Department of Immunology, Institute for Animal Health, Pirbright, Ash Road, Woking, Surrey GU24 0NF, UK1
Author for correspondence: Simon Kollnberger. Fax +44 1865 222502. e-mail simon.kollnberger{at}imm.ox.ac.uk
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Abstract |
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Introduction |
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ASFV was first observed when it emerged causing a typically lethal haemorrhagic disease of domestic pigs (Sus scrofa) (Montgomery, 1921 ). This is in complete contrast to the absence of disease symptoms in the natural hosts of ASFV in sub-Saharan Africa, the bush pig (Potamochoerus porcus) and the warthog (Phacochoerus aethiopicus), where the virus persists. Later, ASFV was shown to infect soft ticks (Ornithodoros moubata and O. erraticus), which are ectoparasites of warthogs and, on occasion, domestic pigs, thereby creating a cycle of infection that maintains the virus in Africa and elsewhere (Costa, 1990
; Viñuela, 1985
; Wilkinson, 1989
). Since its initial isolation, a number of less virulent forms have evolved and a range of isolates, causing from rapidly lethal to very attenuated and chronic infections in domestic pigs, has been described (Mebus, 1988
). The sequences of the virulent Malawi and non-pathogenic Ba71V isolates of the virus share 92% identity and the majority of proteins, including structural subunits encoded by different isolates, are either identical or highly similar (Dixon et al., 1994
; Yañez et al., 1995
).
Swine infected with ASFV develop anti-viral antibodies that can be demonstrated from 7 days post-infection (p.i.) by a wide variety of tests (Cowan, 1961 ; Crowther et al., 1979
; Hamdy et al., 1981
; Malmquist, 1963
; Pan et al., 1972
, 1974
, 1982
; Parker & Plowright, 1968
). Pigs that recover are usually resistant to challenge with homologous, but not heterologous, virus isolates (Ruiz-Gonzalvo et al., 1986
; Schlafer et al., 1984
). This isolate-specific immunity is not understood but it is unlikely to result from variation in antibody responses against different isolates, as both virulent and non-pathogenic isolates have a shared core of serological immunodeterminants.
Although there is no formal evidence for classical anti-virus neutralizing antibodies, antibody responses can clearly contribute to protection. Passive administration of serum or colostrum from recovered, convalescent animals can delay the onset of clinical symptoms in infected pigs, reduce viraemia and enhance survival (Coggins, 1968 , 1974
; Malmquist, 1963
; Parker & Plowright, 1968; Ruiz-Gonzalvo et al., 1986
; Schlafer et al., 1984; Wardley et al., 1985
), suggesting that there is an antibody-mediated component of resistance to virus infection (Knudsen et al., 1987
). This notion is supported by the observation that antibodies to p72 and p54 inhibit virus attachment to cells, whilst antibodies to p30 inhibit virus internalization (Gomez-Puertas et al., 1996
). Moreover, a conformational-dependent neutralizing epitope of the major viral structural protein p73 has been identified (Borca et al., 1994
; Zsak et al., 1993
). Finally, neutralizing antibodies against p54 and p30/32 have also been reported and immunization protocols utilizing a combination of these two proteins have had some success in delaying the clinical onset of the disease after infection with a virulent isolate of the virus (Gomez-Puertas et al., 1996
, 1998
). The molecular characterization of several putative viral proteins that may encode serological epitopes of the virus has been attempted by two-dimensional gel analysis but few of these proteins have been conclusively identified (Alcaráz et al., 1992
; Carrascosa et al., 1985
; Esteves et al., 1986).
In terms of possible cellular mechanisms of resistance to infection, there is some evidence for the participation of cytotoxic T cells (Martins et al., 1993 ; Alonso et al., 1997
; Leitão et al., 1998; Jenson et al., 2000
).
