Evaluation of fowlpox–vaccinia virus prime-boost vaccine strategies for high-level mucosal and systemic immunity against HIV-1

Vaccine 24 (2006) 5881–5895

Evaluation of fowlpox–vaccinia virus prime-boost vaccine strategies for high-level mucosal and systemic immunity against HIV-1 Charani Ranasinghe a,∗ , Jill C. Medveczky a , Donna Woltring a , Ke Gao a , Scott Thomson a , Barbara E.H. Coupar b , David B. Boyle b , Alistair J. Ramsay c , Ian A. Ramshaw a a

Division of Immunology and Genetics, John Curtin School of Medical Research, Australian National University, Canberra, ACT 2601, Australia b CSIRO Livestock Industries, Geelong, Vic. 3220, Australia c Gene Therapy Program, Clinical Sciences Building, Louisiana State University, Health Sciences Center, 533 Bolivar St, New Orleans, LA 70112, United States Received 17 January 2006; received in revised form 16 March 2006; accepted 14 April 2006 Available online 2 May 2006

Abstract We have tested the efficacy of recombinant fowl pox (rFPV) and recombinant vaccinia virus (rVV) encoding antigens of AE clade HIV-1 in a prime-boost strategy, using both systemic and mucosal delivery routes. Of the various vaccine routes tested, intranasal/intramuscular (i.n./i.m.) AE FPV/AE VV prime-boosting generated the highest mucosal and systemic T cell responses. Peak mucosal T cell responses occurred as early as 3 days post-boost vaccination. In contrast only low systemic responses were observed at this time with the peak response occurring at day 7. Current data also revealed that, due to better uptake of the rFPV, intranasal viral priming was much more effective than intranasal rDNA priming tested previously. The i.m./i.m. prime-boost delivery also generated strong systemic but poor mucosal responses to Gag peptides. Interestingly, the oral administration of AE FPV followed by i.m. AE VV delivery elicited strong systemic responses to sub-dominant Pol 1 peptides that were absent in mice that received vaccine by other routes. Moreover, priming with AE FPV co-expressing cytokine IL-12 significantly enhanced the T cell responses to target antigens, whilst co-expression of IFN␥ decreased these responses. The results also indicated that the route of inoculation and the vaccine vector combination could radically influence not only the magnitude but also the antigen specificity of the immune response generated. Further, in contrast to the generally protracted HIV rDNA/rFPV multiple delivery prime-boosting, this single rFPV prime and rVV boost approach was more flexible and generated excellent mucosal and systemic immune responses to HIV vaccine antigens. © 2006 Elsevier Ltd. All rights reserved. Keywords: Poxvirus; Prime-boost; Mucosal immunity; Oral/intranasal rFPV delivery; Co-stimulatory molecules; Immunodominance

1. Introduction Novel easily deliverable vaccine strategies are urgently needed to combat the HIV-1 epidemic that is spreading at an alarming rate in the developing world. Consecutive immunisation with heterologous vectors encoding the same antigen has proven highly immunogenic in models of type 1 human immunodeficiently virus (HIV-1)/simian immunodeficiently virus (SIV) infection. Several heterologous recom∗

Corresponding author. Tel.: +61 2 6125 4704; fax: +61 2 6247 8921. E-mail address: [email protected] (C. Ranasinghe).

0264-410X/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2006.04.023

binant DNA (rDNA) prime-boost vaccine strategies have elicited robust cellular and humoral responses to HIV antigens in animal models [1–6] and phase I/II prime-boost HIV-1 vaccine trials have now been conducted around the world [7–9]. Despite generating good immune responses in mice and non-human primates rDNA prime-boost strategies have so far proven to be disappointing in human clinical trials. In one such trial, using rDNA prime and recombinant modified vaccinia Ankara (rMVA) boost immunisation, only 20% of 205 recipients developed a significant level of immunity to HIV antigens (http://www.iavi.org). In another study, using rDNA/rFPV prime-boosting little or no immunity to

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vaccine antigens was detected [10]. These results possibly indicate that current rDNA vectors may be poor immunogens in humans, in contrast to the responses elicited in other species. Novel vaccines that generate broadly reactive highavidity T cell and humoral immune responses are still in needed for HIV-1 prevention. A compelling body of evidence suggests that virus specific CD8+ cytotoxic T lymphocytes (CTL) play a vital role in containing HIV-1 infection [11,12]. Further, vaccine-induced cytotoxic T cells have also been shown to initially control the replication of pathogenic simian immunodeficiency virus (SIV), and although there is emergence of CTL escape variants, these mutants have a replication disadvantage compared to the wild-type virus [11]. However, new data indicate that the absolute levels, breadth, durability and cytokine secretion profiles of responding CTL, may all be important for protection of humans from HIV-1 exposure [13–15]. The presence of HIV-specific immunity at major sites of virus transmission, i.e. rectal and oral tissues and particularly the cervico-vaginal tissues in females may be critical in the initial control of infection. For diseases such as HIV/AIDS, it is hence important to develop vaccine strategies that elicit immunity at the primary site of mucosal infection [16–18]. In general, systemically administered vaccine antigens, although inducing good systemic T cell responses, rarely induce optimal mucosal immune responses [16,19]. It has also been postulated that application of a vaccine at one mucosal surface can trigger immunity at local and distant mucosa [19,20]. Indeed, a number of mucosal immunisation regimes based on this approach have been tested and shown to be effective in generating HIV-1 specific mucosal cellular and humoral immunity in mice and macaques [21–27]. Mucosal (intra nasal and intrarectal) delivery of rDNA/rFPV prime-boost vaccines in mice and macaques also confirm these findings (Ranasinghe et al., unpublished data) [4]. Different vaccine delivery routes and vectors have proven to induce a diverse range of immune responses to vaccine antigens [28–30]. In recent years, a number of recombinant viral and bacterial vectors that are suitable for mucosal and systemic delivery have been tested, i.e. adenovirus [25,31], poliovirus [24] influenza viral vectors [27], Listeria monocytogenes [32]. More recently, a rFPV/rMVA prime-boost regime was reported to induce strong antigenspecific T cell responses to Plasmodium berghei malaria and Mycobacterium tuberculosis antigens, [30,33]. Also the coexpressions of certain cytokines and chemokines have been shown to enhance immune responses to vaccine antigens [34–39]. These data indicate that a rational combination of vectors, delivery routes and co-stimulatory molecules could play a pivotal role in modulating immune responses to vaccine antigens. In this study we have characterised the use of rFPV and rVV expressing AE clade HIV-1 antigens, in prime-boost vaccine strategies, and have shown that these vaccines generate strong sustained mucosal and systemic T cell immunity, with mucosal responses peaking very early after boost

