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Vaccine 17 (1999) 2836±2843
Intramuscular inoculation of Sin Nombre hantavirus cDNAs induces cellular and humoral immune responses in BALB/c mice Mausumi Bharadwaj a, C. Richard Lyons b, Ivo A. Wortman a, Brian Hjelle a,c,d,* a
Department of Pathology, University of New Mexico School of Medicine, Albuquerque, NM 87131, USA Department of Internal Medicine, University of New Mexico School of Medicine, Albuquerque, NM 87131, USA c Department of Biology, University of New Mexico, Albuquerque, NM 87131, USA d Department of Molecular Genetics and Microbiology, University of New Mexico School of Medicine, Albuquerque, NM 87131, USA b
Received 4 December 1998; received in revised form 17 February 1999; accepted 19 February 1999
Abstract To examine whether genetic immunization with Sin Nombre (SN) hantavirus genes could elicit immune responses, nine fragments spanning the envelope glycoprotein genes G1 and G2, and the complete N gene were cloned into a CMV expression vector. To ensure representation of all potential epitopes, adjacent fragments of the glycoprotein genes overlapped one another by 100 nucleotides. Vectors containing the gene fragments were inoculated intramuscularly into BALB/c mice and splenocyte proliferation and western blot-detectable antibodies and neutralization titers were determined. The N gene and seven of the nine M segment-derived cDNAs tested produced signi®cant speci®c lymphoproliferative responses, and many of the constructs elicited either neutralizing or western blot-detectable antibodies. These promising results encourage the development of infection models for SN virus that will be capable of detecting protective responses. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Sin Nombre hantavirus immune response; DNA vaccine; Genetic immunization; Neutralizing antibodies; Lymphoproliferative response
1. Introduction Hantaviruses comprise a genus of the family Bunyaviridae. At least 10 distinct members of the genus are linked to human disease [1,2]. Each hantavirus is associated with a speci®c rodent reservoir and is transmitted to man by inhalation [2±6]. Sin Nombre (SN) hantavirus has been associated with an outbreak of hantavirus pulmonary syndrome (HPS) in the southwestern United States in 1993 [7,8]. HPS continue to occur sporadically. More than 230 human cases have been reported in North America, causing approximately 100 deaths (Ref. [7] and Artsob H., personal communication). In the last several years,
* Corresponding author. Tel.: +1-505-272-0624; fax: +1-505-2725186. E-mail address: [email protected] (B. Hjelle)
outbreaks of HPS were reported in several regions of South America [9±12]. The genome of hantaviruses consists of three segments of negative-strand RNA encoding three proteins: the L segment an RNA-dependent RNA polymerase, the M segment an envelope protein that is post-translationally processed into the glycoproteins G1 and G2, and the S segment, a nucleocapsid protein (N) [13]. A vaccinia-based preparation using Hantaan (HTN) virus glycoprotein and N antigens protected hamsters from HTN virus challenge [14]. Immunization with the amino-terminal 118 amino acids of Puumala (PUU) virus N protein was sucient to protect bank voles from PUU virus challenge [15]. Although no animal model has been identi®ed for SN virus, it is likely that an infection model can be developed using its natural reservoir, the deer mouse (Peromyscus maniculatus ) [4]. Establishment of such a model will be necessary to evaluate the ecacy of any vaccine preparation.
0264-410X/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 4 - 4 1 0 X ( 9 9 ) 0 0 0 9 6 - 1
Fig. 1. (a) Schematic representation of the location of cDNA fragments along the SN virus M segment (coding strand). Each fragment is 500 nt long with 100 nt overlapping regions. (b) SDS-PAGE. Coomassie blue stain of the partially puri®ed fusion proteins. Lanes: 1 low range SDSPAGE molecular weight standard (Bio-Rad), A±I recombinant expressed peptides A through I encoded by G1 or G2 gene fragments (see Table 1), N (N) protein, vector (V) pATH 23. (c) Western blot. Immunoblot of fragments D, E, G and N protein probed with sera from mice immunized with the corresponding gene or gene fragment. The most strongly-reactive antisera against glycoprotein fragments D, E and G were used for this ®gure, but the N protein reactivity was typical of the 4 N antibody-positive mice. The trpE protein alone was not reactive (data not shown).