The weight of evidence, therefore, indicates an important (but not necessarily exclusive) role for antibodies in the protective antibody response of pigs to ASFV but the exact mechanism and range of critical serological virus immunodeterminants have yet to be defined. Thus, in order to systematically identify potentially protective serological immunodeterminants, we have screened a representative lambda phage cDNA expression library of the ASFV Ba71V isolate with polyclonal immune antisera from susceptible domestic pigs and resistant bush pigs, both recovered from infection with virulent isolates of the virus. In this way, we have identified 14 viral ORFs that encode serological epitopes of the virus. These included four structural proteins, three non-structural proteins and six viral subunits with unassigned functions. Several of these proteins elicited antibody responses comparable to the important structural immunodeterminants p32 and p73 in domestic pigs protected from infection with the virulent OURT88/1 isolate by previous infection with the related non-pathogenic OURT88/3 isolate of the virus. The fact that the majority of these proteins were also recognized by immune antisera from the natural host, the bush pig, suggests that some of these determinants may play a role in protection against virus infection.
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Methods |
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Antiserum from domestic pigs infected with the Malta isolate of ASFV.
An outbred pig (LandraceLarge White cross) was infected with 104 HAD50 (50% haemadsorbing doses) of the virulent Malta isolate of ASFV. Blood for antiserum preparation for ELISA was taken on day 0 and 28 days after infection.
Longitudinal antiserum was collected from a domestic pig infected with the non-pathogenic OURT88/3 isolate of ASFV and subsequently challenged with the virulent OURT88/1 isolate.
An outbred pig was infected with 104 HAD50 of the avirulent Portuguese isolate OURT88/3. Pigs infected with OURT88/3 are subsequently immune to the related highly virulent OURT88/1 isolate. The pig was challenged on day 28 with OURT88/1. ELISAs were performed with serial dilutions of antiserum prepared from blood taken 6 days after challenge with OURT88/1.
Bush pig antiserum.
Bush pig antiserum was obtained from Dr Euan Anderson, Veterinary Research Laboratory, Harare, Zimbabwe. A 6-month-old bush pig, captured from an ASFV-free area in Zimbabwe, was intramuscularly inoculated with 105 HAD50 of the Malawi (SINT90/1) isolate of ASFV (Haresnape et al., 1988 ). Blood for antiserum preparation was taken on day 0, before primary infection with the virus, and 307 days after a secondary challenge with the SINT90/1 isolate of the virus on day 217.
Generation of an ASFV lambda cDNA expression library
Infection of Vero cells with the Ba71V isolate of ASFV and isolation of polyadenylated RNA.
VERO cells (2x107) at 80% confluence were infected (m.o.i. of 10) with the Portuguese Ba71V isolate for 3, 6, 9 and 12 h.
Polyadenylated RNA was isolated from cells at each time-point using the Poly(A) Tract 1000 system (Promega). Samples of 1·25 µg mRNA isolated from each time-point were pooled and the resulting 5 µg was used for cDNA synthesis.
cDNA synthesis and size selection.
Synthesis of cDNA, adaptor ligations, enzyme digestions, phosphorylations and size selection were performed according to the manufacturer's instructions using the Lambda ZAP Library system (Stratagene).
cDNA was pooled into three fractions: high, medium and low, each containing DNA of sizes 2·0, 1·02·0 and 0·41·0 kb, respectively. Each fraction was used to generate a cDNA library in the Lambda-ZAP Phage Expression system.
Each of the three cDNA fractions was ligated into the phage vector, packaged and titred, as described in the Stratagene manual.
Purification of plaques, plaque excision and sequencing
Expression screening.
IPTG induction of phage protein expression and plaque lifts were performed as described previously (see Stratagene manual). After protein induction, membranes (Hybond C) (AmershamPharmacia) were removed from the plates, washed with PBS and blocked with 5% Marvel (w/v) in PBS (blocking buffer). Subsequently membranes were incubated at 4 °C overnight with porcine anti-ASFV Malta serum MI92 (1:2000 in blocking buffer) and developed with DAB (Sigma) (Cedillo-Barron et al., 2001 ) after incubation for 1 h at room temperature with HRP-conjugated rabbit anti-pig immunoglobulins (Dako) at a 1:1000 dilution. Positive plaques were then picked and purified and single clone excisions were performed as outlined in the Stratagene manual.
