Centro de Virología Animal, Serrano 669, 1414 Buenos Aires, Argentina1
Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabellón 2, Piso 4, 1428 Buenos Aires, Argentina2
Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junin 956, 1113 Buenos Aires, Argentina3
Author for correspondence: María Franze-Fernández. Fax +54 11 4825 1863. e-mail mtff{at}cevan.sld.ar
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Abstract |
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Introduction |
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Two groups of arenaviruses are currently recognized (Buchmeier et al., 1995 ). The Old World group, which includes among others lymphocytic choriomeningitis virus (LCMV) and Lassa virus (LASV), and the New World (Tacaribe complex) group. The prototype of the New World group, Tacaribe virus (TACV), seems not to be pathogenic, but the group also includes several viruses associated with severe haemorrhagic disease in humans. Junin virus (JUNV) causes Argentine haemorrhagic fever (AHF) (Parodi et al., 1958
), which has been recognized as a major public health problem in certain agricultural areas of Argentina. Machupo virus (MACV), first recognized in 1965 (Johnson et al., 1965
), has caused periodic outbreaks of haemorrhagic fever in Bolivia. In the 1990s, Guanarito virus (GUAV) and Sabia virus (SABV) have emerged as the aetiological agents of severe haemorrhagic fever in Venezuela and Brazil, respectively (Salas et al., 1991
; Coimbra et al., 1994
).
A phylogeny of New World arenaviruses constructed on the basis of N gene sequences indicated that these viruses formed three lineages. TACV and the highly pathogenic JUNV, MACV, GUAV and SABV are all members of one of the lineages, with JUNV, MACV and TACV included in a sublineage (Bowen et al., 1996 ). The phylogeny correlates well with the close antigenic relationships and cross-protection described among JUNV, MACV and TACV (Peters et al., 1987
; Martinez Peralta et al., 1993
).
Most cross-protection studies of New World arenaviruses have been directed towards TACV and JUNV, using guinea pigs and marmosets as experimental animals. Guinea pigs infected with the prototype JUNV XJ strain develop a lethal disease that shares many clinical and pathological features with AHF. Inoculation with a single dose of TACV protects guinea pigs fully against lethal JUNV infection (Peters et al., 1987 ; Weissenbacher et al., 1987
; Martinez Peralta et al., 1993
).
Although a considerable number of studies on cross-protection have been reported, there is so far no information on the nature of the antigens involved. However, this knowledge could have relevance to the design of a vaccine against multiple New World arenaviruses. In order to get an insight into this point, we constructed vaccinia virus recombinants that express the TACV GPC or N proteins and studied both the immunogenic properties and the ability of the recombinants to elicit a protective immune response against lethal challenge with JUNV in guinea pigs. We also constructed a vaccinia virus recombinant that expresses JUNV GPC, and the immunogenicity and protective efficacy of this recombinant were evaluated.
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Methods |
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Homologous recombination by co-transfection of temperature-sensitive (ts7) vaccinia virus-infected CV-1 cells with chimeric vector and wild-type Copenhagen vaccinia virus DNA was carried out according to protocols described by Kieny et al. (1984) . Recombinant viruses carrying the foreign gene in the TK locus of vaccinia virus were selected by three rounds of plaque-purification on HuTK- 143B cells (ATCC) grown in selective medium (DMEM containing 100 µg/ml 5-bromodeoxyuridine; Mackett et al., 1985
). Homogeneity of virus progeny was monitored by a plaqueimmunoperoxidase assay (Avrameas, 1972
). All virus plaques reacted with hyperimmune serum to either TACV or JUNV. The TK- vaccinia virus recombinants that expressed the glycoprotein or the nucleoprotein of TACV were termed VVGTac and VVN, respectively. That expressing JUNV GPC was named VVGJun. Control non-recombinant TK- vaccinia virus was obtained by plaque purification in selective medium of spontaneous TK- mutants generated in transfections with wild-type vaccinia virus DNA. This TK- mutant virus was termed VV.
Radiolabelling, immunoprecipitation and protein gel electrophoresis.