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The technology of genetic immunization oers an opportunity to respond rapidly to protect populations when little more than a portion of DNA sequence is available from the pathogen [16]. Genetic immunization can overcome some, but not all, of the technical challenges inherent in hantavirus vaccine development. These limitations include (1) limited quantities of virus due to relatively poor viral replication and high biohazard; (2) diculties in the production of protein for immunization due to diculties in attaining high levels of expression of recombinant glycoproteins in native conformation; (3) the lack of an animal model for disease to test viral attenuation and protection. To develop a model system for rational design of DNA vaccines against pathogens with poor growth characteristics, we have prepared a series of overlapping deletion constructs that encode all or part of the N, G1 and G2 proteins of SN virus. Since previous work had shown the full length hantavirus glycoproteins are dicult to express in eukaryotic cells (M. Bharadwaj et al., unpublished), we elected to use smaller fragments of the M genome in the hope of overcoming the problem. Thus, 500-nucleotide (nt) segments representing the M genome of SN virus were cloned into a CMV expression vector. Each segment had a 100-nt overlap with the adjacent segment. The complete N gene (1287 nt) was also inserted into the CMV vector. BALB/c (n=5) mice were immunized intramuscularly with the constructs separately and lymphoproliferative responses were characterized by splenocyte proliferation in the presence of recombinant-expressed peptide. Antibody responses were also examined by western blot analysis. Finally, the immunized mouse serum samples were tested for neutralizing antibody against SN virus by focus reduction neutralization test (FRNT). 2. Methods 2.1. Construction of recombinant plasmids Fig. 1(a) shows a schematic representation of the fragments of the genes G1 and G2 for immunization. cDNAs of SN virus M segment were generated from RNA isolated from virus-infected (strain CC107, generously provided by C. Schmaljohn) Vero E6 cell cultures (ATCC CRL1586) by using the reverse transcription-polymerase chain reaction (RT-PCR) [17]. The N gene construct was prepared from a preexisting full-length clone derived from 3H226 patient lung RNA [18]. This variant diers from that of CC107 by <1% at the amino acid level. Restriction sites for Pst I and Xba I were incorporated into the 5 ' and 3' ends of G1 and G2 primers, and Hind III and Xba I sites were incorporated into the N gene primers
to facilitate cloning into the pCMVi (±H3) UBs vector (provided by S. Johnston). The pCMVi (±H3) UBs uses the human CMV IE promoter to drive expression of the insert. All 5 ' primers intended for eukaryotic expression incorporated the three native upstream nucleotides of the G1 gene (coordinates 49±51 of GenBank accession L33684) along with an ATG initiation codon (i.e., the sequence AGAATG). For insertion into the prokaryote expression vector pATH 23, Sal I/Xho I (G1) or Bam HI/Sal I (G2) were used for ampli®cation followed by cloning into the vector [19]. The primer sequences are available upon request. The N gene had previously been cloned into another bacterial expression system, pET 23b (Novagen, Madison, WI) using Hind III and Xho I sites [21]. The viral antigens were expressed in fusion with bacterial leader sequences derived from trp E (pATH 23, 37 kDa) or T7 gene 10 (pET 23b, 3 kDa). The M gene 500 nucleotide segments were designated A through E (G1 gene) and F through I (G2 gene) (Fig. 1(a)). Unlike the pET 23b system used to express the N gene, the pATH 23 system does not allow anity puri®cation of the expressed fusion proteins. 2.2. Puri®cation of fusion proteins After expression in E. coli, the G1 and G2 protein segments were partially puri®ed from preparative SDSpolyacrylamide gels after electrophoretic separation. The gel fragments were excised with a razor blade and agitated in 10 mM tris±HCl pH 8.0 containing 0.1% sodium dodecyl sulfate (SDS) 8±12 h [20] and ®nally the proteins were concentrated and detergents removed with centricon-30 ®lters according to the manufacturer's instructions (Amicon/Millipore). The N fusion protein was puri®ed by nickel-chelating column according to the manufacturer's instructions (Novagen, Madison, WI) [21]. Finally the protein concentrations were determined by the method of Bradford and the protein purity was examined by SDS-PAGE with coomassie brilliant blue R-250 staining (Fig. 1(b)) [22,23]. 2.3. Genetic immunization Puri®cation of DNA was carried out by standard techniques with an endotoxin-free kit (EndoFree, Qiagen, Valencia, CA). DNA was dissolved in 0.9% saline without adjuvant, at a concentration of 1 mg/ ml. BALB/c mice (6±8 week old) were immunized intramuscularly three times at 4 week intervals, with 50 mg of plasmid into each set of quadriceps muscles (total 100 mg). For each construct ®ve mice were immunized.
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Fig. 2. Dose±response pro®le of lymphocytes in presence of recombinant-expressed partially puri®ed glycoprotein peptides as well as N protein. The lanes V, A, B, C, D, E, F, G, H, I and N used lymphocytes from mice immunized with vector alone (V), glycoprotein gene fragments A, B, C, D, E, F, G, H, or I, or the nucleocapsid gene (N) that were stimulated in vitro with cognate antigen. The stimulation index of Con A-stimulated control was 56.8, p < 0.01 (data not shown). p values were <0.05 for A, B, E, and F; <0.03 for C and G; and <0.01 for N relative to vector control. The error bars represent standard deviations from the mean.