Sequencing and sequence alignment.
Miniprep DNA for sequencing was prepared from excised phagemids using the alkaline lysis method (Sambrook et al., 1989 ). Sequencing was performed with 5 µg DNA template using the Cy5 Autoread Sequencing kit (AmershamPharmacia) and an ALF Automated DNA Sequencing machine (AmershamPharmacia). Sequence analyses and manipulation were performed using the EasyGCG8 program and the NCBI database (http://www.ncbi.nlm.nih.gov).
PCR primer design, PCR and subcloning.
PCR primers for the viral ORFs A151R, C44L, E184L, K78R and K145R were designed with reference to the database, incorporating appropriate restriction sites for subcloning in pGEX 4T-1 (AmershamPharmacia). In the case of ORFs C44L and E184L, a combination of a 3' T7 primer from the parental pBK-CMV vector and a 5' N-terminal primer specific for these ORFs were used. All PCRs were performed with high-fidelity Pfu polymerase (Promega). PCR products were cloned into the pGEX 4T-1 prokaryotic expression vector and the resulting constructs were used to transform competent Escherichia coli, strain DH5- (Gibco), for expression and analysis of reactivity with immune antisera.
Purification of glutathione S-transferase (GST)-fusion proteins.
Overnight cultures of E. coli transformed with ASFV cDNA recombinants in pGEX grown in select medium were diluted 1:50 in 100 ml fresh LB medium containing ampicillin and grown for 35 h to the mid-exponential phase. Expression was induced for 2 h with 0·1 mM IPTG. Cells were harvested by centrifuging at 2500 g for 10 min, resuspended in ice-cold PBS and lysed with B-Per Extraction agent (Pierce). ORFs p54/E183L, p32/CP204L, histone-like protein/A104R, thymidine kinase/K196R, K205R and C44L were purified as soluble proteins on glutathioneSepharose 4B columns (AmershamPharmacia), as described by the manufacturer. The insoluble proteins p73/B646L, p10/K78R, DNA ligase/NP419L, small subunit of ribonucleotide reductase/F334L, B602L and CP312R were solubilized from inclusion bodies with 6 M guanidine hydrochloride prior to resolving by SDSPAGE, as described previously (Katrak et al., 1992 ).
ELISA.
Nunc ELISA plates were coated with purified ASFV fusion proteins or purified Schistosoma japonicum GST (Sigma) (2 µg/ml at 50 µl per well) in coating buffer (0·05 M Na2CO3/NaHCO3, pH 9·6) and incubated overnight at 4 °C. Plates were washed and blocked, as described previously (Cedillo-Barron et al., 2001 ), and incubated for 1 h at 37 °C with serial doubling dilutions of test sera in PBS. Plates were developed, as described previously (Cedillo-Barron et al., 2001
), and absorbances were read at 492 nm on a Titretek Multiskan spectrophotometer.
Analytical SDSPAGE and Western blots.
Proteins were separated by denaturing SDSPAGE using 10 or 15% (v/v) acrylamidebisacrylamide resolving gels and a 4% (v/v) acrylamidebisacrylamide stacking gel. Biorad molecular mass markers were used as standards.
Western blots were performed using Immobilon membrane (Millipore) and a semidry Western blotter (Sartoblot II), as described previously (Cedillo-Barron et al., 2001 ). After blocking, membranes were incubated for 1 h at 37 °C with immune antisera (diluted in blocking buffer) from pigs that had recovered from virus infection, washed with PBStween, incubated with HRP-conjugated rabbit anti-pig immunoglobulins (1:1000 in blocking buffer) for 1 h at 37 °C and developed with DAB.
