Erasmus MC, Institute of Virology, PO Box 1738, 3000 DR Rotterdam, The Netherlands
Correspondence
Willem Huisman
w.huisman.1{at}erasmusmc.nl
Albert D. M. E. Osterhaus
a.osterhaus{at}erasmusmc.nl
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ABSTRACT |
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Present address: UMC Utrecht, Department of Immunology, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands.
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INTRODUCTION |
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The most promising results with candidate vaccines have been obtained using classical approaches such as inactivated whole virus and infected cell vaccines (Yamamoto et al., 1991, 1993
; Matteucci et al., 1999
, 2000
; Pu et al., 2001
; Finerty et al., 2001
). However, from these studies it was not clear what the immune correlates of protection were. It has been reported that protection correlated with the presence of virus-neutralizing antibodies on the day of challenge (Yamamoto et al., 1991
, 1993
), but vaccine-induced protection has also been observed in the absence of virus-neutralizing antibodies (Matteucci et al., 1996
). Candidate FIV subunit vaccines tested to date have failed to induce protective immunity. The majority of these vaccines were based on the use of the FIV envelope glycoprotein (gp130). Vaccines consisted of recombinant bacterial proteins (Siebelink et al., 1995c
; Lutz et al., 1995
; Verschoor et al., 1996
; Leutenegger et al., 1998
), recombinant baculovirus proteins (Lutz et al., 1995
; Leutenegger et al., 1998
), recombinant vaccinia virus protein (Siebelink et al., 1995c
; Huisman et al., 1998
), peptides representing parts of the envelope protein (Lombardi et al., 1994
; Finerty et al., 2000
, 2001
; Flynn et al., 1995
) or recombinant vectors expressing the envelope protein or parts thereof (Gonin et al., 1995
; Tijhaar et al., 1997
). Recently, partial protection was reported using an envelope-encoding DNA vaccine co-injected with an IL12 expression plasmid (Boretti et al., 2000
; Leutenegger et al., 2000
).
In contrast, acceleration rather than reduction of FIV replication was observed upon challenge of vaccinated cats in a number of studies (Hosie et al., 1992; Lombardi et al., 1994
; Siebelink et al., 1995c
; Richardson et al., 1997
, 2002
; Karlas et al., 1999
; Giannecchini et al., 2002
). This phenomenon is characterized by an accelerated viraemia and sometimes increased virus loads in FIV-vaccinated animals compared with mock-vaccinated animals. The accelerated virus replication correlated with the presence of envelope-specific antibodies at the time of challenge in some studies (Lombardi et al., 1994
; Siebelink et al., 1995c
; Karlas et al., 1999
; Giannecchini et al., 2002
), but not in others (Hosie et al., 1992
; Richardson et al., 1997
, 2002
). Hence, the mechanism underlying this enhancement phenomenon has not been fully elucidated. Transfer studies using plasma of vaccinated animals strongly suggested the involvement of plasma-associated factors, like virus-specific antibodies or cytokines (Siebelink et al., 1995c
). Moreover, the accelerated viraemia also correlated with the presence of antibodies specific for the hypervariable regions HV3, HV4 and HV5 (Siebelink et al., 1995c
). We showed that cat antisera raised against FIV molecular clone 19k1 neutralized this virus but enhanced the replication of a closely related molecular FIV clone, 19k32, in vitro (Siebelink et al., 1992
; unpublished results). Since these two clones differ in their envelope amino acid sequence at only four positions, all located in regions HV4 and HV5, we hypothesized that antibody responses against these regions are involved in antibody-mediated acceleration of infection.
Here, we report on the in vivo evaluation of a recombinant FIV envelope glycoprotein candidate vaccine from which the regions HV35 were deleted. The results show that antibodies directed against HV35 are not solely responsible for vaccine-induced acceleration of infection.
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METHODS |
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Crandel feline kidney (CrFK) cells originated from an FIV-susceptible clone, CrFK 1D10, originally provided by N. Pedersen (Yamamoto et al., 1988) and were cultured in Dulbecco's modified Eagle's medium (Cambrex Bioscience) supplemented with penicillin (100 IU ml1), streptomycin (100 µg ml1), L-glutamine (2 mM),
-mercaptoethanol (2x105 M) and 10 % foetal bovine serum (D10F medium).
FIV AM19 was used as the challenge virus, as described previously (Siebelink et al., 1995c). This virus was originally isolated from PBMCs of a naturally infected cat, and an in vitro-grown virus stock was subsequently titrated in vivo (Siebelink et al., 1995c
).