2.4. Cellular proliferation assay The mice were sacri®ced two weeks after the third injection and spleens were collected. The spleens were transferred to 100 mm sterile tissue culture dishes containing RPMI medium with 10% fetal bovine serum and rubbed in between frosted microscope slides to disperse cells. Cell suspensions were treated with erythrocyte lysing solution (0.155 M NH4Cl, 0.001 M KHCO3, 0.1 M NaEDTA pH 7.4) for 2 min and then centrifuged for 10 min at 200 g. The cell pellet was resuspended in the medium and was used as the source of splenocytes [24]. For proliferation assays, 3 105 splenocytes/well were aliquoted in 96-well u-bottom plates in triplicate. Extra aliquots were frozen in cryopreservation medium containing 10% DMSO and stored in liquid nitrogen. Recombinant peptides were added to the cells at a ®nal concentration of 10, 20 and 50 mg per ml. Cells were allowed to proliferate for 5 d. Eighteen h before harvesting 1 mCi of methyl 3 Hthymidine was added per well. Cultures were harvested with a Cambridge Technology cell harvester and radioactivity incorporated into DNA was measured by scintillation counting (Beckman LS 6500). To assure that cells were viable with proliferative potential, 10 mg/ml of Concanavalin A was used as a polyclonal stimulator. The stimulation index was determined from the formula: stimulation index=(experimental count ÿ spontaneous count)/spontaneous count [25]. Note that this formula for stimulation index produces a lower
value than those that do not subtract the spontaneous count in the numerator. 2.5. Western blot analysis We used 0.8±1 mg of gel puri®ed G1 and G2 protein segments or anity-puri®ed N fusion protein per lane to make western blots using Mini-Protean gels as described previously [19,21] (Bio-Rad, Richmond, CA). Mouse serum samples were incubated at a 1:40 dilution (glycoprotein fragments) or 1:500 (N-protein) for 16 h with western blot strips. Antigen±antibody complexes were detected by incubating the blots with alkaline phosphatase-conjugated goat anti-mouse IgG antiserum (Boehringer Mannheim) at a 1:1000 dilution for 4 h. Alkaline phosphatase activity was detected with an alkaline buer containing nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate [19]. 2.6. Focus reduction neutralization test (FRNT) FRNT is used widely for hantaviruses instead of the plaque reduction neutralization test since hantaviruses plaque somewhat inconsistently [26,27]. The serum samples from immunized mice were examined by FRNT individually in 48 well tissue culture plates. Samples were serially diluted (1:10, 1:20, 1:50, 1:100) and mixed with equal volumes of approximately 45 u of SN virus (strain CC107) for 1 h at 378C before incubation on Vero E6 cells. After adsorption for 4 h at
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Table 1 Immunization with Sin Nombre virus cDNA constructsa; p < 0.05; Gene
G1
G2
N
Immunization construct
A B C D E F G H I N
Genetic and amino acid coordinate
49±549/1±167 448±948/132±299 847±1347/265±432 1246±1746/399±565 1507±2007/486±652 2008±2508/1±167 2392±2907/134±300 2509±3006/266±433 2902±3474/400±488 43±1330/1±428
p < 0.03;
p < 0.01
Splenocyte proliferation with:
Number of mice antibody-positive (Western blot)
Cognate antigen (CPM2S.D.)
Other M segment antigen (CPM2S.D.)/ control peptide
CMV vector only (CPM2S.D.)
62872521 67012478 652522283 17035218267 66232181 68562739 895721473 534822087 478721008 32,02828367
30212574/eb 28792619/f 24132448/g 48422465/h 29452245/a 31022553/b 25632844/c 48562439/e 45782249/a 37742731/g
27292374c 26982397 20482266 31562683 25032172 29282298 22892442 335121232 30172729 25372556
2 0 2 4 2 2 3 0 0 4
Number of mice FRNTpositive 1:10
1:20
5/5 0 2/5 5/5 3/5 4/5 5/5 0 0 0
3/5 0 0 2/5 0/5 0/5 2/5 0 0 0
a
Results shown here are the mean of ®ve mice per group, each assayed in triplicate. The lower-case letter refers to the heterologous glycoprotein peptide that was used as a control antigen in the proliferation assay. c CMV immunized splenocytes were stimulated with the indicated peptide antigen.