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Results |
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Three fractions of cDNA in the size ranges of 0·21·0, 1·02·0 and >2·0 kb were used separately to generate three corresponding cDNA libraries with 176120, 199060 and 185738 recombinants, respectively. These three fractions were chosen in order to include a representative range of viral ORFs, taking into account that ASFV ORFs range from 182 bp to 7·4 kb in size, with an average size of 987 bp.
Between 6 and 10% of plaques from each of the unamplified phage libraries were shown to contain virus inserts by colony filter hybridization using Malawi isolate viral DNA as a probe (Table 1).
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As a further assessment, DNA isolated from the phage library by PEG precipitation was screened by PCR with specific primers for the viral ORFs. Full-length PCR products were obtained using primers for ORFs A238L and Xp124L, which code for two early genes of 800 bp and 500 bp, respectively (results not shown).
Sequencing of purified viral cDNAs isolated from the library
A total of 50 plaques was arbitrarily purified from each of the three different cDNA libraries (low, medium and high) and miniprep DNA from excised phagemids was prepared for sequencing. Clones were identified by sequencing from the N- and C-terminal ends of the cDNA and viral ORFs were assigned with reference to the database. The contiguity of intervening sequence was confirmed by restriction mapping, comparing the pattern of restriction fragments observed with that predicted from the published sequence of the Ba71V isolate (results not shown).
Following this analysis, 18 unique viral cDNAs were defined. Six were fragments of other larger cDNAs that were independently isolated. The remaining 12 cDNAs incorporated sequences from 18 viral ORFs, with the size of these cDNAs ranging from 0·35 to 2·9 kb (Table 2).
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After preabsorption against bacterial lysate, polyclonal antisera from recovered domestic pigs consistently showed no cross reaction with IPTG-induced bacterial lysates from the XLOLR host strain used for phagemid excision and E. coli strain DH5 used for subcloning (Fig. 2B
, C
, lanes 2 and 4). In addition, preabsorbed antisera failed to react with lysates from several ASFV cDNA constructs generated by restriction digestion (Fig. 3
). Fusion proteins with the predicted molecular masses from pBK-CMV were identified for each of the cDNAs encoding B602L (
85 kDa), p32/CP204L (
33 kDa), CP312R (
37 kDa), K205R (
24 kDa) and DNA ligase/NP419L (
42 kDa) (Fig. 2
). ORFs small ribonucleotide reductase subunit/F334L and p73/B646L were not clearly recognized by Western blot when production of bacterial proteins was induced by IPTG in the pBK-CMV phagemid vector used to generate the library (results not shown). However, both of these viral subunits were clearly identified by ELISA (see below and Figs 5 and 6) after subcloning of the respective cDNAs (clones 5 and 6) in the pGEX expression system and sensitization of ELISA plates with purified GST fusion proteins. In addition, a 100 kDa GST fusion protein of the viral p73 structural protein was also identified by immune antisera (results not shown). Antisera from immune domestic pigs and bush pigs clearly recognized both these proteins when protein expression was induced in the purified phage corresponding to these two ORFs (results not shown). The absence of a positive Western blot with the ribonucleotide reductase subunit/F334L may be explained by a requirement for conformationally dependent epitopes.
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Thus, clone 1 (A104R, A118R, A151R and A276R) was digested with XmnI/XhoI to remove all of the ORFs downstream of A104R. A 15 kDa -galactosidase fusion protein of the predicted size of ORF A104R expressed from the resulting construct was positively identified by Western blot with MI92 antiserum (Fig. 3
).
An 85 kDa -galactosidase fusion protein of the predicted molecular mass of ORF B602L was expressed from clone 2 (B602L and B354L) and identified by Western blot (Fig. 2
).
Clone 10 (K196R and K145R) was digested with XhoI to remove the downstream K145R ORF and the resulting construct obtained after religation and transformation was digested with EcoRI/EcoRV to generate a truncated cDNA encoding ORF thymidine kinase/K196R in-frame with N-terminal -galactosidase from the pBK-CMV vector. In this way, a 15 kDa
-galactosidase fusion protein of the correct size of the truncated C-terminal domain of thymidine kinase/K196R was positively identified by Western blot with MI92 antiserum (Fig. 3
).