immunisation. We have also studied cytokine co-expression to enhance immunogenicity of vaccine antigens. Moreover, we have evaluated the efficacy of oral delivery of rFPV, an important vaccine delivery approach for implementation in the developing world.

2. Materials and methods 2.1. Recombinant poxvirus vaccines The AE FPV contained modified AE clade gag, pol, env, rev and tat genes. The AE IL-12-AE and AE IFN␥-AE constructs expressed Gag, Pol, Env and IL-12 or IFN␥, respectively (Table 1). Whereas, AE VV contained modified gag and pol genes only as indicted in Table 1. These recombinant viruses were constructed as described elsewhere [40–42]. 2.2. Immunisation of mice Groups of (4–5) 5–7 weeks old BALB/c (H-2d ) mice were primed with rFPV followed by a booster 2 weeks later of rVV (1 × 107 pfu) both expressing AE clade HIV-1 antigens (Table 1). Mice were sacrificed at different time intervals to evaluate immune responses to vaccine antigens. Initially, varying amounts of AE FPV (5 × 104 , 5 × 105 , 5 × 106 , 1 × 107 pfu) were administered to the mice, to measure the dose response. However, in the subsequent experiments 1 × 10 pfu of AE FPV were given to each mouse in a final volume of 20–25 ␮l (intranasal, i.n.; intradermal, i.d.), 100 ␮l (intramuscular, i.m.) or 200 ␮l (intravenous, i.v.), except mice immunised orally received 2 × 107 pfu in a 20–40 ␮l volume. Prior to each immunisation the rFPV or rVV was diluted in phosphate buffered saline (PBS) and sonicated 30–40 s to obtain a homogeneous viral suspension. Note that most of these experiments were repeated over three times and the data are a representative of one experiment. 2.3. Sample collections and preparation of lymphocytes Sera were collected from pre-immune and immunised mice. Blood was collected from tail vein or heart bleed, serum separated by centrifugation and stored at −20 ◦ C until assayed. To measure systemic and mucosal T cell responses Table 1 Recombinant viruses used in this study [40,41] Recombinant

FPV-106 (AE IFN␥-FPV) FPV-107 (AE IL-12-FPV) FPV-117 (AE FPV) VV-336 (AE VV)

Insertion sites F6,7-9

TK-ORFX or TK

REV

AE gag/pol(m) AE gag/pol(m) AE gag/pol(m)

muIFN␥ muIL-12 AE tat-rev AE gag/pol(m)

AE env(m) AE env(m) AE env(m)

TK: thymidine kinase, ORFX: uncharacterised gene, and REV: reticuloendotheliosis provirus.

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mice were sacrificed 2–13 weeks post-booster immunisation, spleen and genito-rectal nodes draining the genito-rectal mucosa were removed and single cell suspensions prepared. The splenocytes but not lymph nodes were treated with red cell lysis buffer. 2.4. HIV-1 p24 specific serum ELISA HIV-1 p24 Gag specific serum antibody titres were determined by enzyme-linked immunosorbent assay (ELISA). Falcon Microtest III plates (Becton Dickinson, Oxnard, CA) were coated with HIV-1 p24 at 1.5 ␮g/ml (50 ␮l/well) in Borate buffer (Pierce, USA) overnight at 4 ◦ C. These plates were washed with 0.05% Tween 20 in PBS (PBST), and non-specific binding sites were blocked, and serum samples diluted in 5% skim milk/PBST were added in a 50 ␮l volume to each well. All plates were covered and incubated for 1.5 h at 37 ◦ C and washed as indicated. Secondary antibody, biotin-conjugated anti-mouse IgG (Southern Biotechnology Associates, Birmingham, AL) diluted tol:1000 in 1% bovine serum albumin/PBST (Sigma) (BSA/PBST) was added to respective wells in a 50 ␮l volume, and incubated overnight at 4 ◦ C. Plates were washed, and 50 ␮l of streptavidin alkaline phosphatase (Amersham Life Science, Australia) diluted 1:1000 in 1% BSA/PBST was added to each well and incubated at 37 ◦ C for 1.5 h. The antibodies were detected using BCIP/NBT substrate (Sigma, USA). Optical densities (OD) of each plate were read at wavelength of 405 nm. To determine endpoint titres, serum from unimmunised mice was titrated across an ELISA plate beginning at the same dilution as the samples. The optical density values for each titration point were added together and the average and standard deviation were calculated. The endpoint titre was defined as the mean of the optical densities plus three standard deviations. The endpoint titre value was applied to each sample with the last dilution being recorded as the reciprocal of the dilution. 2.5. IFNγ ELISpot assay HIV-specific CD4+ and CD8+ responses were measured by IFN␥ capture ELISpot. Mouse anti-IFN␥ capture antibody (BD PharMigen, San Diego, CA), was diluted to 5 ␮g/ml in PBS and 96-well Millipore PVDF plates (Millipore Corporation, USA) were coated with 50 ␮l of diluted antibody. Plates were incubated overnight at 4 ◦ C or 4–5 h at 37 ◦ C. Plates were washes four times with 0.05% PBST, once with PBS, and were blocked with 100 ␮l of complete RPMI-1640 (GIBCO-BRL) containing 10% fetal calf serum (FCS) for 40–60 min at 37 ◦ C. Splenocytes or lymphocytes were added in duplicate or in triplicate to appropriate wells at a final concentration of 2 × 105 per well in a final volume of 100 ␮l. These cells were stimulated with HIVspecific 15 mer overlapping Gag, Pol specific peptide pools (kindly supplied by the NIH AIDS Research and Reference Reagent Program) or BALB/c specific 9 mer peptide