b
378C, the cells were overlaid with media containing 1.2% methyl cellulose for 7 d. The methyl cellulose layer was removed and the cells were ®xed with methanol containing 0.5% hydrogen peroxide [28]. Viral antigen was visualized by addition of rabbit anti-SN virus nucleocapsid protein serum followed by peroxidase-conjugated goat anti-rabbit IgG and DAB/metal concentrate as substrate (Pierce). The neutralization activity of an antibody was expressed as the serum dilution necessary to reduce the number of foci by 50%. 2.7. Statistical analysis Unpaired data were analyzed with a 2-way ANOVA test, using Statview statistical software on a Macintosh PowerPC computer. 3. Results 3.1. Cellular proliferation Two weeks after the last booster inoculation, spleens were collected from the immunized as well as control (vector-DNA immunized or unimmunized) mice and single cell suspensions were prepared. The single cell suspensions were incubated in the presence or absence of their cognate antigens and examined for proliferative responses. Initially, three dierent antigen concentrations (10, 20 and 50 mg/ml) were tested to determine the optimal dose for proliferation assays. In most cases 10 mg/ml of the speci®c antigen produced the strongest proliferative response (Fig. 2). A total of 10
mg/ml of Con A was used as a positive control for stimulation. As shown in Fig. 2, most of the glycoprotein fragments, particularly A, C, E and G, showed signi®cantly higher stimulation (SI 1 6.8±12.5, p < 0.03±0.05) relative to the vector control. For fragment D cDNA-immunized mice, two out of ®ve showed dramatically higher proliferation than the remaining three. In the presence of N protein, N cDNA immunized mouse lymphocytes showed the highest proliferation overall, with a stimulation index of 15.9, p < 0.01. We wished to establish that the observed proliferation responses were antigen-speci®c and were not caused by contaminating E. coli antigens in the viral antigen preparations. Since all recombinant M segment peptides were of approximately the same molecular mass and were produced in an identical fashion, we used a nonoverlapping M segment peptide as a speci®city control in proliferative assays. For this con®rmatory testing we repeated the proliferation assays using exclusively cryopreserved splenocytes. We found that the use of cryopreserved cells was acceptable but resulted in 2±3-fold reductions in the stimulation indices and Con A responses. Nevertheless, the N gene and each of seven M segment-derived cDNAs produced signi®cant speci®c T cell responses (Table 1). In the presence of N antigen, splenocytes from mice immunized with the N gene cDNA proliferated almost nine times as well as did cells from mice immunized with an M segment derived cDNA ( p < 0.01). Stimulation of splenocytes with the peptide encoded by a given immunizing glycoprotein gene segment produced in several cases a 02±3-fold stimulation compared with the same splenocytes exposed to another
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nonoverlapping M segment peptide or cells from mice immunized with CMV vector alone. For example, in the presence of fragment B peptide (G1 amino acids 132±299), from mice immunized with segment B cDNA, splenocytes proliferated about 2.4 times higher than did splenocytes from mice immunized with F cDNA (G2 aa 1±167). As with the non-cryopreserved splenocytes, cryopreserved cells from mice immunized with fragment D cDNA (G1 aa 399±565), three mice had little or no proliferative responses (mean 5893.532 482.9) but two other showed 19,000 CPM and 47,921 CPM ( p>0.3 for overall stimulation compared to controls). Two antigen preparations elicited small, apparently nonspeci®c responses: splenocytes from mice immunized with gene segment H proliferated in response to E antigen, and I-segment-immunized splenocytes proliferated in response to A antigen (Table 1). 3.2. Western blot analysis Antibody responses of the cDNA-immunized mice were evaluated by western blot analysis. Of the 50 immunized mice tested nineteen mice produced detectable antibody responses against A, C, D, E, F, or G peptides as well as against N protein (Table 1, Fig. 1). Fig. 1(b) showed the partially puri®ed Coomassiestained fusion proteins (fragments A through I, and N protein). Note that the 428-aa N antigen migrates at the same position as do the glycoprotein antigens because the N antigen has only approximately 3 kDa of ¯anking fusion protein compared to 37 kDa for the glycoprotein antigens. Fig. 2(c) showed selected western blots using mouse sera that showed positive antibody responses. All responses against M-segment antigens were dicult to detect, but the four mice that produced antibodies to N protein had easily detectable responses even at 1:1500 serum dilutions (data not shown). The trpE protein alone did not react with any of the immunized mouse sera (data not shown). 3.3. Focus reduction neutralization test The 50 mouse serum samples were tested individually for their neutralization activities against SN virus (strain CC 107). Sera from most mice immunized against peptides A, C, D, E, F and G cDNAs showed neutralization activity at a 10-fold serum dilution. Serum from only two mice of ®ve immunized with fragment C cDNA immunized showed signi®cant neutralization activity against SN virus. Three mice genetically immunized against peptide A and two mice immunized against peptide G had 50% neutralizing activity at a 20-fold dilution Table 1. Two mice immunized with the D glycoprotein gene construct showed both exceptionally high lymphoproliferation and higher (20-fold dilution) neutralization titer than did
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other mice immunized with the same construct Table 1. Immunization with the N gene did not produce any neutralizing antibodies. 4. Discussion Vaccination of BALB/c mice with Sin Nombre viral DNA resulted in speci®c immune responses in some mice. G1, G2 and N cDNA fragments induced speci®c lymphoproliferative responses in the presence of their cognate antigens. Fragments C and D elicited the highest speci®c proliferative responses of the envelope gene fragments, although three mice inoculated with the D gene fragment apparently did not generate any immune response. The N gene cDNA elicited stronger proliferative and antibody responses than did any glycoprotein fragment but as has been noted previously, such antibodies were not neutralizing [14,15]. Several envelope fragments induced antibody responses against the cognate antigens, albeit inconsistently. As might be expected with a strategy that uses small portions of genes, the neutralization responses to the glycoprotein antigens were of modest titer. We believe that selected high-risk groups could bene®t by development of a vaccine against SN virus. These include certain Native American groups, forestry and agricultural workers, and ®eld biologists. Currently there is no infection or disease model available to evaluate the