ORFs A151R, E184L, K78R and K145R were all subcloned as PCR products from clones 1, 7 and 9, respectively, in pGEX. GST fusion proteins of K78R, K145R and E184L of the predicted sizes of 36, 44 and 46 kDa were all positive when probed with porcine anti-ASFV MI92 antiserum by Western blot (Fig. 3). The A151R GST fusion protein was not recognized, although a GST fusion protein of the predicted size of ORF A151R was identified by Coomassie blue staining (Fig. 3
).
Recognition of isolated recombinant viral proteins by antibodies from ASFV-infected domestic pigs and bush pigs
In order to determine which isolated viral subunits may play a key role in protective immunity against the virus, ELISAs were performed with purified recombinant GST fusion proteins. Sensitised ELISA plates were developed with sera from: (1) a pig infected with the Malta isolate of ASFV (this pig was a different animal from the donor of serum MI92 used to screen the library); (2) pigs sequentially infected with non-pathogenic OURT88/3 (day 0) and with OURT88/1 (day 28) and bled 6 days later; and (3) a bush pig that had been infected with the Malawi isolate of the virus and bled 90 days after a secondary challenge with virus.
All of the expressed proteins recognized by serum MI92 from Malta-infected domestic pigs were recognized by ELISA with immune antiserum from a different domestic pig that had also been infected with the Malta isolate of ASFV (Fig. 5). In addition, the small ribonucleotide reductase subunit/F334L was clearly recognized by ELISA with immune antiserum from this Malta-infected pig (Fig. 5
), although it was not identified by Western blot, suggesting that this protein may encode conformational epitope(s) recognized by the antisera used in these experiments.
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As this was a preliminary experiment, quantification and reproducibility of these responses to viral antigens must await a more systematic study. Nonetheless, sera from both domestic pigs and the bush pig studied were very clearly positive by ELISA when tested against the structural immunodeterminants p73/B646L, p32/CP204L and p10/K78R and the non-assigned subunit CP312R. The observation of clearly positive ELISA titres against these subunits in domestic pigs both 28 days after primary infection with the OURT88/3 isolate and 6 days after secondary challenge with the virulent OURT88/1 isolate is consistent with their possible involvement in protective humoral immunity.
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Discussion |
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It is probable that effective elimination of ASFV requires both serological and cellular immunity. Direct evidence consists of: (1) delayed outset of symptoms in infected pigs that have received a transfusion of recovered pig serum (Ruiz-Gonzalvo et al., 1986; Schlafer et al., 1984 ); (2) some success in vaccination with the purified viral structural proteins p54 and p32 (Gomez-Puertas et al., 1998
); (3) demonstration of cytotoxic T cells in ASFV-infected pigs (Martins et al., 1993
; Alonso et al., 1997
; Leitão et al., 1998
; Jenson et al., 2000
); and (4) abrogation of established protective immunity by administration of monoclonal anti-porcine CD8 antibodies in vivo (M. S. Denyer, C. Oura, H. Takamatsu and R. M. E. Parkhouse, unpublished work).
The prime objective achieved in this work is the identification of the principal serological immunodeterminants of ASFV in both the susceptible domestic pig and the resistant wildlife host, the bush pig. This was initially done by antibody screening of a viral cDNA expression library and then confirmed by demonstrating similar reactivities in sera from infected recovered domestic pigs and one infected resistant bush pig. The minor differences observed between the infected bush pig and the domestic pigs may result from differences in virulence and/or immunogenicity of the virus isolates employed.
These results support the notion that there may be a key core of serologically dominant virus immunodeterminants, regardless of the virus isolate (summarized in Table 2), consisting of five structural, three non-structural and six proteins with unassigned functions. It is possible that some of these subunits will prove to play a role in immunity and this is now open to direct experimental testing. Although an effective vaccine relying solely on the stimulation of serological immunity is unlikely to provide effective protection, the definition of protective serological virus immunodeterminants does, nevertheless, provide useful information relevant to the eventual construction of a vaccine.