Infected CV-1 cells were labelled for 1 or 4 h (200 µCi/ml) or for 16 h (50 µCi/ml) with Express(35S) (New England Nuclear). Labelled TACV particles were obtained from the supernatant medium of TACV-infected cells labelled for 16 h. The medium was clarified by centrifugation at low speed and virus particles were pelleted through a sucrose cushion (5%, w/v, sucrose; 100 mM NaCl; 1 mM EDTA; 10 mm TrisHCl, pH 7·4) by centrifugation (35000 r.p.m., 3 h) in a Beckman SW 65 rotor. Labelled polypeptides from cell lysates, TACV particles or supernatant medium from JUNV-infected cells were immunoprecipitated as described previously (Iapalucci et al., 1994 ) with the following modifications: incubation with the antisera was for 1 h at 4 °C and the immune complexes were precipitated by incubation for 1 h at 4 °C with Protein ASepharose CL-4B (Sigma). Hyperimmune serum to TACV or to JUNV was raised in rabbits and used at 1:100 dilution. Monospecific sera to recombinant TACV proteins GPC and N, obtained as described previously (Rossi et al., 1996
), were used at 1:50 dilution. Monoclonal antibodies to JUNV glycoproteins were GB03-BE08, GDO1-AG02, QC03-BF11 and ODO1-AA09; those to JUNV N were SA02-BG12 and ICOG-BE10 (Sanchez et al., 1989
). All were obtained from A. Sanchez (Centers for Disease Control, Atlanta, GA, USA) and were used at 1:200 dilution. Analysis of the proteins by SDSPAGE was performed as described previously (López & Franze-Fernández, 1985
). Gels were dried and exposed to BioMax films by using the TranScreen-LE intensifying screen (Kodak).
Virus neutralization assay.
Neutralizing antibodies (NA) against JUNV, TACV and vaccinia viruses in sera were determined by a plaque-reduction assay on Vero cells as described in Candurra et al. (1989) except that, in vaccinia virus-neutralization assays, plaques were developed at day 2. Neutralization titres are expressed as the reciprocal of the serum dilution that resulted in a 50% reduction in the number of plaques with respect to control serum.
Immunization and challenge of guinea pigs with JUNV.
Outbred male guinea pigs weighing 300400 g were inoculated subcutaneously on days 0 and 68 with 1x108 p.f.u. of the vaccinia recombinants or VV. The single inoculation (day 0) with 1x105 p.f.u. TACV was performed intramuscularly. On day 87, animals were challenged with 1x103 p.f.u. JUNV XJ strain by intramuscular inoculation. Clinical signs, weight and mortality were recorded for 30 days after challenge. Blood samples were collected by cardiac puncture.
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Results |
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In order to characterize the TACV-specific proteins, CV-1 cells were infected with the vaccinia virus recombinants or with TACV, cells were pulse-labelled and proteins were immunoprecipitated with TACV-specific immune sera and resolved by gel electrophoresis. As seen in Fig. 2(A), the TACV GPC (molecular mass 69 kDa) and N (68 kDa) proteins migrated very closely when TACV-infected cell lysates were immunoprecipitated with hyperimmune serum to TACV (lane 3) and were identified with monospecific sera (lanes 2 and 4). Vaccinia virus-expressed TACV GPC and N proteins were recognized by hyperimmune serum to TACV and showed identical mobility in gel electrophoresis to that of authentic TACV proteins (lanes 1 and 6). TACV GPC is processed to the structural proteins G1 and G2, which co-migrate and are detected as a diffuse band of between 35 and 39 kDa (Rossi et al., 1996
). In order to compare the processing of recombinant and authentic GPC, TACV- and VVGTac-infected cells were labelled for 16 h prior to immunoprecipitation. Analysis of the proteins showed that GPC from both TACV- and VVGTac-infected cells was processed similarly (Fig. 2B
).