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AMQMLKETI (synthesised at the Bio-Molecular Resource Facility at John Curtin School of Medical Research, Australia) [43,44] ConA (Sigma, USA) was used as the positive control. Plates were then incubated for 20–24 h at 37 ◦ C presence of 5% CO2 . Cells were removed and any bound cells were lysed by adding ice-cold sterile water for 10 min and washing as described above. Subsequently, 50 ␮l biotin-labelled secondary antibody (BD PharMigen, San Diego, CA) diluted 1:2000 in 1% BSA/PBS was added to each well, plates were incubated at room temperature (RT) for 2 h. Plates were washed and 50 ␮l streptavidin alkaline phosphatase (Amersham, Life Science, Australia) diluted 1:1000 in 1% BSA/BPS was added, and incubated at RT for 1–2 h and the spots were developed by adding BCIP/NBT developer (Moss, USA) to each well, for 15–40 min at RT. Plates were washed in water to stop reaction, plate backings were removed and plates were dried at RT before counting the spots using an ELISpot Bio Reader4000 (BIOSYS, GmbH, Germany). Unstimulated cells from each sample were used as the background control and this value was subtracted from each sample before plotting the data. 2.6. Intracellular cytokine staining Initially 1 × 106 lymphocytes were stimulated for 12–14 h with 15 mer Gag peptide pool and for further 4–5 h in the presence of 2 ␮M monensin. Cells were transferred into U-bottom 96-well plates and were washed once with PBS prior to staining. CD8+ and CD4+ cells were surface stained, respectively, with anti-CD8 APC and with anti-CD4 PerCp (BD PharMigen, San Diego, CA), diluted in FACS buffer (2% heat inactivated FCS in PBS + 0.1% sodium azide) for 30 min at 4 ◦ C, followed by washing and fixing the cells with IC fix (Bio Source, Belgium). These cells were permeablised with 1× IC-Perm (Bio Source, Belgium) for 10 min at RT, and stained for IFN␥ or TNF␣ using anti-mouse IFN␥-FITC or TNF␣-PE (BD PharMigen, San Diego, CA), diluted in IC-perm buffer for 30 min at 4 ◦ C. Cells were then washed twice in PBS and were resuspended in PBS for analyse on a four-colour FACSCalibur flow cytometer (Becton-Dickinson) using Cell Quest Pro analysis software. During analysis unstimulated cells from each sample were used as the background control and where appropriate this value was subtracted from each sample before plotting the data. Pooled cell samples were used in this assay. 2.7. Tetramer staining Allophycocyanin (APC) or R-phycoerythrin (PE) conjugated Gag AMQMLKETI BALB/c specific H-2Kd MHC class I restricted tetramers were synthesised at the BioMolecular Resource Facility at John Curtin School of Medical Research, Australia. Between 2 and 5 × 106 splenocytes or lymphocytes from genito-rectal nodes were transferred into U-bottom 96-well plates and were washed

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with PBS and resuspended in 20 ␮l FACS buffer, 10 ␮l of 1/50 diluted anti-CD8 FITC antibody (BD PharMigen, San Diego, CA) and 0.5–1.0 ␮l of APC or PE labelled tetramer diluted in FACs buffer were added to each sample to a final volume of 40 ␮l and were incubated at room temperature, protected from light, for 30–40 min. Cells were washed twice with FACS buffer and resuspended in 100 ␮l of FACS buffer containing 0.5% paraformaldehyde. These samples were then analysed on a FACSCalibur flow cytometer (Becton-Dickinson) using Cell Quest Pro analysis software. During tetramer staining splenocytes and genito-rectal nodes from unimmunised animals were used as background controls. 2.8. Statistics Where appropriate p-values were calculated using a twotailed two sample equal variance Student’s t-test.

