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1 Department of Virology, Biomedical Primate Research Center (BPRC), 2288 GH Rijswijk, The Netherlands
2 GlaxoSmithKline Biologicals, Rixensart, Belgium
3 GlaxoSmithKline Biopharmaceuticals CEDD Biology, Stevenage, UK
4 Swedish Institute for Infectious Disease Control, Karolinska Institutet, Stockholm, Sweden
5 Department of Veterinary Medicine, University of Cambridge, UK
Correspondence
Gerrit Koopman
koopman{at}bprc.nl
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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A supplementary figure showing antigen-specific T-cell cytokine responses as measured by intracellular cytokine staining is available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Heeney, 2004; Letvin et al., 2002; Pantaleo & Fauci, 1996). However, despite these obstacles, examples from long-term non-progressor human cohorts and proof-of-principle studies in non-human primates have revealed that control of virus load and protection from disease can be achieved in different settings (Heeney & Plotkin, 2006; Jin et al., 1999; Mascola et al., 2000; Montefiori et al., 1996; Schmitz et al., 1999). Several lines of evidence suggest that, similar to protection against retroviral infection in mice, an efficacious prophylactic HIV-1 vaccine may need to elicit both humoral and cell-mediated immune (CMI) responses (Desrosiers, 2004; Heeney, 2006; Mascola et al., 2005; Messer et al., 2004). DNA vaccines were shown to induce both humoral and cellular immune responses in rodents, but in non-human primates and in humans, only low-level immunogenicity has been reported (Estcourt et al., 2004; Robinson, 1999). Efforts have been directed at improving these responses by boosting with viral vectors (Amara et al., 2002; Brave et al., 2007; Casimiro et al., 2005; Doria-Rose et al., 2003; Hel et al., 2006; Koopman et al., 2004; Letvin et al., 2004). DNA vaccines are generally effective at stimulating CD8 responses, whilst subunit vaccines are more effective at eliciting antibody responses (Barouch et al., 2000; Earl et al., 2001, 2002; Patterson et al., 2004; Robinson, 1999). Combined-modality DNA prime–protein boost vaccination strategies have been evaluated in the past (Cristillo et al., 2006; Letvin et al., 1997; Mooij et al., 2004; Otten et al., 2005; Pal et al., 2005, 2006; Robinson et al., 1999). As with single-modality envelope protein-based vaccines (Berman et al., 1990; Bruck et al., 1994; Girard et al., 1991; Hu et al., 1992; Mooij et al., 1998; Verschoor et al., 1999), protection against homologous, non-pathogenic simian–human immunodeficiency virus (SHIV) can be obtained (Letvin et al., 1997; Pal et al., 2006; Robinson et al., 1999). However, protection from heterologous, pathogenic SHIV challenge has proven to be much more difficult to achieve (Mooij et al., 2000). Unfortunately, none of these previous studies has compared the immunogenicity and efficacy of single- and mixed-modality immunization in a comprehensive way.
Strategies to improve the efficacy of HIV-1 Env single-component vaccines have been directed at inclusion of gag/pol structural and/or tat, rev and nef regulatory gene products (Calarota et al., 1998; Hel et al., 2006; Stittelaar et al., 2002; Wilson et al., 2006). Whilst immunization with simian immunodeficiency virus (SIV) Gag, alone or in combination with HIV-1 Env or Nef and Tat, has already yielded promising results, the data must be interpreted with caution as, in most cases, Mamu A*01-positive animals, which mount a particularly robust response to highly conserved Gag peptides, were used (Amara et al., 2001, 2002; Barouch et al., 2001; Casimiro et al., 2005; Egan et al., 2004; Montefiori et al., 1996; Mooij et al., 2004; Seth et al., 2000; Shiver et al., 2002). Importantly, previous work using an adjuvanted protein vaccine based on Env, Nef and Tat antigens suggested protection from SHIV-induced sustained virus load and CD4 T-cell decline in rhesus monkeys (Voss et al., 2003). However, analysis of T-cell responses was not performed. Here, we extend this work by evaluating different modes of delivery of the Env, Tat and Nef antigens, using the rhesus macaque pathogenic SHIV challenge model. Antigens were delivered either epidermally as plasmid DNA or intramuscularly as adjuvanted recombinant proteins in single-modality or in DNA prime–protein boost or protein prime–DNA boost vaccine regimes. The study provides a comparative analysis of the induction of cellular and humoral immune responses, as well as efficacy against SHIV89.6p challenge induced by these different immunization strategies.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Vaccines.