FIV AM6c was used in the CrFK-based virus neutralization assay (see below). This virus was also isolated from a naturally infected cat and subsequently adapted to replicate on CrFK 1D10 cells (Siebelink et al., 1995b). The env sequence homology of FIV AM6c and the molecular clone FIV 19k1 is 94·8 %.
Vaccines: recombinant vaccinia virus (rVV) constructs.
The candidate vaccines used in this study were either identical to or derivatives of the vGR657x15 vaccine described previously (Rimmelzwaan et al., 1994), which has been shown to be responsible for vaccine-induced enhancement of challenge infection (Siebelink et al., 1995c
). vGR657x15 consists of a VV-expressed, lentil lectinSepharose column-purified FIV envelope protein. The cleavage site between the surface and transmembrane protein has been deleted through site-directed mutagenesis to facilitate incorporation into immune-stimulating complexes (iscoms) (Rimmelzwaan et al., 1994
). For clarity, this vaccine is referred to as FIV-Env-iscom.
A schematic representation of the regions deleted from the Env construct is provided in Fig. 1
. To generate the FIV-
Env vaccines, the envelope of FIV 19k1 was amplified using two different primer sets [
ENV1 (5'-GGGTACCTGGAATAACAC-3') with
ENV2 (5'-GGTCGACCCACCATCCACATTTTGG-3', SalI), and
ENV3 (5'-GGTCGACGGCATCTTAAGAAATTGG-3') with
ENV4 (5'-CCTACCCAATCTTCCCAC-3', SalI) (restriction sites underlined)] generating two fragments, 5'
Env and 3'
Env. These fragments were linked by the introduced SalI site. This resulted in an envelope sequence missing aa 358567 (based on the envelope sequence of FIV 19k1; GenBank accession no. M73964), which constitute HV35. Internal KpnI sites were used to exchange
Env with the original envelope sequence in pGR657x15, the plasmid originally used to generate vGR657x15 (Rimmelzwaan et al., 1994
). Subsequent generation of rVV-GR657x15-
Env, production, isolation, purification and incorporation into iscoms was done as described for rVV-GR657x15 (Rimmelzwaan et al., 1994
). The obtained vaccine is referred to as FIV-
Env-iscom.
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Animals, vaccination and challenge.
The specific-pathogen-free (SPF) cats used in this study were obtained from Harlan (Horst). An external ethical committee approved the protocol used in this study. Twelve animals were randomly distributed into groups: group 1 (n=3) was vaccinated with the original FIV-Env-iscom preparation; group 2 (n=4) was vaccinated with the FIV-Env-iscom preparation; group 3 (n=5) received PBS (non-vaccinated controls). Originally, group 1 consisted of four animals; however, one animal died during the vaccination scheme due to reasons not related to the experimental set-up of this study. Cats were inoculated with the respective vaccines at weeks 0, 6 and 10, identical to the previous study (Siebelink et al., 1995c
). Each vaccine dose contained 1·5 µg envelope protein. Animals were challenged intramuscularly with 20 CID50 FIV AM19 2 weeks after the last vaccination. PBMCs and plasma samples were obtained before and 1, 2, 3, 4, 6 and 8 weeks post-challenge.
Virus neutralization assay.
A virus neutralization assay based on the inhibition of FIV AM6c infection of CrFK cells was performed as described previously (Siebelink et al., 1995c).
Env ELISA
Cloning into prokaryotic expression vectors.
A modified version of the pThio plasmid (Invitrogen) was used to generate expression vectors containing FIV 19k1 (Siebelink et al., 1992) env sequences. The modifications were introduced for detection and purification purposes. Using primers ThioHisfor (5'-GCGTCTAGAATCCCTAACCCTCTCC-3', XbaI) and ThioHisback (5'-CGCGCTGCAGTCAATGGTGATGGTGATGATG-3', PstI) and pMT/V5-His (Invitrogen) as a template, the V5 epitope and the hexahistidine (6xHis)-tag were amplified, after which the PCR product was cloned into XbaI/PstI-digested pThio. The obtained expression vector was named pThio/V5-His. Expression products from this expression vector were thioredoxin fusion proteins, containing a C-terminal V5 epitope for detection purposes and a 6xHis-tag for purification.