3. Results 3.1. Evaluation of vaccine routes and regime Mice were primed with 1 × 107 pfu of AE FPV and boosted with 1 × 107 pfu AE VV as described in the methods section. Several purely systemic (i.m./i.m., i.m./i.v., i.v./i.v., i.d./i.d.), purely mucosal (i.n./i.n.) and combination mucosal and systemic (i.n./i.m.) vaccination routes were tested. As shown in Fig. 1, all routes induced substantial systemic T cell responses as measured by IFN␥ ELISpot assay, against vaccine antigens. However, i.m./i.m. and i.n./i.m. routes induced the most robust systemic responses, generating over six hundred IFN␥ spot forming units (SFU) per 106 splenocytes (Fig. 1). Also, when splenocytes and genitorectal lymphocytes from these mice were stimulated with, serial dilutions (1 ␮mol to 1 pmol) of BALB/c specific 9 mer Gag peptide AMQMLKETI, similar ELISpot IFN␥ counts were observed across the dilutions (results not shown) suggesting that i.n./i.m. regime generates high-avidity mucosal CD8+ T cell responses to vaccine antigens. Our data also indicated that i.m./i.v., i.d./i.d. and i.n./i.m. strategies induced good CD4+ and CD8+ mucosal IFN␥ responses in the draining genito-rectal lymph nodes (Table 2) to Gag peptides. In contrast i.m./i.m. showed low mucosal IFN␥ T cell counts and also, i.n./i.n. pure mucosal route generated lower than expected mucosal IFN␥ responses in these mice (Table 2). 3.2. Assessment of the optimal rFPV dose As described in the methods, mice were immunised i.n. with varying concentrations of AE FPV to establish the optimal priming dose of AE FPV, followed by an i.m. booster of 1 × 107 pfu of AE VV. Results indicated that 1 × 107 pfu of AE FPV generated the highest systemic T cell response and halving the AE FPV priming dose dramatically reduce the overall splenic T cell responses generated to vaccine antigens (Fig. 2B). In contrast, reducing the priming dose of AE FPV (l × 107 to 5 × 105 pfu) did not significantly alter the mucosal T cell responses generated (Fig. 2A). Moreover, no signifiTable 2 Mucosal T cell responses measured in genito-rectal lymph nodes post AE FPV/AE VV prime-boost immunisations by IFN␥ ELISpot Vaccine route

Mucosal IFN␥ responses AE 15 mer Gag peptide pool (SFU per 106 cells)

Fig. 1. Evaluation of vaccine routes and regimes. Systemic T cell responses in BALB/c (H-2d ) splenocytes 2 weeks post 1 × 107 pfu AE FPV priming and 1 × 107 pfu AE VV boosting were measured by IFN␥ ELISpot (n = 5 mice/group). Pooled spleen suspensions were stimulated with AE clade 15 mer overlapping Gag peptide pool as indicated in the materials and methods. The unstimulated cells from each sample were used as the background control and this value was subtracted from each sample before plotting the data. The data represent mean + standard deviation (S.D.) of inter sample variation.

i.m./i.m. i.m./i.v. i.d./i.d. i.n./i.m. i.n./i.n.

87.5 167.5 175.0 220.0 95.0

± ± ± ± ±

14.1 7.1 88.4 3.5 21.5

9 mer peptide AMQMLKETI (SFU per 106 cells) 140.0 222.5 157.5 232.5 85.0

± ± ± ± ±

3.5 7.1 35.4 42.2 21.2

n = 5 mice/group. The data represent counts from pooled cells samples and mean ± S.D. of triplicates samples. The mucosal route that generated the highest systemic and mucosal ELISpot counts is indicated in italic letters.

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Fig. 2. Assessment of optimal rFPV dose. Groups of four BALB/c (H-2d ) mice were primed i.n. with different doses of AE FPV and boosted i.m. with 1 × 107 pfu of AE VV to determine the optimal rFPV dose required for priming. Mice were sacrificed 2 weeks post-boost and single cell suspensions from genito-rectal nodes (A) and spleens (B) were stimulated with AE clade specific 15 mer overlapping peptide pool and the mucosal and systemic T cell responses were measured by IFN␥ ELISpot. The data represent mean + S.D. of four individual mice. Unstimulated cells from each sample were used as the background control and this value was subtracted from each sample before plotting the data. Percentage of to Gag specific AMQMLKETI H-2Kd MHC class I restricted tetramer positive CD8+ T cells observed in spleen with different priming doses of AE FPV and 1 × 107 pfu AE VV (C). The unimmunised animals were used as background controls. IgG antibody responses in sera against the P24 gag protein observed with different priming doses of AE FPV and 1 × 107 pfu AE VV (D). Known positive and negative sera were used in these assays. The data represent mean + S.D. of four individual mice.

cant IFN␥ responses were observed in mice that received only AE FPV or AE VV i.n. or i.m. When splenocytes from mice primed i.n. with AE FPV doses 1 × 107 to 5 × 107 pfu and boosted i.m. with 1 × 107 pfu AE VV, were monitored for binding to Gag specific AMQMLKETI H-2Kd MHC class I restricted tetramer, relatively similar numbers of tetramer positive CD8+ T cells were observed between the various groups (Fig. 2C). In the genito-rectal lymphocytes the overall CD8+ tetramer binding was significantly less (>2%) compared to the spleen. AMQMLKETI H-2Kd is a dominant Gag specific epitope recognised by Balb/c mice.

In general the prime-boost vaccine regimes elicited only low levels of p24 Gag specific IgG antibody responses and the titre of response was not directly related to the dose of AE FPV administered, with 5 × 10 giving the highest IgG antibody response (Fig. 2D). 3.3. Determination of the minimum time interval required for an optimal viral boost Mice were i.n. primed with AE FPV, boosted i.m. with AE VV as presented in Table 3, and sacrificed 2 weeks post prime-boosting as described in methods. The IFN␥

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Table 3 Systemic T cell responses generated at different boosting intervals measured using IFN␥ ELISpot

3.4. Assessment of the order of recombinant pox viral vector administration

Interval between priming and boosting

AE 15 mer Gag peptide pool (SFU per 106 cells)

14 days 6 days 3 days Same day VV boost only

1117 1007 452.4 117.5 110

Our results indicated that in some instances the sequence in which the pox viral vectors were administered influenced the immune responses elicited to the vaccine. The i.m., AE VV priming followed by i.n. AE FPV boosting induced weaker systemic and mucosal T cell responses compared to i.n. AE FPV priming and i.m. AE VV boosting, as shown by IFN␥ ELISpot analysis (Fig. 3A) and intracellular IFN␥ and TNF␣ cytokine staining (Fig. 3B). These results indicate that priming with AE FPV generated better T cell responses than priming with AE VV. Moreover, when i.m. AE VV priming was followed by i.m. AE FPV boosting there was no significant difference in the IFN␥ ELISpot responses (Fig. 3A) compared to the i.m. AE FPV/i.m. AE VV. In contrast, i.m. AE FPV priming elicited different cytokine profiles to i.m. AE VV priming regime as shown by intracellular IFN␥ and TNF␣ cytokine staining (Fig. 3B). The results show that the order in which poxvirus vectors were delivered influenced the cytokine profiles and magnitude of the immune responses generated.