The pRix57 vector was used for DNA immunizations. pRix57 was derived from pRix28 (Rollman et al., 2007) and is a pUC-based plasmid with a kanamycin selection marker. It encodes HIV-1W6.1D Env (gp120), SIV Nef and HIV-1 Tat, which are expressed as a single fusion protein from a cytomegalovirus promoter. The Tat gene possesses three point mutations to inactivate the transactivation function. The gp120 gene was constructed not to encode the signal peptide, allowing the protein to remain cell-associated with little glycosylation. For protein immunization, the HIV-1W6.1D Env protein, together with the HIV-1 NefTat and the SIV Nef protein, were formulated in the AS02A adjuvant system as described previously (Voss et al., 2003).
Immunization and challenge schedule.
Five groups of six rhesus macaques were immunized at weeks 0, 8, 16 and 24 with either the pRix57 DNA vector or the HIV-1W6.1D Env, HIV-1 NefTat or SIV Nef proteins. Group 1 received DNA at all time points, group 2 received protein at all time points, group 3 received protein twice followed by DNA twice and group 4 received DNA twice followed by protein twice. The control group (5) received empty DNA vector plus AS02A at all time points. The DNA encoding HIV-1W6.1D Env, SIV Nef and HIV-1 Tat was given with a Bio-Rad DNA delivery device (0.5 µg per shot, four shots). The Bio-Rad gene gun delivers DNA-coated gold particles to the skin at high speed. These target mainly the cells in the epidermis, but some cells in the dermis also receive the DNA plasmids. For protein immunization, macaques were immunized intramuscularly at two injection sites. The first injection site received 20 µg HIV-1W6.1D Env+20 µg HIV-1 NefTat in AS02A (500 µl final volume); the second received 20 µg SIV Nef in AS02A (500 µl final volume).
At week 32, 8 weeks after the last immunization, animals were challenged with 20 MID50 of the heterologous, pathogenic, cell-free SHIV stock ‘89.6p’ by the intravenous route (1 ml per monkey). The chimeric SHIV strain SHIV89.6p expresses the genes tat, rev, vpu and env of HIV-189.6 in the genomic background of SIVmac239. This pathogenic variant was isolated after serial in vivo passage of the original SHIV89.6 from rhesus macaques (Reimann et al., 1996). The challenge virus stock has been propagated in rhesus peripheral blood mononuclear cells (PBMCs) and was titrated intravenously in rhesus macaques at the BPRC. This virus has been shown to infect readily via the intravenous route and to establish high virus loads and AIDS-like disease in outbred rhesus macaques.
Cellular immunology assays.
Induction of gamma interferon (IFN-
), interleukin-2 (IL-2) and IL-4 cytokine responses was measured by using an ELISpot assay, which was performed 2 and 6 weeks after each immunization and after challenge (Koopman et al., 2004). In brief, 2.5x106 PBMCs ml–1 were stimulated for 24 h with peptides (1 µg ml–1) in 600 µl RPMI 1640 medium supplemented with 5 % pooled rhesus serum, in a 24-well tissue-culture plate. Separate peptide pools covering HIV-1W6.1D gp120, HIV-1 Nef, HIV-1 Tat, SIVmac239 Nef (NIH catalogue no. 8762) and SIVmac239 Gag (NIH catalogue no. 6204) were used, consisting of 15mers with an 11 aa overlap. Medium alone was used as a negative control, whilst PMA (20 ng ml–1) plus ionomycin (1 µg ml–1) stimulation was used for the positive control. For the enumeration of antigen-specific cytokine production, non-adherent cells were collected and plated at 2x105 cells per well in a 96-well ELISpot plate with the same antigens added again. The microtitre plates were pre-coated with monoclonal antibodies (mAbs) specific for the lymphokine of interest, i.e. anti-IFN- mAb MD-1 (U-CyTech), anti-IL-4 mAb QS-4 (U-CyTech) and anti-IL-2 mAb B-G5 (Diaclone Laboratories).
