particularly in the group vaccinated with pHA+ pNP. A total of 2 weeks after virus boost, we have noted significant increase of antibody titers against WSN virus in sera from baboons immunized at birth with DNA vaccine or inoculated with CP (Table 3). This increase of virus-specific IgG, as determined by ELISA, was more pronounced in the group vaccinated at birth with pHA +pNP as compared to CP (Table 3). A different method to assess antibody responses, namely HI that measures the titer of virus-neutralizing antibodies, also indicated a similar memory response. The baboons immunized as neonates with pHA+ pNP displayed modest HI titers against homologous virus

before the boost (only three out of four animals, Table 2). In contrast, none of the control baboons displayed detectable HI titers before exposure to WSN virus. Significant increase (1–2 logs) in the HI titers against homologous virus were noted in all the baboons instilled with WSN influenza virus. However, again, the titers against homologous virus were higher in the baboons primed as neonates with pHA+ pNP as compared to the controls (the geometric mean of titers in the group C was fourfold higher; PB 0.05 by Wilcoxon rank-sum test; Table 2). Four months after the challenge, however, the level of HI antibodies declined to similar levels in both the primed and non-primed baboons (Table 2). Modest HI titers were measured against influenza virus strains of similar subtype (PR8 and P50-H1N1) but no detectable titers were measured against viruses of different subtype (Jap H2N2 or HK H3N2) (Table 2). A slight but not significant increase in the antibody response against variant strains was noted in the case of baboons primed with pHA+ pNP (Table 2). The IgG subtype analysis showed that IgG1 was the dominant component in 3-month old baboons vaccinated with pHA+ pNP (Table 4). Subsequent virus boost, around 1 year and a half, resulted in the increase of IgG1 antibodies (four out of four baboons) as well as IgG2 antibodies (in two out of four baboons) in the animals primed with pHA+ pNP. The control baboons showed lower titers of IgG1 antibodies after virus challenge (one out of four). Lower binding of WSN-specific antibodies in Table 4 (subtyping) versus the data presented in Table 2 (HI assay) and Table 3 (IgG ELISA), may be explained by differences in the sensitivity of the assays. Finally, the levels of WSN-specific antibodies in the nasal washes of DNA vaccinated baboons, were determined before and at various intervals after viral boost. By ELISA assay, a transient increase of IgG antibodies specific for the homologous virus was observed in the nasal washes harvested from the baboons vaccinated with pHA+ pNP as neonates and boosted 1 year and a half later with virus (Table 3). On day 12 after the virus boost, the baboons from group C displayed IgG titers in the nasal wash between 10 and 40. The IgG titers declined by day 14 after the administration of virus. In contrast, no specific reactivity was detected in nasal washes from control baboons that received CP as neonates (Table 3). No viral-specific IgA antibodies were detected (data not shown). This shows that systemic DNA vaccination of newborn baboons resulted in enhanced local immunity upon exposure with viral antigen at the level of respiratory tract.

3.3. Cellular immune response of baboons primed with DNA 6accine as newborns We have addressed the question if neonatal vaccina-

A. Bot et al. / Vaccine 19 (2001) 1960–1967

tion of baboons with pHA+pNP modified the responsiveness to influenza virus, in terms of cellular immunity. PBMC were obtained from baboons immunized with pHA + pNP (group C, highest dose) or injected with control plasmid (group D) and subsequently challenged with influenza virus via tracheal route. The PBMC were harvested at 4.5 months after the instillation with virus. At that time, the baboons were older than 1 year and a half. In parallel experiments, the PBMC were individually stimulated with killed or live homologous virus, or with syngeneic stimulator cells infected with recombinant vaccinia virus (vacc-NP) expressing the type A/subtype H1N1 nucleoprotein. Negative controls were simultaneously run: incubation in the absence of virus or with syngeneic cells infected with a recombinant vaccinia virus expressing the T7 protein (Vacc-T7). The production of IFN-g was assessed at 72 h after incubation. As shown in the Table 5, in vitro stimulation with either killed or live WSN virus triggered significant production of IFN-g by PBMC from challenged mice, irrespective of their priming status. There were no clear-cut differences among the animals from group C and D regarding IFN-g production when PBMC were stimulated with whole virus. However, when in vitro stimulated with Vacc-NP, two animals primed with pHA +pNP as neonates exhibited significant IFN-g production. None of the baboons injected with control plasmid and subsequently instilled with WSN virus exhibited IFN-g production upon in vitro stimulation of PBMC with Vacc-NP infected syngeneic