ecacy of vaccine preparations for SN virus. Pending the development of such animal models, it is helpful to determine whether BALB/c (Mus musculus ) mice could be used to screen genetic vaccine candidates for SN virus. We showed here that most of the glycoprotein fragments studied induced antibodies that can neutralize SN virus at modest dilution. We choose to introduce no non-native secretory signal upstream of our gene fragments, because viral peptides produced by the transfected tissues may be expressed as antigens at least in the class I pathway even when the peptides lack such signals [29,33]. Choi et al. showed that in case of rotavirus capsid protein VP7-containing plasmid gene immunized mice, had no dierence in immune responses regardless of the presence of a secretory signal peptide [30]. Several recent reports indicate immunization that resulted in both cellular proliferation and antibody production with or without neutralizing antibody can be associated with protection from virus challenge [31,32]. In Aujeszky's disease, the glycoprotein construct gC elicited very low virus-neutralizing activity, but gave the greatest protection against virus challenge. In contrast, a gD gene construct gave no protection, even though neutralizing activity was demonstrable [31]. Our tests did not discern which population(s) of lymphocytes were actually
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stimulated to proliferate. Nevertheless, glycoprotein cDNA fragments A, C, D, F and G, which induced speci®c splenocyte proliferation and neutralizing activity may be candidates for further study. Nucleocapsid gene immunization induced good lymphoproliferative responses to the N antigen as well as a strong non-neutralizing antibody response. Such responses may nevertheless result in protection from challenge, at least when administered as exogenous peptide [15]. Although Mus musculus mice such as BALB/c are not the natural carriers of SN virus, these inbred mice help de®ne possibly important epitopes of the viral antigens and allow us to preliminarily characterize cellular and humoral responses where no eective animal models exist. At present, little is known about the nature of protective immunity to hantaviruses, and the relative roles of cell-mediated immunity and antibody responses in such protection. We believe that the demonstration of detectable immune responses elicited by naked DNA vaccines in the BALB/c system oers hope that such vaccine candidates may also elicit protective responses in an infection model. These data thus should encourage the development of such models using the deer mouse (P. maniculatus ). We recently established a deer mouse colony for that purpose.
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Acknowledgements We thank S. Johnston for providing valuable technical advice, the pCMVi (±H3) UBs vector and unpublished data; C. Schmaljohn for providing the CC107 virus and helpful discussions; H. Artsob for providing unpublished data, and the UNM protein chemistry laboratory for technical assistance. This work was supported by the Department of Defense Advanced Projects Agency (DARPA) to CRL and S. Johnston and Public Health Services grants RO1 AI 41892 and RO1 AI 36336 to BH (National Institute of Allergy and Infectious Diseases). CRL is a Charles E. Culpeper Foundation Medical Scholar and the work was supported in part by the Charles E. Culpeper Foundation.
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Intramuscular inoculation of Sin Nombre hantavirus cDNAs induces cellular and humoral immune responses in BALB/c mice Mausumi Bharadwaj a, C. Richard Lyons b, Ivo A. Wortman a, Brian Hjelle a,c,d,* a
Department of Pathology, University of New Mexico School of Medicine, Albuquerque, NM 87131, USA Department of Internal Medicine, University of New Mexico School of Medicine, Albuquerque, NM 87131, USA c Department of Biology, University of New Mexico, Albuquerque, NM 87131, USA d Department of Molecular Genetics and Microbiology, University of New Mexico School of Medicine, Albuquerque, NM 87131, USA b
Received 4 December 1998; received in revised form 17 February 1999; accepted 19 February 1999
Abstract To examine whether genetic immunization with Sin Nombre (SN) hantavirus genes could elicit immune responses, nine fragments spanning the envelope glycoprotein genes G1 and G2, and the complete N gene were cloned into a CMV expression vector. To ensure representation of all potential epitopes, adjacent fragments of the glycoprotein genes overlapped one another by 100 nucleotides. Vectors containing the gene fragments were inoculated intramuscularly into BALB/c mice and splenocyte proliferation and western blot-detectable antibodies and neutralization titers were determined. The N gene and seven of the nine M segment-derived cDNAs tested produced signi®cant speci®c lymphoproliferative responses, and many of the constructs elicited either neutralizing or western blot-detectable antibodies. These promising results encourage the development of infection models for SN virus that will be capable of detecting protective responses. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Sin Nombre hantavirus immune response; DNA vaccine; Genetic immunization; Neutralizing antibodies; Lymphoproliferative response
1. Introduction Hantaviruses comprise a genus of the family Bunyaviridae. At least 10 distinct members of the genus are linked to human disease [1,2]. Each hantavirus is associated with a speci®c rodent reservoir and is transmitted to man by inhalation [2±6]. Sin Nombre (SN) hantavirus has been associated with an outbreak of hantavirus pulmonary syndrome (HPS) in the southwestern United States in 1993 [7,8]. HPS continue to occur sporadically. More than 230 human cases have been reported in North America, causing approximately 100 deaths (Ref. [7] and Artsob H., personal communication). In the last several years,
* Corresponding author. Tel.: +1-505-272-0624; fax: +1-505-2725186. E-mail address: [email protected] (B. Hjelle)
outbreaks of HPS were reported in several regions of South America [9±12]. The genome of hantaviruses consists of three segments of negative-strand RNA encoding three proteins: the L segment an RNA-dependent RNA polymerase, the M segment an envelope protein that is post-translationally processed into the glycoproteins G1 and G2, and the S segment, a nucleocapsid protein (N) [13]. A vaccinia-based preparation using Hantaan (HTN) virus glycoprotein and N antigens protected hamsters from HTN virus challenge [14]. Immunization with the amino-terminal 118 amino acids of Puumala (PUU) virus N protein was sucient to protect bank voles from PUU virus challenge [15]. Although no animal model has been identi®ed for SN virus, it is likely that an infection model can be developed using its natural reservoir, the deer mouse (Peromyscus maniculatus ) [4]. Establishment of such a model will be necessary to evaluate the ecacy of any vaccine preparation.