Detection of cytokine-secreting cells took place after either 15 h for IL-4 or 4 h for IFN- and IL-2. The cells were lysed and the debris was washed away before adding detector antibodies. IFN-, IL-2 and IL-4 were detected by using biotinylated rabbit anti-rhesus IL-2, biotinylated rabbit anti-rhesus IFN- or biotinylated mouse anti-rhesus IL-4. Spots were visualized by using a gold staining/silver enhancement technique (U-CyTech). IFN-, IL-2 or IL-4 ELISpot results are expressed as the number of spot-forming cells per 106 PBMCs minus the background (mean of medium control+2SD). The assay was discarded if PMA/ionomycin stimulation gave no response.
Intracellular cytokine staining (ICS).
PBMCs (5x106 ml–1) were incubated at 37 °C for 2 h with anti-CD28 and anti-CD49d antibodies (2 µg of each antibody; BD Pharmingen) and either staphylococcal enterotoxin B (1.25 µg ml–1; Sigma), pooled peptides (1.25 µg of each peptide ml–1) or medium only. Peptides used were HIV-1W6.1D gp120 peptide pool, HIV-1 Nef peptide pool, HIV-1 Tat peptide pool, SIVmac239 Nef peptide pool (NIH catalogue no. 8762) and a SIVmac239 Gag peptide pool (NIH catalogue no. 6204).
Cells were treated with brefeldin A (Golgiplug 1 : 1000; BD Pharmingen) to inhibit protein trafficking and incubated for 12 h at 37 °C. Cells were then washed with PBS/1 % BSA solution and stained for surface markers by using ECD-labelled anti-CD14 (clone RM052; Beckman Coulter), ECD-labelled anti-CD20 (clone B9E9; Beckman Coulter), peridinin chlorophyll protein (PerCP)-labelled anti-CD4 (clone L200; BD Pharmingen), allophycocyanin (APC)-labelled anti-CD3 (clone SP34; BD Pharmingen), APC–cytochrome 7 (Cy7)-labelled anti-CD8 (clone SK1; BD Pharmingen), for 30 min at 4 °C in the dark. Subsequently, cells were washed with PBS/BSA and fixed with cytofix/cytoperm solution (BD Pharmingen) for 20 min at 4 °C. Then, the cells were washed twice with permeabilization buffer (diluted 10x in H2O) and resuspended in permeabilization buffer containing 5 % normal human serum (Sanquin), phycoerythrin (PE)-labelled anti-IL-2 (clone MQ1-17H12; BD Pharmingen), PE–Cy7-labelled anti-tumour necrosis factor alpha (TNF-
) (clone mAb 11; BD Pharmingen) and fluorescein isothiocyanate (FITC)-labelled anti-IFN- mAb (clone B27; BD Pharmingen). After 30 min incubation at 4 °C, cells were washed twice with permeabilization buffer and fixed in 2 % paraformaldehyde solution (in PBS) for 16 h. Acquisition was performed on a FACSAria flow cytometer (BD Pharmingen), collecting 100 000–200 000 lymphocyte-gated events per sample. For analysis of cytokine-producing cells, first CD3-positive cells that were negative for CD14 and CD20 were selected and CD4 was plotted against CD8 to select CD4 and CD8 T cells. Subsequently, IL-2 was plotted against IFN- and subsequently against TNF- to select seven cytokine-producing subpopulations, i.e. IL-2, IFN- or TNF- single cytokine-producing cells, cells that make IL-2 plus IFN-, IL-2 plus TNF-, or IFN- plus TNF-, and finally a subset that produced all three cytokines together.
Humoral responses.
Antibodies specific for HIV-1W6.1D Env, HIV-1 Nef, HIV-1 Tat and SIVmac239 Nef were measured by ELISA as described previously (Voss et al., 2003). Virus-neutralization capacity was measured against the homologous SHIVW6.1D virus (Ranjbar et al., 1997) and the heterologous SHIV89.6p challenge virus (Reimann et al., 1996), using a luciferase reporter-gene assay in JC53-BL (TZM-bl) cells (Li et al., 2005). Both viral stocks were prepared on human PBMCs and titrated on JC53-BL (TZM-bl) cells. For the neutralization assay, 50 TCID50 virus stock was incubated with a 1 : 10 dilution of either pre-immune serum or serum obtained 6 weeks after the fourth immunization, in duplicate for 1 h at 37 °C in a total volume of 150 µl Dulbecco's medium with 10 % fetal calf serum (Invitrogen) in 96-well flat-bottom culture plates. Freshly trypsinized cells (10 000 cells in 100 µl medium containing 37.5 µg DEAE–dextran ml–1) were added to each well. One set of eight control wells received cells plus virus (virus control), and another set of eight wells received cells only (background control). After a 48 h culture period, 150 µl culture medium was removed from each well and 100 µl Bright-Glo reagent (Promega) was added to the cells. After 2 min incubation at room temperature to allow cell lysis, 150 µl cell lysate was transferred to 96-well black/white solid plates for measurement of luminescence using a Victor 3 luminometer. The percentage neutralization was calculated as follows: [(mean of virus control–mean of background control)–(mean of test serum–mean of background control)]/(mean of virus control–background control). Heat-inactivated sera (56 °C, 30 min) were used for all neutralization assays.