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cells (Table 5). The specificity of IFN-g production is supported by the fact that PBMC from naı¨ve baboons did not produce significant amounts of cytokine upon in vitro stimulation with viral antigens (group N, Table 5). The data are consistent with the induction of NP-specific memory T cells upon neonatal DNA vaccination, that were further expanded above the threshold of detection by the virus boost.

4. Discussion In this study, we have addressed the issue of immune memory persistence triggered by neonatal DNA vaccination in a non-human primate model. A mixture of plasmids expressing HA and NP of A/WSN/32 (H1N1) influenza virus was used as a prototype vaccine. The baboons inoculated with DNA vaccine or control plasmid during the neonatal stage, were exposed to WSN virus delivered by intratracheal instillation, at the age of  1 year and a half. Antibody and cellular responses were measured. Antibody titers at various intervals after the initiation of the DNA vaccination regimen, showed a clearcut dependency of the magnitude and persistence of virus-specific antibodies on the dose of vaccine (Fig. 1(A –B)). The vaccinated baboons mounted significant virus-specific responses without detectable antibody responses to the vector (plasmid DNA), consistent with previous studies carried out in adult non-human pri-

Table 4 The isotype profile of virus-specific antibodies induced by neonatal DNA vaccination of baboons Timing

Group/baboon number

IgG subtypesa IgG1

IgG2

IgG3

IgG4

Age of 3 monthsb

C1 C2 C3 C4

0.317(+) 0.319(+) 0.413(+) 0.308(+)

0.084 0.084 0.117 0.101

0.050 0.066 0.076 0.074

0.056 0.036 0.051 0.039

At virus challenge (12–18 months)b

C1 C2 C3 C4

0.172 0.108 0.167 0.058

0.116 0.070 0.100 0.051

0.128 0.063 0.107 0.045

0.065 0.054 0.079 0.050

14 days after virus challenge

C1 C2 C3 C4 D1 D2 D3 D4

0.434(+) 0.413(+) 0.547(+) 0.429(+) 0.297(+) 0.203 0.221 0.126

0.117 0.093 0.156(+) 0.134(+) 0.060 0.037 0.043 0.115

0.093 0.082 0.107 0.095 0.040 0.051 0.036 0.032

0.053 0.047 0.056 0.060 0.049 0.035 0.049 0.059

a

The values correspond to average optical densities of triplicate determinations after subtraction of background, using sera diluted at 1:50. The values higher than 3× background were labeled as (+). b Baboons from group D were not included since they did not display detectable IgG antibodies in a pilot screening.

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Table 5 The cellular immune response of baboons primed as neonates with DNA and boosted with influenza virus Baboons

Response to whole virus Killed WSN virus

Live WSN virus

C1 C2 C3 C4

101 93a 3293 54917 92 946

130 95 64 9 10 42 9 14 379 9

D1 D2 D3 D4

199 931 55 913 56 93 73 94

5059 11 3598 206 95 1379 23

N1c N2 N3 N4

– – – –

– – – –

Response to NPb

– 64 9 10 – 65 9 14 – – – –d – – – –

a Data expressed as pg/ml of IFN-g at 72 h in cell culture supernatants. The background in the absence of virus was 11 9 10 pg/ml. Values less than mean 9 2×SD of background were expressed as ‘–’. b The response of baboons from group C, D and N was analyzed following stimulation with Vacc-NP infected syngeneic cells. In parallel, the stimulation was carried out with control Vacc-T7. The background after stimulation with vaccinia-T7 control was 14 9 13 pg/ml. c Naı¨ve baboons. d D4 exhibited significant reactivity to Vacc-T7 but not Vacc-NP and was excluded from the assessment of reactivity to NP.