Using Pwo Taq polymerase (Stratagene), env sequences were directly amplified from a 3'-19k1 pUC19 subclone (Siebelink et al., 1992). For the Env-SU (surface subunit region) construct, the primers 5'-CGGGATCCGACCCATTACAAATCCCACTG-3' (BamHI) and 5'-CGTCTAGATCTTTTTCTTCTAGGTTTATATTC-3' (XbaI) were used. For the Env-TM (transmembrane region) construct, the primers 5'-CGGGATCCGGCATCTTAAGAAATTGGTAT-3' (BamHI) and 5'-CGTCTAGATTGATTACATCCTAATTCTTGC-3' (XbaI) were used. The obtained amplicons were subsequently digested with BamHI and XbaI and cloned into BamHI/XbaI-digested pThio/V5-His.
The Env-SU construct incorporated Env 19k1 aa 322611 (Env19k1, NCBI nucleotide accession no. M73964), containing HV3, HV4 and HV5 and flanking regions that were not deleted from the Env constructs. The Env-TM construct incorporated Env 19k1 aa 567701, containing the principal immunodominant domain.
The generated plasmids were checked by restriction enzyme analysis and sequencing.
Expression and purification of bacterial recombinant fusion proteins.
For expression of the fusion proteins, plasmid DNA was transfected into BL21pLysS bacteria (Stratagene). After culturing a single colony in 10 ml SOB medium [20 g Tryptone Peptone l1 (Difco), 5 g yeast extract l1 (Difco), 0·5 g NaCl l1, 2·5 mM KCl, 10 mM MgCl2, pH 7·0] with 100 µg ampicillin ml1 for 2 h, the bacteria were subsequently cultured in 500 ml SOB/ampicillin until an OD600 of 0·50·6 was reached. Expression was induced by adding IPTG (Roche) to a final concentration of 1 mM. After 4 h of induction at 2037 °C, depending on the construct, the bacteria were pelleted by centrifugation at 900 g for 15 min and immediately used for purification or frozen overnight at 20 °C.
Subsequently, the pelleted bacteria were solubilized overnight in 5 ml lysis buffer (6 M guanidine/HCl, 0·1 M NaH2PO4, 0·01 M Tris/HCl pH 8·0) per 500 ml bacterial culture. The resulting lysate was centrifuged for 15 min at 10 000 g at 4 °C. The supernatant was then incubated with ProBond resin (Invitrogen) for 1 h at room temperature. After stacking and washing of the column material, guanidine/HCl was replaced with urea by washing the column with 10 ml wash buffer (8 M urea, 0·1 M NaH2PO4, 0·01 M Tris/HCl pH 6·3). After an additional washing step with wash buffer (pH 5·9), elution was carried out with elution buffer (8 M urea, 0·1 M NaH2PO4, 0·01 M Tris/HCl pH 4·5). Washing and elution steps were carried out at 4 °C. Purification was checked by SDS-PAGE on 15 % polyacrylamide gels and Western blot analysis using an anti-V5 monoclonal antibody (Invitrogen). Elution fractions containing the highest concentration of fusion protein were pooled and used in the ELISA.
ELISA.
ELISA plates (96-well; Corning) were coated with 100 ng of recombinant protein in PBS (Gibco) per well. As a control antigen, an FIV Orf Athioredoxin fusion protein was used, expressed and purified as for the envelope fusion proteins. After blocking with ELISA buffer consisting of Meddens reagent (Meddens, Woerden, The Netherlands) supplemented with 0·5 % BSA and 0·05 % Tween 20, the plasma samples were incubated in a 1 : 100 dilution in ELISA buffer for 1 h at 37 °C. Mouse anti-cat IgG (1 : 300; Serotec) was used as a first conjugate, followed by HRP-conjugated rabbit anti-mouse IgG (1 : 1000; DAKO). 3,3',5,5'-Tetramethylbenzidine (TMB) in TMB diluent (Meddens) was used as substrate. The reaction was stopped after 5 min by adding an equal volume of 2 M H2SO4. Background values against the Orf A fusion protein were subtracted from the Env-SU and Env-TM values to obtain specific OD450 values.
Virus loads
Plasma virus load.
Plasma virus loads were determined with a real-time PCR (TaqMan) assay according to a protocol described previously (Klein et al., 1999; Leutenegger et al., 1999
) on an ABI prism 7700 Sequence Detection System (Applied Biosystems). Primers and probe sequences (Klein et al., 1999
; Leutenegger et al., 1999
) were adapted to the FIV 19k1 sequence when different. Primer concentrations of 15 pmol (300 µM) and 30 pmol (600 µM) were found to be optimal for the forward and reverse primers, respectively. The optimal probe concentration was 10 pmol (200 µM). The real-time PCR was performed using the EZ-core kit (Applied Biosystems). The RT-PCR cycling programme was initiated with a 2 min 50 °C uracyl amperase step, followed by a 30 min 60 °C RT step, 5 min denaturation at 95 °C and 45 cycles of a two-step PCR consisting of 20 s at 95 °C and 1 min at 62 °C. Data were collected during the annealing step (62 °C). A diluted 19k1 virus stock was used in each run to obtain a standard curve. Feline plasma (190 µl) was spiked with a known phocine distemper virus stock that was used as an internal control to control for efficiency of RNA isolation (unpublished data). RNA was extracted from plasma (total 200 µl input) and concentrated fourfold (50 µl output) with a Magnapure LC Isolation Station (Roche), using the Magna Pure LC Total Nucleic Acid Isolation kit (Roche).