Determination of virus load.
A quantitative competitive RNA PCR was used to estimate the virus load in plasma as described previously (Ten Haaft et al., 1998).
Statistical analysis.
A non-parametric ANOVA Kruskal–Wallis test was used to compare virus load between the control group of unvaccinated monkeys and the different vaccinated groups. Statistical analysis was performed for peak virus load, measured at week 2 after infection, and the steady-state plasma virus load, measured at week 28 after infection. Correlation between ELISpot responses and steady-state virus load (week 28 after infection) was calculated by using a two-tailed Spearman correlation test. At 2 and 8 weeks after challenge, a one-tailed non-parametric Mann–Whitney test was used to compare IFN- ELISpot responses between the control-group animals and a combined group of all immunized animals.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, the animals immunized with DNA only (group 1) did not mount antibody responses, whilst in all other groups, where protein immunization was included, antibodies against HIV-1 Env, SIV Nef, HIV-1 Tat and HIV-1 Nef were generated. Interestingly, in group 2 (protein only), the magnitude of the response was already at its peak after the second immunization and persisted at a high level, whilst in group 3 (protein/DNA), antibody titres declined as soon as the DNA immunizations were started. In group 4, where animals were immunized with DNA first, antibodies were absent until the monkeys were boosted with adjuvanted protein. In addition to the presence of binding antibodies, capacity to neutralize the homologous SHIVW6.1D strain was observed in the sera of almost all animals that had received protein immunization at some time point (Table 1), with >95 % inhibition of virus infection being achieved in all six animals of the protein-only group, in all five animals of the DNA/protein group and in five of six animals of the protein/DNA group. Here, the one animal that was negative (Ri12175) also had the lowest anti-HIV-1 Env antibody titre. None of the six DNA-immunized animals was able to neutralize SHIVW6.1D. In contrast to these results, >95 % inhibition of the heterologous SHIV89.6p challenge strain was never observed (Table 1).
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-, IL-2- and IL-4-secreting cells. The biggest difference among the vaccine groups was observed for IL-4, which was seen in both the protein and the DNA prime–protein boost animals (groups 2 and 4) and, to a lesser extent, also in animals that had received a protein prime–DNA boost (group 3). IL-4-secreting cells were not detected in the DNA-vaccinated animals (group 1). Induction of IL-2 and IFN- responses was detected to some extent in all four vaccine groups (Fig. 2). Responses were directed predominantly against HIV-1 Env, HIV-1 Nef and SIV Nef, whilst HIV-1 Tat-specific responses were low.
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and IL-2 production were seen in the ELISpot assay. This analysis revealed that antigen-specific CD4 cells were induced in all vaccine groups. The highest responses were observed in the protein alone-immunized animals (group 2). CD8 cells were virtually absent in the vaccinees, except for two weak responders in the DNA-alone group (group 1) (Fig. 3a). The cytokine-expression pattern of the CD4 T cells was largely comparable between the groups, with a strong contribution of IL-2 observed, mostly in combination with either IFN- or TNF-. In contrast, the CD8 T cells in the DNA-immunized animals displayed less IL-2 production and more IFN- production. However, as only two of the six animals in group 1 were able to mount a CD8 response, expression of cytokines in CD8 T cells must be interpreted with caution. Multifunctional CD4 T cells that produced all three cytokines were seen in all vaccine groups (Fig. 3b). The antigen-specific responses seen in ICS were directed mainly against HIV-1 Env and SIV Nef, whilst HIV-1 Tat elicited only low responses.