0264-410X/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 4 - 4 1 0 X ( 9 9 ) 0 0 0 9 6 - 1
Fig. 1. (a) Schematic representation of the location of cDNA fragments along the SN virus M segment (coding strand). Each fragment is 500 nt long with 100 nt overlapping regions. (b) SDS-PAGE. Coomassie blue stain of the partially puri®ed fusion proteins. Lanes: 1 low range SDSPAGE molecular weight standard (Bio-Rad), A±I recombinant expressed peptides A through I encoded by G1 or G2 gene fragments (see Table 1), N (N) protein, vector (V) pATH 23. (c) Western blot. Immunoblot of fragments D, E, G and N protein probed with sera from mice immunized with the corresponding gene or gene fragment. The most strongly-reactive antisera against glycoprotein fragments D, E and G were used for this ®gure, but the N protein reactivity was typical of the 4 N antibody-positive mice. The trpE protein alone was not reactive (data not shown).
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The technology of genetic immunization oers an opportunity to respond rapidly to protect populations when little more than a portion of DNA sequence is available from the pathogen [16]. Genetic immunization can overcome some, but not all, of the technical challenges inherent in hantavirus vaccine development. These limitations include (1) limited quantities of virus due to relatively poor viral replication and high biohazard; (2) diculties in the production of protein for immunization due to diculties in attaining high levels of expression of recombinant glycoproteins in native conformation; (3) the lack of an animal model for disease to test viral attenuation and protection. To develop a model system for rational design of DNA vaccines against pathogens with poor growth characteristics, we have prepared a series of overlapping deletion constructs that encode all or part of the N, G1 and G2 proteins of SN virus. Since previous work had shown the full length hantavirus glycoproteins are dicult to express in eukaryotic cells (M. Bharadwaj et al., unpublished), we elected to use smaller fragments of the M genome in the hope of overcoming the problem. Thus, 500-nucleotide (nt) segments representing the M genome of SN virus were cloned into a CMV expression vector. Each segment had a 100-nt overlap with the adjacent segment. The complete N gene (1287 nt) was also inserted into the CMV vector. BALB/c (n=5) mice were immunized intramuscularly with the constructs separately and lymphoproliferative responses were characterized by splenocyte proliferation in the presence of recombinant-expressed peptide. Antibody responses were also examined by western blot analysis. Finally, the immunized mouse serum samples were tested for neutralizing antibody against SN virus by focus reduction neutralization test (FRNT). 2. Methods 2.1. Construction of recombinant plasmids Fig. 1(a) shows a schematic representation of the fragments of the genes G1 and G2 for immunization. cDNAs of SN virus M segment were generated from RNA isolated from virus-infected (strain CC107, generously provided by C. Schmaljohn) Vero E6 cell cultures (ATCC CRL1586) by using the reverse transcription-polymerase chain reaction (RT-PCR) [17]. The N gene construct was prepared from a preexisting full-length clone derived from 3H226 patient lung RNA [18]. This variant diers from that of CC107 by <1% at the amino acid level. Restriction sites for Pst I and Xba I were incorporated into the 5 ' and 3' ends of G1 and G2 primers, and Hind III and Xba I sites were incorporated into the N gene primers
to facilitate cloning into the pCMVi (±H3) UBs vector (provided by S. Johnston). The pCMVi (±H3) UBs uses the human CMV IE promoter to drive expression of the insert. All 5 ' primers intended for eukaryotic expression incorporated the three native upstream nucleotides of the G1 gene (coordinates 49±51 of GenBank accession L33684) along with an ATG initiation codon (i.e., the sequence AGAATG). For insertion into the prokaryote expression vector pATH 23, Sal I/Xho I (G1) or Bam HI/Sal I (G2) were used for ampli®cation followed by cloning into the vector [19]. The primer sequences are available upon request. The N gene had previously been cloned into another bacterial expression system, pET 23b (Novagen, Madison, WI) using Hind III and Xho I sites [21]. The viral antigens were expressed in fusion with bacterial leader sequences derived from trp E (pATH 23, 37 kDa) or T7 gene 10 (pET 23b, 3 kDa). The M gene 500 nucleotide segments were designated A through E (G1 gene) and F through I (G2 gene) (Fig. 1(a)). Unlike the pET 23b system used to express the N gene, the pATH 23 system does not allow anity puri®cation of the expressed fusion proteins. 