± ± ± ± ±

28.3 21.2 35.4 3.5 35.4

9 mer Gag peptide AMQMLKETI (SFU per 106 cells) 997.5 ± 42.4 815.5 ± 35.4 0 0 0

n = 4 mice/group. The data represent counts from pooled cells samples and mean ± S.D. of triplicates samples.

ELISpot results indicated that optimal T cell responses (Table 3) were obtained when the interval between priming and boosting was over 6 days. Furthermore, ELISpot data with 9 mer Gag peptide clearly indicated that when sub-optimal boosting intervals were employed, weak or no CD8+ memory responses were observed. Hence, the timing between priming and boosting seems to be a critical factor in generating optimum memory T cell responses to vaccine antigens.

Fig. 3. Assessment of the order of pox viral vector delivery. Systemic and mucosal T cell responses generated (n = 4 mice/group) against 15 mer AE clade overlapping Gag peptide pool (black bars — spleen; stripped bars-genito-rectal nodes) and the AMQMLKETI Gag peptide (grey bars-spleen, white bars-genitorectal nodes), 2 weeks post i.n. or i.m. 1 × 107 pfu AE FPV priming or boosting, and i.m. 1 × 107 pfu AE VV priming or boosting measured by IFN␥ ELISpot (A). The x-axis represent the order in which the poxvirus vectors were delivered. The rFPV only was delivered i.n. or i.m. once or twice (note that i.n. or i.m. rFPV results were identical) and rVV only was at all times given i.m. in a similar manner. The data represent mean + S.D. of mice and (* ) indicates the SFU were significantly different. The data are representative of two experiments. Systemic T cell responses generated against 15 mer AE clade overlapping Gag peptide pool measured by intra cellular staining of IFN␥ and TNF␣ (B). The number of CD8+ and CD4+ T cells producing IFN␥ and TNF␣ are indicated in grey bars. Unstimulated cells from each sample were used as the background control and this value was subtracted from each sample before plotting the data. This data represent a pooled value.

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3.5. Evaluation of mucosal T cell responses To monitor the time course of the mucosal responses, the mice were primed i.n. or i.m. with 1 × 107 AE FPV and boosted i.m. with 1 × 107 AE VV 2 weeks a part as described in methods. These mice were sacrificed at 3, 7 and 90 days, genito-rectal nodes and spleens were collected to measure mucosal and systemic responses, respectively. Interestingly, the highest mucosal responses were observed at 3 days postboost in the combined mucosal/systemic regime (i.n./i.m.) as shown by IFN␥ ELISpot (Fig. 4) and intracellular cytokine staining (Fig. 5). In contrast, both i.n./i.m. and i.m./i.m. vaccine regimes induced low systemic T cell responses in these animals at day 3 (less than 100 IFN␥ SFU) but peaked at day 7 and were maintained at the same level up to 90 days (Fig. 4). Moreover, i.n./i.m. regime showed increased num-

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ber of CD8+ cells producing IFN␥ and/or TNF␣ at day 7 compared to i.m./i.m. by intracellular staining (Fig. 6A and B). The current data suggest that unlike systemic responses, mucosal responses should be measured at a much early stage in vaccination. The unexpected rapid mucosal response may explain some of the difficulties encountered when monitoring mucosal responses in the past [4]. 3.6. Evaluation of long-term T cell memory responses Five to six weeks old BALB/c mice were primed i.n. with AE FPV followed by i.m. AE VV boost, and were sacrificed at 1, 2, 6 and 13 weeks. One group of mice were given a second AE VV boost at 12 weeks and were sacrificed 1 week later. IFN␥ ELISpot and tetramer analysis, all indicated that the i.n./i.m. pox viral prime-boost regime generated robust sustained CD8+ memory T cell responses to gag peptides in both the systemic (Figs. 4 and 7A) and mucosal compartments (Figs. 4 and 7B). 3.7. Oral delivery of rFPV induce T cell responses to a range of HIV peptides

Fig. 4. Evaluation of mucosal T cell responses at different time intervals. BALB/c (H-2d ) mice (n = 4 mice/group) were primed i.n. with 1 × 107 pfu AE FPV and boosted i.m. with 1 × 107 pfu of AE VV. At 3, 7 and 90 days IFN␥ ELISpot analysis was performed using AMQMLKETI Gag peptide. Also the last group of mice were given a 2nd i.m. AE VV boost at 84 days and were sacrificed at 90 days. The data represent mean + S.D., the grey bars indicate the responses in the spleen and the black bars represent the genitorectal node responses. Spleen and genital node responses were significantly different at 3 and 7 days (* p < 0.05) and also 7 days compared to the boosted animals (** p < 0.05). The x-axis indicates the time post AE VV boost. The unstimulated cells from each sample were used as the background control and this was subtracted from each sample before plotting the data. The data are representative of three experiments.