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, all animals became infected and there was no statistical difference in peak viraemia between the groups at 2 weeks after infection. In the control group, three of the six animals subsequently reached steady-state virus levels of between 104 and 105 copies ml–1, whereas the other three animals partially suppressed replication to 103 copies ml–1. None of the control animals exhibited a virus load reduction to undetectable levels. In contrast, animals from all four vaccine groups showed some enhanced degree of protection from high virus load. Four of six DNA-immunized animals (group 1) and five of six of the protein/DNA-vaccinated animals (group 3) suppressed virus replication to below the detection limit. However, the animals remained virus-positive as determined by DNA PCR (not shown). Only the protein/DNA-immunized animals (group 3) had a statistically significantly lower steady-state virus load than the control group (P<0.01). Statistical significance was not reached for the DNA-only group (group 1). In the other two vaccine groups, only one of 11 animals exhibited a high virus load. All other animals from these two groups showed some degree of virus-load control, with two animals in the protein-only group and one animal in the DNA/protein group showing undetectable virus load (groups 2 and 4, respectively).
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). Two weeks after challenge, a marked increase was seen in antigen-specific IFN- production in the DNA group (group 1) and, to a lesser extent, in the protein/DNA group (group 3), whereas such an anamnestic response was absent in the other vaccine groups. However, this difference was not maintained over time and, at week 72, all vaccine groups and the control animals had similar levels of IFN- response. In contrast, IL-2 responses were lost almost immediately in all animals and IL-4 responses disappeared gradually. Interestingly, the post-challenge IFN- responses were directed in good part against SIV Gag, which was not included in the vaccine (Fig. 6). These responses were already detected 2 weeks after challenge at comparable magnitude both in the control group and in the vaccine groups (Fig. 7a). At this time point, responses against SIV Nef or other vaccine antigens were absent in the control group, whereas strong SIV Nef-specific responses were seen in a large proportion of the immunized animals, reaching statistical significance when the combined group of immunized animals was compared with the group of control animals (Fig. 7a). Eight weeks following challenge, the IFN- responses were even more dominated by SIV Gag and SIV Nef (Fig. 6) and there was no significant difference between the vaccine groups and the control animals (Fig. 7b). Similar results were obtained by ICS analysis performed 8 weeks after challenge, showing that, at this time point, CD8 T cells also contributed to the response, particularly against SIV Gag and SIV Nef (see Supplementary Fig. S1, available in JGV Online). Finally, in the chronic phase of the infection, at week 40, the pattern of predominant IFN- responses against the SIV Gag and Nef proteins was maintained (Fig. 6).
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responses immediately (2 weeks) after challenge showed a clear inverse correlation with virus load, whereas no correlation could be drawn with any of the pre-challenge IFN-, IL-2 or IL-4 immune responses [see Fig. 7c
, where IFN- responses measured immediately before challenge (6 weeks after the last immunization) and 2 weeks after challenge are depicted]. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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responses was observed predominantly in the vaccinated animals that controlled virus load best.
Whilst protein-based vaccination strategies have been shown to be partly effective against challenge with homologous, neutralization-sensitive viral strains (Letvin et al., 1997; Otten et al., 2005; Pal et al., 2006; Verschoor et al., 1999), a direct comparison with DNA was often hampered due to the weak responses generated by this approach (Estcourt et al., 2004; Robinson, 1999). However, by using the new-generation pRix57 vector delivered by gene gun, it was possible to induce clearly measurable antigen-specific IFN- and IL-2 responses. Although these were still somewhat lower than in protein-immunized animals, they were comparable to those in the two other vaccine groups (those immunized with protein/DNA or DNA/protein). Importantly, the DNA-immunized animals differed from all other groups with regard to the development of CD8 responses (in two of six animals) and the complete absence of antibody responses (in all animals). This strong Th1-biased response was somewhat surprising, as gene-gun immunization has been described to elicit Th2 responses in mice (Robinson, 1999). Of note is that the closely related pRix28 vector, which differs from pRix57 only in that it contains HIV Nef, rather than SIV Nef, was shown to induce similar Th1-biased responses when given to mice with the same gene gun apparatus and protocol (Rollman et al., 2007). Although not investigated in our study, the exact nucleotide composition may have an impact on the array of innate receptors that are triggered, thus affecting the outcome of immune induction (Estcourt et al., 2004).
The sequence in which DNA and protein immunizations were given was found to have little impact on the magnitude of the cellular immune responses achieved after the last immunization (Figs 2, 3, 5). However, the kinetics of the antibody responses were clearly different, being strictly dependent on the use of proteins. Importantly, only in the protein-immunized animals were antibody levels consistently high, which could imply that protein must be given at least twice for robust B-cell memory and that the combined-modality vaccine groups may experience a loss of antibodies over the long term. Strikingly, IFN-, IL-2 and IL-4 production also tended to be consistently high in the protein-only group and more variable or somewhat lower in the other groups. No clear reversion in Th1/Th2 cytokine patterns was seen in the combined-modality vaccine groups, indicating that, with regard to induction of immune responses, the order in which the DNA and protein are given may not be essential.