Cell-associated virus load.
Provirus loads or cell-associated virus loads were determined using a slightly modified infectious centre test, described previously (Siebelink et al., 1995c). Briefly, threefold serially diluted PBMC samples obtained after challenge infection were co-cultured in 96-well U-bottom plates (Greiner Bio-One) with a concanavalin A- and rhuIL2-stimulated mixture of PBMCs from two SPF cats in 10 wells. Culture medium containing rhuIL2 was added weekly to maintain the cultures. After 4 weeks, the culture supernatants were analysed for the presence of FIV antigen by ELISA (Siebelink et al., 1990
). The number of infected cells in the PBMCs was calculated from the results of the ELISA by assuming that one infected cell gave rise to FIV antigen production after co-cultivation with the stimulated PBMCs, when one or more cultures tested in the 10 wells were negative for FIV antigen.
Statistical analyses.
For statistical analysis of the data between two groups, the MannWhitney test was used.
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RESULTS |
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Outcome of challenge infection
Plasma virus loads.
Plasma virus load kinetics in the respective groups of vaccinated cats were similar, irrespective of the vaccine used (Env vs Env, Fig. 4
a). At two weeks post-challenge, plasma virus loads were significantly higher in group 2 (median 22 400 copies ml1) compared with control group 3 (median 0, P=0·041). Group 1 (FIV-Env-iscom, median 26 000 copies ml1) also exhibited a trend towards higher virus loads in circulation (P=0·167). Peak virus loads ranged from 60 000 to 4 600 000 copies ml1, with no significant differences between groups. However, envelope protein-vaccinated animals reached peak plasma virus loads 13 weeks earlier than animals in the control group. These plasma virus load kinetics suggested vaccine-induced acceleration of infection in envelope-vaccinated animals.
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Post-challenge virus-neutralizing antibody kinetics
Upon infection, all cats vaccinated with FIV-Env-iscoms developed an anamnestic antibody response. Virus-neutralizing antibody titres 5120 were measured in individual cats from 3 weeks post-challenge onward (Table 1
). The FIV-
Env vaccinated cats (group 2) exhibited faster VNA titre kinetics compared with the mock-vaccinated cats (group 3), as two out of four cats developed VNA titres from 2 weeks post-challenge onward (all cats were positive at 3 weeks post-challenge), while most mock-vaccinated cats did not develop VNA titres until week 6 post-challenge.
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DISCUSSION |
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Post-challenge serum VNA responses in the FIV-Env-iscom-vaccinated group developed more rapidly than in the control group. This may have been caused by the accelerated kinetics of virus replication, by a priming effect of the FIV-
Env-iscom vaccine on the induction of VNA specific for epitopes outside HV35, or a combination of both mechanisms. HIV-1 vaccination studies in guinea pigs have demonstrated that masking of the envelope V3 region by the introduction of N-linked glycosylation sites and reduction of the net positive charge resulted in the induction of antibodies specific for the V1 region and with a broader neutralizing capacity in vitro than antibodies induced by the original protein (Garrity et al., 1997
). Furthermore, in an attempt to generate immunogenically superior HIV-1 envelope proteins, it has been shown that removal of one or more of the V1, V2 or V3 loops can result in the exposure of normally obscured, conserved neutralization epitopes (Sanders et al., 2000
). Thus, the antibody response induced by FIV-
Env-iscoms may have been redirected towards regions on the envelope protein other than HV35 and have primed for the induction of post-challenge antibodies that can neutralize FIV in vitro but may predispose for accelerated infection in vivo. This is in agreement with the more efficient induction of antibodies specific for epitopes present in the TM region of the envelope glycoprotein observed after vaccination with FIV-
Env-iscoms (see Fig. 3b, E
nv-TM response). As in HIV-1 neutralization, these epitopes may also be involved in in vitro virus neutralization of FIV.