To assess whether orally delivered AE FPV could induce T cell immunity to HIV-1 vaccine antigens, groups of mice were primed with 2 × 107 pfu AE FPV i.m., i.n. or orally and boosted with 1 × 107 pfu AE VV i.m. as described in Section 2. IFN␥ ELISpot analysis was performed using a range of HIV peptide antigens, and the number of T cells expressing IFN␥ measured (Fig. 8A). i.m./i.m. i.n./i.m. regimes induced strong systemic T cell responses to all Gag specific peptide pools including consensus including consensus Gag A–C (Fig. 8A). i.n./i.m. regime also induced strong mucosal responses in the draining lymph nodes to Gag peptide pools (Fig. 8B) but not to other peptides used. Interestingly, although orally primed mice generated systemic Gag specific IFN␥ responses similar to i.m. or i.n. immunised mice, systemic T cell responses to Pol 1 peptides were also greatly enhanced. Such immunodominant responses were not observed in i.m./i.m. group, even though low systemic Pol 1 responses were associated with i.n./i.m. regime (Fig. 8A). Orally immunised mice demonstrated low mucosal T cell responses in the genito-rectal draining lymph nodes to Gag peptides (Fig. 8B) whilst no responses to Pol 1 were detected. Splenocytes from all three immunisation regimes were subjected to PE conjugated BALB/c specific Gag AMQMLKETI H-2Kd MHC class I restricted tetramer staining, in each group over 3–4% of the CD8+ cells were found to be tetramer positive (Fig. 8C). 3.8. Effect of cytokine co-expression by rFPV vectors and CD8+ T cell response Mice were primed i.m. or i.n. with 1 × 107 pfu of AE FPV, AE IL-12-FPV and AE IFN␥-FPV, respectively, and 2 weeks

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Fig. 5. Mucosal T cell responses generated against 15 mer AE clade overlapping Gag peptide pool measured at 3 (left) and 7 (right) days by IFN␥ intra cellular staining. The FACS plots represent (a) i.n./i.m. unstimulated cells, (b) i.n./i.m. stimulated cells, and (c) i.m./i.m. stimulated cells. Note that the i.m./i.m. unstimulated % were similar to i.n./i.m. The values in the upper right quadrants indicate the percentage of CD8+ T cells producing IFN␥. The y-axis indicates the CD8-APC channel and x-axis the IFN␥-FITC channel.

later boosted i.m. with 1 × 107 of AE VV. To evaluate T cell responses, mice were sacrificed 3 weeks post AE VV and cells were prepared as described in the methods. Comparable systemic T cell IFN␥ ELISpot responses were obtained from i.m. and i.n. AE FPV and AE VV boosted mice. Mice that received AE FPV expressing IL-12 elicited significantly enhanced T cell response, compared to the mice given AE FPV (Fig. 9A). In contrast, mice receiving AE IFN␥-FPV showed reduced T cell responses to the Gag peptides. Moreover, i.n./i.m. immunised groups showed similar IFN␥ ELISpot responses in the mucosal compartment (results not shown) compared to spleen, but no significant IFN␥ ELISpot responses were observed in genito-rectal draining lymph nodes of mice that received i.m./i.m. immunisation. When mice primed i.m. AE IL-12-FPV and boosted i.m. AE VV, were subjected to Gag specific PE labelled AMQMLKETI H-2Kd MHC class I restricted tetramer staining, 9.3% of CD8+ T cells recognised the BALB/c epitope (Fig. 9B), whereas AE FPV and AE IFN␥-FPV primed mice showed tetramer positive populations of 7.55 and 4.03%, respectively (Fig. 9B). All three vaccines showed low (0.4–0.7%) tetramer positive CD8+ cell frequencies in the draining genito-rectal

lymph nodes (results not shown), further substantiating the premise that a pure systemic immunisation strategy does not induce good T cell mediated mucosal immune responses.

4. Discussion Numerous studies have illustrated that simultaneous generation of systemic and mucosal memory T cell responses may be critical for controlling HIV-1 [16–18]. In the current study we have investigated a range of vaccination regimes using poxvirus expressing AE HIV antigens. We have shown that intranasal AE FPV prime followed by intramuscular AE VV boost elicited the greatest long-term CD4+ and CD8+ systemic and mucosal T cell responses to HIV-1 antigens in BALB/c mice (Figs. 4 and 7). These responses were generally better than those elicited by the lengthy mucosal systemic rDNA/rFPV prime-boost regime previously tested. Our previous studies in mice and macaques have clearly shown that the mucosal uptake of rFPV vectors is more efficient than the mucosal uptake of plasmid vectors (Ranasinghe et al., unpublished data and [4]). The delivery of plasmid vectors via the

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Fig. 6. Intracellular cytokine staining post i.n./i.m and i.m./i.m. immunisations. Post prime-boost vaccination (n = 4 mice/group) systemic T cell responses generated against 15 mer AE clade overlapping Gag peptide pool was measured at 3 (left) and 7 (right) days by IFN␥ intra cellular staining (6A). The FACS plots represent (a) i.n./i.m. and (b) i.m./i.m. The values in the upper right quadrants indicate the percentage of CD8+ T cells producing IFN␥ (unstimulated background % subtracted). They axis indicates the CD8-APC channel and x-axis the IFN␥-FITC channel. IFN␥ and TNF␣ measured at 7 days in splenocytes by intra cellular cytokine staining (B). The FACS plots represent left i.m./i.m. regime and right i.n./i.m. (a) unstimulated cells, (b) 15 mer AE clade overlapping Gag peptide pool stimulated cells. The values in the upper right quadrants indicate the percentage of CD8+ and CD4+ T cells producing IFN␥ and TNF␣. The y-axis indicates the TNF␣-PE channel and x-axis the IFN␥-FITC channel.