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Immunogenicity of the expressed TACV proteins
The immunogenicity of vaccinia virus-expressed TACV proteins was assessed in guinea pigs. Animals were divided into four groups and were immunized with 108 p.f.u. of either VVN (six animals), VVGTac (10 animals) or VV (six animals) or with 105 p.f.u. TACV (four animals). At 21 and 59 days after infection, all animals that had been immunized with VV or with the vaccinia virus recombinants displayed low but reproducible NA titres to vaccinia virus, indicating that they had all been successfully infected (not shown). At these times post-infection, all animals immunized with VVGTac exhibited NA to TACV, although titres were lower than in TACV infections. None of the animals immunized with TACV or with VVGTac elicited NA to JUNV (Fig. 3). As expected, none of the animals vaccinated with VVN developed NA to TACV (not shown).
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The immunogenicity of the recombinant proteins after secondary immunization was also assessed by immunoprecipitation, with labelled TACV particles as antigen. Analysis was performed with pooled sera, which reflected results obtained with individual samples. Animals inoculated with VVN or TACV displayed antibodies to N (Fig. 4A, lanes 25). Those inoculated with VVGTac or TACV elicited antibodies to the processed glycoproteins G1/G2. The latter were detected after longer exposure of the film (Fig. 4B
, lanes 58). Each of the recombinant viruses appeared to generate amounts of antibody comparable to the TACV infection.
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We also analysed whether VVGTac-inoculated animals that survived JUNV challenge generated specific antibodies to JUNV N protein. To this end, labelled TACV particles were used as antigen for immunoprecipitation, as it is known that antibodies to JUNV N recognize TACV N (Damonte et al., 1986 ; Sanchez et al., 1989
). Analysis of the proteins by gel electrophoresis showed that animals inoculated with VVGTac that had antibodies only to TACV glycoproteins prior to challenge elicited antibodies to N after challenge (Fig. 4 A
and B
, lanes 8 and 9).
Protection of guinea pigs against experimental AHF by vaccinia virus recombinants expressing JUNV glycoproteins
Having established that vaccinia virus expressing TACV glycoprotein but not N induced protection against JUNV challenge, we studied the capacity of vaccinia virus expressing JUNV glycoprotein (VVGJun) to provide protection against experimental AHF. For immunization with VVGJun, we followed a protocol identical to that used previously with vaccinia virus expressing TACV antigens, as this has proved to induce levels of antibodies comparable to those in infection by the virus. Two groups of animals were vaccinated with 1x108 p.f.u. of either VVGJun (18 animals) or VV (six animals) and, 68 days after initial inoculation, animals were boosted with the same doses of the corresponding virus. To demonstrate a serological response to immunization, animals were bled at day 82 after primary inoculation and NA titres to vaccinia virus and to JUNV were determined (Table 1). Titres of antibodies to vaccinia virus revealed that all animals had been successfully infected, with values ranging from 1200 to 5000. The mean titre to vaccinia virus of 2490 after two immunizations with VVGJun compared well with that of 2640 after double immunization with VVGTac (see legend to Fig. 3
). Titres of NA to JUNV were low (values ranged from 13 to 250) or undetectable (four animals of 18). Antibodies specific to JUNV in pre-challenge sera could be detected by immunofluorescence (not shown), but attempts to immunoprecipitate radiolabelled JUNV glycoproteins were unsuccessful (Fig. 4C
, lane 2).
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Discussion |
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In order to evaluate the ability of the recombinant viruses to protect against experimental AHF, we used the guinea pig model under conditions that led to death of all unprotected animals. Of the two recombinant vaccinia viruses tested, that which expressed TACV GPC conferred protection upon 50% of the animals. The recombinant expressing TACV N failed to afford protection.
The level of protection afforded by vaccinia virus expressing TACV GPC is less than the 100% protection found when TACV is used as vaccine. Full protection by whole virus might reflect the optimum presentation of antigens in the virus particle and/or the requirement for other viral proteins. However, at least for LASV, simultaneous inoculation of vaccinia virus recombinants expressing GPC or N has been shown to be less protective than single immunization with each recombinant virus (Morrison et al., 1989 ). It should be remarked that correct presentation of viral antigens seems critical for protection against diseases caused by arenaviruses. This point is highlighted by the success of live-virus vaccines such as TACV and Mopeia virus for protection against JUNV and LASV infections and the failure of killed-virus vaccines (Clegg, 1992
). On the other hand, the protective role played by viral proteins encoded in the L RNA segment should not be overlooked. The p11/Z protein, for instance, has been found in equimolar proportions with G1/G2 in TACV and LCMV particles and might be associated with N and G2 (Salvato, 1993
; Rossi et al., 1996
).