It has been suggested that antibodies directed to the principal immunodominant domain (PID), located in the extracellular part of the TM region, are involved in enhancement of HIV-1 infection of cell cultures in vitro (Robinson et al., 1990). The high similarity between HIV-1 and FIV PID structures (Pancino et al., 1995
) suggests that this region may be involved in the induction of antibodies mediating enhancement of FIV infection. However, the observation that immunization of cats with a peptide containing the FIV PID resulted in reduced virus loads upon challenge infection compared with control animals (Richardson et al., 1998
) does not support this option. Collectively, these results point towards major differences between virus neutralization in vitro and in vivo, and it should be stressed that data generated in vitro should be extrapolated cautiously to the in vivo situation. Indeed, the physiological microenvironment has been shown to have a major effect on the infectivity of HIV-1 isolates in PBMCs and monocyte-derived macrophages (Wu et al., 1995
). Considering the outcome of this study, epitope deletion as an approach to improve the efficacy of candidate lentiviral vaccines is not likely to result in a successful vaccine. Although it has been shown for HIV-1 infection in vitro that antibody-mediated enhancement by a monoclonal antibody could be abrogated by mutating the targeted epitope (Mitchell et al., 1998
), this is less likely to occur in the context of a polyclonal immune response in vivo.
In vitro infection experiments have revealed possible mechanisms involved in enhancement of HIV-1 infection besides virus-specific antibodies. For example, it has been shown that more efficient virus entry was mediated by components of the complement system and their receptors (Boyer et al., 1991; Tacnet-Delorme et al., 1999
). These mechanisms may also act in vivo. Since in most studies cats are challenged shortly (within 24 weeks) after the last booster vaccination, an additional role for other mechanisms, such as immune activation, in vaccine-induced acceleration of infection cannot be excluded (Richardson et al., 1997
, 2002
; Karlas et al., 1999
). We have attempted to address the role of immune activation in another group of cats that was vaccinated with FIV-Env-iscoms by challenging these animals 12 weeks after the last booster vaccination instead of 2 weeks. Despite this adjourned challenge, both provirus and plasma virus load kinetics developed similar to groups 1 and 2 of the current study (data not shown). Both virus-neutralizing titres as well as envelope-specific antibody responses (ELISA) were similar to those described for the FIV-Env-iscom-vaccinated animals in the current study. Therefore, it was suspected that antibodies, not vaccine-induced immune activation, were at the basis of the observed accelerated virus replication. However, a control group vaccinated with simian immunodeficiency virus (SIV)-Env-iscoms and challenged 2 weeks after the last booster vaccination exhibited virus load kinetics in between those of the PBS controls and the groups with accelerated virus replication. Since an SIV-Env-iscom-vaccinated group challenged 12 weeks after the last booster vaccination was not included, we could not accurately assess the potential involvement of immune activation in this set-up and these groups were therefore not included in this study.
The difference in virus replication kinetics in vaccinated compared with control cats was more pronounced when virus loads were tested as the number of infected cells in the infectious centre test than when plasma loads were tested by real-time PCR (compare Fig. 4a and b). This suggests that in the vaccinated cats a more efficient entry of virus into susceptible cells took place. Recent studies suggest that the specific activation of lentivirus-specific CD4+ T cells results in an increased susceptibility to infection of these cells (Richardson et al., 1997
, 2002
; Douek et al., 2002
). Accordingly, the induction of FIV-specific CD4+ T cells by vaccination could have supported a more efficient infection of these cells upon challenge infection, possibly through an increased expression of CXCR4 (Willett et al., 1997b
). However, earlier results showing that a factor responsible for vaccine-induced enhancement of virus replication could be transferred to naïve cats by plasma obtained from cats vaccinated with an FIV-Env-iscom vaccine (Siebelink et al., 1995c
) argue against a role for virus-specific CD4+ T cells as a single determinant responsible for vaccine-induced enhancement of virus replication. Still, it cannot be ruled out that multiple vaccine-induced mechanisms are involved in the observed accelerated virus replication.
The present study has shown that vaccine-induced enhancement of FIV challenge infection is not exclusively mediated by antibody responses against epitopes within HV35 of the envelope glycoprotein. Since the envelope iscom vaccine from which these regions were deleted retained its property to induce an antibody response that predisposed for accelerated viraemia upon challenge infection, other regions of the envelope glycoprotein may be involved in antibody-mediated acceleration of infection as well. Until those regions are identified, the use of the envelope glycoprotein as a candidate subunit vaccine against FIV infection of cats should be considered with caution.
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ACKNOWLEDGEMENTS |
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Received 5 January 2004;
accepted 2 March 2004.
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