Clearly, the structural proteins comprising the virus capsid constitute a logical target for protective antibodies and so it may be significant that three of the five structural proteins identified in this study (p32/CP204L, p54/E183L and p73/B646L) have been shown previously to encode important neutralizing epitopes of ASFV (Gomez-Puertas et al., 1996 , 1998
). Our studies also show that the structural proteins bacterial histone-like protein/A104R and the virus DNA-binding protein/K78R are recognized by antisera from recovered domestic pigs and bush pigs.
Three of the isolated cDNAs encoded non-structural proteins: small subunit of ribonucleotide reductase/F334L, thymidine kinase/K196R and DNA ligase/NP419L. Antibodies to non-protective immunodeterminants of pathogens are a common occurrence in infectious diseases and ASFV is unlikely to be different. A direct role for humoral responses against such non-structural subunits of the virus in protective immunity is not immediately apparent. Without definitive evidence, however, we cannot exclude the possibility that some of the viral enzyme subunits we have isolated are incorporated into the virion, especially as there is already evidence for this in the case of the ASFV ubiquitin-conjugating enzyme (Hingamp et al., 1995 ). Immunization with non-structural proteins can protect against virus infection (Lin et al., 1998
). Indeed, in some cases, incorporation of epitopes from non-structural proteins within multicomponent peptide or DNA vaccines has been demonstrated to be beneficial (Cedillo-Barron et al., 2001). In so far as the presence of antibody against these subunits is an indicator of a helper T cell response, responses against these particular immunodeterminants may be of some relevance, since T cells may also contribute to immunity by providing a source of cytokines to help the generation of antibody against protective immunodeterminants and cytotoxic CD8+ T cell generation. Finally, there are two principal routes for stimulation of antibodies to non-structural and/or inaccessible, internal structural virus proteins: (1) the lysis of infected cells releasing soluble virus proteins and virus particles at all stages of assembly and (2) the uptake of apoptotic cells by antigen-presenting cells followed by antigen presentation to the immune system (Albert et al., 1998
; Bellone et al., 1997
).
Interestingly, three of the six serologically dominant immunodeterminants with unassigned function (C44L, CP312R and E184L) have not been identified previously. The other isolated subunits included B602L, shown previously to be recognized by antisera from convalescent pigs (Irusta et al., 1996 ), K145R and K205R (Yañez et al., 1995
). ORFs K205R and K145R encode early and late proteins of the virus, respectively (Yañez et al., 1995
). Viral subunits encoded by ORFs CP312R and K205R were strongly recognized by antisera from immune bush pigs and domestic pigs, raising the possibility that they may be important in the protective antibody response.
In conclusion, these results support the notion that the antibody response against different isolates of ASFV is directed against multiple but shared virus determinants in both the natural bush pig host and domestic pigs and, thus, there may be a key core of serological virus immunodeterminants, regardless of the virus isolate. Which of the viral subunits we have isolated is involved in the serological component of a protective immune response against the virus awaits further study but the list of viral proteins to investigate is relatively short and the relevant experiments are practically feasible. Immunolocalization and protection studies may permit a characterization of the minimum number of viral subunits necessary for incorporation into a multicomponent vaccine. Alternatively, vaccination with a disabled recombinant virus, as has been reported by Moore et al. (1998) , may offer a more successful approach.
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Acknowledgments |
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Footnotes |
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b Present address: Centro de Investigacion y de Estudios Avanzados, IPN, Departamento de Genetica y Biología Molecular, Av. IPN # 2508 Col. Zacatenco 07360, Mexico.
c Present address: Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK.
d || Present address: Gulbenkian Institute for Science, Rua da Quinta Grande 6, Apartado 14 P-2781, Oeiras Codex, Portugal.
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Received 1 November 2001;
accepted 29 January 2002.
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