2.2. Puri®cation of fusion proteins After expression in E. coli, the G1 and G2 protein segments were partially puri®ed from preparative SDSpolyacrylamide gels after electrophoretic separation. The gel fragments were excised with a razor blade and agitated in 10 mM tris±HCl pH 8.0 containing 0.1% sodium dodecyl sulfate (SDS) 8±12 h [20] and ®nally the proteins were concentrated and detergents removed with centricon-30 ®lters according to the manufacturer's instructions (Amicon/Millipore). The N fusion protein was puri®ed by nickel-chelating column according to the manufacturer's instructions (Novagen, Madison, WI) [21]. Finally the protein concentrations were determined by the method of Bradford and the protein purity was examined by SDS-PAGE with coomassie brilliant blue R-250 staining (Fig. 1(b)) [22,23]. 2.3. Genetic immunization Puri®cation of DNA was carried out by standard techniques with an endotoxin-free kit (EndoFree, Qiagen, Valencia, CA). DNA was dissolved in 0.9% saline without adjuvant, at a concentration of 1 mg/ ml. BALB/c mice (6±8 week old) were immunized intramuscularly three times at 4 week intervals, with 50 mg of plasmid into each set of quadriceps muscles (total 100 mg). For each construct ®ve mice were immunized.
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Fig. 2. Dose±response pro®le of lymphocytes in presence of recombinant-expressed partially puri®ed glycoprotein peptides as well as N protein. The lanes V, A, B, C, D, E, F, G, H, I and N used lymphocytes from mice immunized with vector alone (V), glycoprotein gene fragments A, B, C, D, E, F, G, H, or I, or the nucleocapsid gene (N) that were stimulated in vitro with cognate antigen. The stimulation index of Con A-stimulated control was 56.8, p < 0.01 (data not shown). p values were <0.05 for A, B, E, and F; <0.03 for C and G; and <0.01 for N relative to vector control. The error bars represent standard deviations from the mean.
2.4. Cellular proliferation assay The mice were sacri®ced two weeks after the third injection and spleens were collected. The spleens were transferred to 100 mm sterile tissue culture dishes containing RPMI medium with 10% fetal bovine serum and rubbed in between frosted microscope slides to disperse cells. Cell suspensions were treated with erythrocyte lysing solution (0.155 M NH4Cl, 0.001 M KHCO3, 0.1 M NaEDTA pH 7.4) for 2 min and then centrifuged for 10 min at 200 g. The cell pellet was resuspended in the medium and was used as the source of splenocytes [24]. For proliferation assays, 3 105 splenocytes/well were aliquoted in 96-well u-bottom plates in triplicate. Extra aliquots were frozen in cryopreservation medium containing 10% DMSO and stored in liquid nitrogen. Recombinant peptides were added to the cells at a ®nal concentration of 10, 20 and 50 mg per ml. Cells were allowed to proliferate for 5 d. Eighteen h before harvesting 1 mCi of methyl 3 Hthymidine was added per well. Cultures were harvested with a Cambridge Technology cell harvester and radioactivity incorporated into DNA was measured by scintillation counting (Beckman LS 6500). To assure that cells were viable with proliferative potential, 10 mg/ml of Concanavalin A was used as a polyclonal stimulator. The stimulation index was determined from the formula: stimulation index=(experimental count ÿ spontaneous count)/spontaneous count [25]. Note that this formula for stimulation index produces a lower
value than those that do not subtract the spontaneous count in the numerator. 2.5. Western blot analysis We used 0.8±1 mg of gel puri®ed G1 and G2 protein segments or anity-puri®ed N fusion protein per lane to make western blots using Mini-Protean gels as described previously [19,21] (Bio-Rad, Richmond, CA). Mouse serum samples were incubated at a 1:40 dilution (glycoprotein fragments) or 1:500 (N-protein) for 16 h with western blot strips. Antigen±antibody complexes were detected by incubating the blots with alkaline phosphatase-conjugated goat anti-mouse IgG antiserum (Boehringer Mannheim) at a 1:1000 dilution for 4 h. Alkaline phosphatase activity was detected with an alkaline buer containing nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate [19]. 2.6. Focus reduction neutralization test (FRNT) FRNT is used widely for hantaviruses instead of the plaque reduction neutralization test since hantaviruses plaque somewhat inconsistently [26,27]. The serum samples from immunized mice were examined by FRNT individually in 48 well tissue culture plates. Samples were serially diluted (1:10, 1:20, 1:50, 1:100) and mixed with equal volumes of approximately 45 u of SN virus (strain CC107) for 1 h at 378C before incubation on Vero E6 cells. After adsorption for 4 h at
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Table 1 Immunization with Sin Nombre virus cDNA constructsa; p < 0.05; Gene
G1
G2
N
Immunization construct
A B C D E F G H I N
Genetic and amino acid coordinate
49±549/1±167 448±948/132±299 847±1347/265±432 1246±1746/399±565 1507±2007/486±652 2008±2508/1±167 2392±2907/134±300 2509±3006/266±433 2902±3474/400±488 43±1330/1±428
p < 0.03;
p < 0.01
Splenocyte proliferation with:
Number of mice antibody-positive (Western blot)
Cognate antigen (CPM2S.D.)