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Fig. 7. Systemic and mucosal memory T cell responses. Gag specific AMQMLKETIH-2Kd MHC class I restricted tetramer responses were measured at different time intervals post i.n. 1 × 107 pfu AE FPV priming and i.m. 1 × 107 pfu AE VV boosting (n = 4 mice/group). The graph with black bars (A) represents the systemic responses in spleen and the graph in grey (B) indicates the mucosal responses in genito-rectal nodes. The y-axis represents the sacrifice time and x-axis the % of tetramer positive CD8+ T cells. The unimmunised animals were taken as the controls and the background CD8+ counts for spleen and the genito-rectal nodes were less than 0.5 and 0.1%, respectively.

intranasal route even enclosed in lipid-based agents such as lipofectamine (Invitrogen, Carlsbad, CA) or DOTAP (Roche, Indianapolis, IN) was not very efficient and hence led to the generation of poor mucosal responses [4] (Ranasinghe et al., unpublished data). The current study further substantiates our earlier findings suggesting that the inherent properties of FPV to enter the mucosal cells is largely responsible for the generation of enhanced mucosal T cell responses observed in this study. The pure mucosal prime-boost immunisation regime, i.n. AE FPV/i.n. AE VV yielded lower mucosal T cell responses to Gag peptides by ELISpot compared to the combined mucosal systemic prime-boost immunisation (i.n. AE FPV prime/i.m. AE VV boost) (Table 2). In a HIV-1 heterologous prime-boost immunisation regime using recombinant influenza and rMVA, intranasal delivery of influenza and rMVA was reported to induce systemic but low mucosal T cell responses to vaccine antigens in mice, which is consistent with our own findings [27]. Moreover, i.n. AE VV prime/i.m. AE FPV boosted mice elicited lower T cell responses suggesting that intranasal delivery of rVV was not as effective as the rFPV vector (Fig. 3A and B). Our results indicate, based on immunogenicity and safety, that rFPV’s could be utilised as an excellent ‘mucosal delivery vector’ for humans. Although i.v. and i.d. immunisations elicited good mucosal responses (Table 2), compared to i.m. immunisation possibly due to rVV draining from systemic circulation into the mucosa, we believe that placing emphasis on developing easily deliverable vaccines such as i.m., i.n. or oral would be advantageous in situations where low cost and large cohorts are involved.

Ariyoshi et al. reported that chickens could be immunised against FPV by delivering FPV in drinking water [45]. The oral delivery of another non-replicating poxvirus was also clinically trialed with no adverse effects noted but was shown to induce poor mucosal immune responses [46]. Although AE FPV expressing HIV antigens is a poor immunogen on its own, our results suggest that it is a powerful priming vector when used with a consecutive boosting with another poxvirus expressing the same vaccine antigens. Interestingly in our study, mice primed orally showed increased systemic responses to both Gag and Pol 1 epitopes (Fig. 8A). This suggested that the route of vaccine delivery could markedly affect the immunodominance hierarchy. Many factors may contribute to immunodominance [47] including intrinsic efficiency of antigen presentation such as competition for MHC binding or interaction between epitope specific T cells for access to antigen presenting cells [48]. IFN␥ also can drastically influence dominance hierarchy, IFN␥ knock out mice were shown to produce strong responses to a normally subdominant LCMV epitope after infection with virus [49]. Whether presence of IFN␥ can explain the changed hierarchy dominance observed here with oral delivery, where Th2 cytokines such as IL-4 and IL-10 are prominent remains to be determined. These elevated normally sub-dominant Pol 1 responses associated with the oral delivery could also be due to differential uptake and processing of antigens at mucosal sites [20,50]. Interestingly, we have made similar observations with vaccines where the epitopes have been scrambled in such a way to retain all T cell epitopes, even though this may be a distinctly different process rendering sub-dominant epitopes more immunogenic [51].

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Fig. 8. Oral rFPV delivery and systemic responses. Groups of five BALB/c (H-2d ) mice were primed with 2 × 107 pfu AE FPV, boosted with 1 × 107 pfu AE VV and systemic T cell responses to a range of peptides were measured using IFN␥ ELISpot, 2 weeks post-boost (A). The AE FPV prime was performed via intranasal (grey bars), intra muscular (black bars) and oral (white bars) routes. Oral immunisation the Pol 1 responses were significantly different (* p < 0.005) to the other two regimes. The data represent mean + S.D. of five individual mice, and the x-axis indicates the different peptide(s) used in the stimulation of splenocytes. Mucosal T cell responses from pooled genito-rectal nodes were measured against 15 mer AE clade overlapping Gag peptide pool by IFN␥ ELISpot (B), after intranasal (grey bars), intra muscular (black bars) and oral (white bars) immunisation. Percentage of AMQMLKETI H-2Kd MHC class I restricted tetramer positive systemic CD8+ T cells were measured (C), after intranasal (grey bars), intra muscular (black bars) and oral (white bars) immunisation. The unimmunised mice were taken as the controls and the background CD8+ counts for spleen was less than 0.5%. The data are representative of three experiments.

Contrary to what has been reported in some studies following oral immunisation poor T cell responses were observed in the genito-rectal draining lymph nodes as assessed by IFN␥ ELISpot (Fig. 8B). In an oral HIV envelop glycoprotein (gpl60) rDNA prime/rVV boost immunisation study Wierzbicki et al. observed good mucosal responses in Peyer’s patch and lamina propria [52]. Also, oral delivery of Tat protein was shown to induce good mucosal IgA responses in faeces and sera [53]. In general however, it is thought that distinct cell migration pathways exist within the mucosal immune system and we postulate that the weak genito-rectal

T cell responses observed in our study could also be due to preferential homing of T cells to other mucosal sites [19]. The capacity to administer a vaccine using a simple oral delivery route, such as demonstrated using rFPV, would greatly facilitate the delivery of an HIV-1 vaccine to a large target population. Hence, a more comprehensive study into the nature and dissemination of immune responses to oral rFPV vaccines is warranted. Interestingly, we also demonstrated that mucosal responses developed earlier after vaccination compared to the systemic responses. The data showed that i.n. or