mates [26]. The lack of clinical symptoms together with the absence of infectious virus in the nasal washes of control baboons inoculated with mock plasmid and subsequently instilled with virus, was consistent with the fact that the respiratory tract of baboons was not permissive for replication of MDBK-adapted WSN virus. However, the robust immunity noted in control baboons subsequent to virus instillation (IgG ELISA titers \ 10 000 and HI titers \1000), strongly suggests that an abortive infection of the epithelial respiratory cells had occurred. This allowed us to assess the immune memory triggered by neonatal DNA vaccination, by comparing the magnitude of secondary responses in primed versus non-primed animals. At all tested dilutions of sera, stronger binding of specific IgG antibodies to WSN virus was measured in the samples from baboons primed with DNA vaccine and boosted with virus, as compared to controls (ELISA titers equal or \ 25 600; Table 3). Similarly, the average HI titer was fourfold higher in the sera from baboons vaccinated with DNA and boosted with virus, as compared to baboons inoculated with control plasmid and subsequently exposed to virus (Table 2). Only the baboons primed with DNA vaccine displayed detectable levels of virus specific IgG antibodies in the nasal washes, at 12 and 14 days after the virus boost (Table 3). The assessment of ex vivo virus-neutralizing titers of sera har-

vested from DNA-immunized/virus-boosted baboons lead to concordant results (data not shown). Only modest cross-reactivity to other strains of the same subtype was noted, that was not statistically significant (Table 2), suggesting that the immunogenic B cell epitopes were strain-specific. In conclusion, the DNA vaccination during the neonatal stage leads to humoral immunologic memory demonstrated by the enhanced secondary responses occurring beyond the infant stage (Table 2). Secondly, intramuscular priming with DNA vaccine of infants resulted in increased local antibody titers upon subsequent delayed secondary exposure to influenza virus via the respiratory tract. However, in the absence of detectable IgA antibodies in the nasal wash, we could not rule out at this point that the detected IgG resulted by transudation. The analysis of IgG subtypes showed that DNA vaccination of newborn baboons induced IgG1 antibodies (Table 4). In primates, IgG1 and IgG3 antibodies are associated with excellent ability to activate complement and with strong binding affinity to Fcg receptors on mononuclear cells [27,28]. Induction of virus-specific IgG and neutralizing antibodies strongly suggested the activation of specific T cells. The analysis of the cellular immune response (Table 5) revealed that two out of four baboons primed with DNA vaccine displayed reactivity to nucleoprotein, a conserved element of influenza viruses that expresses major CTL epitopes. In contrast, none of the three baboons inoculated with control plasmid and four naı¨ve baboons tested displayed reactivity to NP (Table 5). Together with the IgG response, these results are consistent with initial priming of long-lived memory T cells by neonatal DNA vaccination, followed by their expansion triggered by virus boost. Based on the data provided by this study, one may propose a scenario consisting of neonatal priming with DNA vaccine followed up by subsequent boosts with live vaccine at older age, when such vaccines can be safely and effectively administered. Together with reducing the risks associated with live vectors in infants [29], such regimens may be more effective in generating sustained, enhanced immunity [30,31] and avoid inhibition by maternal antibodies [18]. In summary, we have shown the induction of immune memory by neonatal DNA vaccination of nonhuman primates. Enhanced immunogenicity of neonatal DNA vaccines may be achieved by using more effective vectors and/or DNA priming, conventional boosting regimens.

Acknowledgements This work was partially supported by NIH grants awarded to Alliance Pharmaceutical Corp. (Adrian

A. Bot et al. / Vaccine 19 (2001) 1960–1967

Bot), to Constantin Bona and Adolfo Garcia-Sastre (Mount Sinai School of Medicine).

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