Immunization of guinea pigs with recombinant vaccinia virus expressing the glycoproteins of TACV greatly increased the possibility of survival of experimental AHF in this animal model. Animals immunized with VVGTac generated high levels of NA to TACV but undetectable or very low levels of NA to JUNV. After challenge, the surviving animals developed a strong antibody response to JUNV glycoproteins, as detected by the appearance/increase of NA to JUNV and to JUNV nucleoprotein as assayed by immunoprecipitation. This indicated that guinea pigs survived after an actual infection by the challenge virus.
Several lines of evidence support the notion that, at variance with the Old World arenaviruses, NA can play a role in protection against infection with New World arenaviruses (Peters et al., 1987 ; Weissenbacher et al., 1987
). It might therefore be expected that generation of NA to JUNV by immunization with the homologous glycoprotein should reduce replication of the challenge virus initially, leading to improved protection in comparison with the heterologous TACV glycoprotein. We therefore constructed a recombinant vaccinia virus that expressed the JUNV glycoprotein (VVGJun). Recombinant JUNV glycoprotein was found to resemble authentic JUNV glycoprotein closely, retaining antigenic sites defined by monoclonal NA to JUNV.
Guinea pigs were next immunized with VVGJun by using a protocol identical to that used previously for inoculation with vaccinia virus expressing TACV antigens. When sera collected 82 days after primary inoculation with VVGJun were analysed, a clear difference was noted in antibody response between the guinea pigs immunized with VVGJun and those previously inoculated with VVGTac, as detected both by homologous NA titres (Table 1 and Fig. 3
) and by immunoprecipitation of the corresponding glycoprotein (Fig. 4
).
We have as yet no explanation for the low level of antibodies directed to JUNV glycoproteins in guinea pigs immunized with VVGJun compared with the high efficacy of VVGTac in generating antibodies to TACV glycoproteins. The same vaccinia virus strain and similar constructs were used for generation of each recombinant and the levels of glycoprotein expression were comparable, as detected by immunoprecipitation of lysates from cells infected with VVGJun or VVGTac. Furthermore, infection of guinea pigs with vaccinia virus that expressed either JUNV or TACV glycoproteins was equally successful, as indicated the NA titres to vaccinia virus in each experiment (Table 1 and legend to Fig. 3
). In addition, special attention was devoted to ensuring that the gene sequence of the JUNV GPC molecular clone inserted in vaccinia virus corresponded exactly to that of the JUNV XJ strain used for challenge and in neutralization tests (see Methods). The capacity of VVGJun to generate antibodies to JUNV glycoproteins in guinea pigs could not be compared with that of JUNV XJ strain as animals died before eliciting detectable NA (Weissenbacher et al., 1987
; our own unpublished observations).
Although VVGJun, contrary to our expectations, failed to generate high levels of NA to JUNV, immunization of guinea pigs with this recombinant virus protected 13 of 18 animals against lethal JUNV challenge. The surviving animals, however, were susceptible to JUNV replication, as antibodies to JUNV N protein (not encoded in the recombinant) were detected after challenge. Whether animals immunized with the homologous or the heterologous glycoproteins survived or died appeared to be defined early after challenge, as clinical signs and loss of body weight started at days 79 and death occurred at about day 20, as in the VV-inoculated controls. Meanwhile, those animals that survived JUNV challenge showed none of the symptoms of experimental AHF.
As in vaccination with the heterologous glycoprotein, animals protected with VVGJun developed a significant increase of NA titres to JUNV. It might be that rapid generation of NA due to a B cell-memory response in guinea pigs inoculated with VVGJun or VVGTac provides the critical protective component and/or that a cell-mediated response is involved in protection. The mechanism(s) underlying protection requires further research; however, our data indicate that, whatever the mechanism(s), vaccinia virus recombinants expressing the glycoprotein, whether homologous or heterologous, can protect guinea pigs against lethal JUNV infection.
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Acknowledgments |
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References |
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Received 25 October 1999;
accepted 19 January 2000.