Whilst all protein-immunized animals developed antibodies directed against HIV-1 Env, as well as the regulatory HIV-1 Nef, HIV-1 Tat and SIV Nef proteins, these were effective at neutralizing the homologous SHIVW6.1D strain, but not the heterologous SHIV89.6p challenge strain (Mooij et al., 2000; Voss et al., 2003).
In the absence of pre-existing neutralizing antibodies, all animals became infected after challenge. However, of the 23 immunized animals that were challenged, 12 were able to control virus replication to below the detection limit of 40 copies ml–1, whereas all six control animals remained plasma viral RNA-positive (Fig. 4). The highest proportion of virus control was seen in the protein prime–DNA boost vaccine group, followed closely by the group that received DNA only, where the immunization procedure had induced only Th1 plus CD8 responses, but no IL-4 and no antibodies. Therefore, it seems unlikely that antibodies against Tat and Nef, which could inhibit their immunosuppressive effects, play a role in containment of virus replication (Cafaro et al., 2001; Pauza et al., 2000). Similar results were obtained recently when the efficacy of these vaccine candidates was tested in an HIV-1/murine leukemia virus pseudotype challenge model in mice (Rollman et al., 2007). Interestingly, Dale et al. (2004) also reported recently that macaques immunized with DNA encoding SIVmac gag and pol and HIV-1AD8 tat, rev, vpu and env could suppress SHIVmn229 effectively, despite the relative lack of vaccine-induced immune responses. However, this was associated with the strong induction of SIV Gag-specific CD8- and CD4-mediated IFN- production immediately after challenge. Here, in the absence of Gag in our vaccine, we observed a marked increase in IFN- production immediately after challenge, in both the DNA-immunized and the protein prime/DNA-boosted animals. The importance of this post-challenge response was illustrated by a clear negative correlation with steady-state virus load, in contrast to the pre-challenge vaccine-induced immune responses, where no correlations were found. It could be hypothesized that the DNA-immunization procedure has been able to prime the immune system to mount the correct CD4 and CD8 responses necessary for early eradication of infected cells. Indeed, 2 weeks following infection, SIV Nef-specific responses were significantly higher in the immunized animals than in the control group. In contrast, the SIV Gag-specific responses, which were not included in the vaccine preparation, were induced at comparable levels in the immunized and control animals. These results suggest an important role for Nef and Gag cellular immune responses that appear to be driven by virus replication in the early phase of the infection. Similar results have been reported for anamnestic responses against Gag (Dale et al., 2004; Hel et al., 2006). However, for SIV Gag, pre-challenge, vaccine-induced responses were also found to correlate with suppression of virus replication when a DNA prime/adenovirus vector or DNA prime/poxvirus vector boost strategy was used (Casimiro et al., 2005; Hel et al., 2002). This may imply that similar correlations may also be found for SIV Nef, with more potent boosting.
Interestingly, animals immunized with protein only or with DNA prime–protein boost were less efficient at controlling virus replication, despite the induction of both T-helper and antibody responses. However, the antibodies generated were unable to neutralize the heterologous SHIV89.6p challenge virus. Probably, improved Env proteins that are capable of presenting broader neutralizing epitopes may be required for greater efficacy of protein-based immunization strategies (Heeney, 2006). Previously, control of SHIV89.6p virus replication was reported in four of four animals that had been immunized with the same HIV-1 Env, HIV-1 Tat/Nef and SIV Nef proteins as used in the current study (Voss et al., 2003). Whilst those results seem to be more clear-cut than the data reported here, application of a detection limit of 1500 copies ml–1, as used in the previous study, would have yielded quite similar outcomes.
In conclusion, this study has demonstrated clear differences in DNA- versus adjuvanted protein-induced immune responses. Whilst these responses did not correlate specifically with efficacy, the early appearance of IFN- responses to the SIV Gag and Nef antigens predicted a more favourable adaptive challenge outcome.
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ACKNOWLEDGEMENTS |
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Received 16 August 2007;
accepted 15 October 2007.
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, Animal developed AIDS during the study.