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Fig. 9. The effect of co-stimulatory molecules. BALB/c (H-2d ) mice (n = 5 mice/group) were primed with 1 × 107 pfu AE FPV with or without co-stimulatory molecule, boosted with 1 × 107 pfu AE VV and systemic T cell responses were measured in spleen against 15 mer B clade overlapping Gag peptide pool (grey bars), 15 mer AE clade overlapping Gag peptide pool (black bars), AE clade 15 mer overlapping Pol 1 peptide pool (white bars) AMQMLKETI Gag peptide (stripped bars) measured by IFN␥ ELISpot 3 weeks post prime-boosting (A). The data represent mean + S.D. and (* ) indicates that the gag peptide responses were significantly different between AE FPV/AE VV and AE FPV-1L-12/AE VV groups, p < 0.05. The data was plotted similar to other ELISPOTs. Pooled splenocytes from AE FPV, AE IL-12-FPV and AE IFN␥-FPV primed and AE VV boosted BALB/c (H-2d ) mice were stained with PE labelled AMQMLKETI H-2Kd MHC class I restricted tetramer and anti-CD8-FITC (9B). The y-axis indicates the PE channel and x-axis the FITC. The unimmunised mice were taken as the controls and the background CD8+ counts for spleen was less than 0.5%.

i.m. rFPV priming followed by i.m. rVV boosting can elicit responses in the genito-rectal nodes within 3 days post-booster immunisation, although these responses are not sustained at the same level over time. Indeed, the emergence of the peak mucosal and systemic IFN( T cell responses was inversely proportional over time and suggests that the kinetics of cytokine expression in the two compartments may be distinctly different. An AE FPV priming dose below 1 × 107 drastically reduced the systemic T cell responses measured by IFN␥ ELISpot assay (Fig. 2B). In contrast, lower doses of AE FPV (5 × 105 to 1 × 107 ) did not affect the number of tetramer positive CD8+ cells in the systemic compartment nor the general

mucosal T cell response measured by IFN␥ ELISpot assay (Fig. 2C and A). The differences in ELISpot and tetramer responses to the different rFPV doses may relate to the epitope recognition or functional state of T cells in different assays. The nature of T cells in different compartments may explain the difference observed with mucosal and systemic IFN␥ ELI Spot responses. Our data suggest that the order, site and dose of poxvirus vector delivery are pivotal for eliciting optimum mucosal and systemic immune responses to encoded vaccine antigens. From the perspective of making vaccine regimes more amenable for use in the developing world, experiments were also undertaken to determine whether simultaneous delivery

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of the poxvirus vectors could elicit good immune responses. However, our model clearly indicated that a minimum of 6 days is imperative between priming and boosting for effective CD8+ immunity to vaccine antigens (Table 3). Furthermore, following AE FPV i.n. priming, it was shown that the AE VV i.m. boosting can be given at 2, 4, 6 or 10 weeks, without affecting the level of T cell responses generated (results not shown), suggesting that this poxvirus prime-boost regime is much more flexible and practical than the DNA prime-boost regime tested previously. The poxvirus prime-boost vaccine strategy did not induce elevated antibody responses to p24 Gag (Fig. 2D), similar to that observed with rDNA/rFPV vaccines tested previously (Ranasinghe et al., unpublished data). However, it would be desirable to develop HIV vaccines that enhance both cellular and humoral immunity, especially mucosal IgA and neutralising antibodies [53]. Further protein boosting may be one approach to enhance antibody responses [54]. Modulating the immune system to generate Thl and Th2 responses by co-expressing selected immuno-modulatory molecules, would also offer some prospects in overcoming this barrier [37,44]. In our study the co-expression of cytokine IL-12 was found to enhance T cell immunity (Fig. 9A and B). We have shown that to generate enhanced responses the IL-12 needed to be delivered in the priming vector but did not enhance responses when expressed at the time of boosting, which is consistent with our previous rDNA/rFPV prime-boost studies (Ranasinghe et al., unpublished data) [55]. Moreover, it is note worthy that these enhanced T cell responses were only observed following 3 weeks post-boost and no marked difference in T cell responses were elicited when animals were sacrificed at 2 weeks or less. Several other vaccine studies also have shown that IL-12 can be used as a co-stimulatory molecule to enhance mucosal and systemic immune responses [36,56,57]. In summary our combined mucosal systemic (i.n. AE FPV/i.m. AE VV) poxvirus prime-boost regime elicited superior mucosal and systemic responses to HIV-1 antigens compared to the other regimes tested. The mucosal T cell responses peaked at a very early stage in the immunisation and were maintained at low levels compared to the initial response. In contrast systemic responses appeared later and was sustained at the same level to memory phase. Also, the co-expression of IL-12 in the prime enhanced the immune responses in a time dependent manner. Moreover, these studies clearly indicated that rFPV vector could be used as a powerful mucosal delivery vaccine when administered via intranasal or oral routes. When AE FPV vaccines were delivered via the oral route both Gag and Pol 1 responses were observed suggesting a differential antigen processing mechanism in the gastrointestinal mucosa. From the perspective of creating easy to use, high quality HIV-1 vaccines that generate both mucosal and systemic immunity, the oral rFPV studies merit further investigation. Our experience with mucosal studies suggests a need for more improved techniques for monitoring post vaccination mucosal T cell immunity.

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Acknowledgements This work was supported by Australian National Health and Medical Research Council Grants 299907, 960338, NIH Contract N01-A1-05395, and NIH 5R21-AI054172. The HIV-specific 15 mer overlapping peptide pools were kindly supplied by the NIH AIDS Research and Reference Reagent Program. Authors would like to thank Dr. Robert De-Rose, Melbourne University for reconstituting most of the HIV peptide pools and Terri Sutherland, Bio-Molecular Resource Facility at John Curtin School of Medical Research, Australian National University for synthesizing the HIV tetramers.

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