Other M segment antigen (CPM2S.D.)/ control peptide
CMV vector only (CPM2S.D.)
62872521 67012478 652522283 17035218267 66232181 68562739 895721473 534822087 478721008 32,02828367
30212574/eb 28792619/f 24132448/g 48422465/h 29452245/a 31022553/b 25632844/c 48562439/e 45782249/a 37742731/g
27292374c 26982397 20482266 31562683 25032172 29282298 22892442 335121232 30172729 25372556
2 0 2 4 2 2 3 0 0 4
Number of mice FRNTpositive 1:10
1:20
5/5 0 2/5 5/5 3/5 4/5 5/5 0 0 0
3/5 0 0 2/5 0/5 0/5 2/5 0 0 0
a
Results shown here are the mean of ®ve mice per group, each assayed in triplicate. The lower-case letter refers to the heterologous glycoprotein peptide that was used as a control antigen in the proliferation assay. c CMV immunized splenocytes were stimulated with the indicated peptide antigen.
b
378C, the cells were overlaid with media containing 1.2% methyl cellulose for 7 d. The methyl cellulose layer was removed and the cells were ®xed with methanol containing 0.5% hydrogen peroxide [28]. Viral antigen was visualized by addition of rabbit anti-SN virus nucleocapsid protein serum followed by peroxidase-conjugated goat anti-rabbit IgG and DAB/metal concentrate as substrate (Pierce). The neutralization activity of an antibody was expressed as the serum dilution necessary to reduce the number of foci by 50%. 2.7. Statistical analysis Unpaired data were analyzed with a 2-way ANOVA test, using Statview statistical software on a Macintosh PowerPC computer. 3. Results 3.1. Cellular proliferation Two weeks after the last booster inoculation, spleens were collected from the immunized as well as control (vector-DNA immunized or unimmunized) mice and single cell suspensions were prepared. The single cell suspensions were incubated in the presence or absence of their cognate antigens and examined for proliferative responses. Initially, three dierent antigen concentrations (10, 20 and 50 mg/ml) were tested to determine the optimal dose for proliferation assays. In most cases 10 mg/ml of the speci®c antigen produced the strongest proliferative response (Fig. 2). A total of 10
mg/ml of Con A was used as a positive control for stimulation. As shown in Fig. 2, most of the glycoprotein fragments, particularly A, C, E and G, showed signi®cantly higher stimulation (SI 1 6.8±12.5, p < 0.03±0.05) relative to the vector control. For fragment D cDNA-immunized mice, two out of ®ve showed dramatically higher proliferation than the remaining three. In the presence of N protein, N cDNA immunized mouse lymphocytes showed the highest proliferation overall, with a stimulation index of 15.9, p < 0.01. We wished to establish that the observed proliferation responses were antigen-speci®c and were not caused by contaminating E. coli antigens in the viral antigen preparations. Since all recombinant M segment peptides were of approximately the same molecular mass and were produced in an identical fashion, we used a nonoverlapping M segment peptide as a speci®city control in proliferative assays. For this con®rmatory testing we repeated the proliferation assays using exclusively cryopreserved splenocytes. We found that the use of cryopreserved cells was acceptable but resulted in 2±3-fold reductions in the stimulation indices and Con A responses. Nevertheless, the N gene and each of seven M segment-derived cDNAs produced signi®cant speci®c T cell responses (Table 1). In the presence of N antigen, splenocytes from mice immunized with the N gene cDNA proliferated almost nine times as well as did cells from mice immunized with an M segment derived cDNA ( p < 0.01). Stimulation of splenocytes with the peptide encoded by a given immunizing glycoprotein gene segment produced in several cases a 02±3-fold stimulation compared with the same splenocytes exposed to another
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nonoverlapping M segment peptide or cells from mice immunized with CMV vector alone. For example, in the presence of fragment B peptide (G1 amino acids 132±299), from mice immunized with segment B cDNA, splenocytes proliferated about 2.4 times higher than did splenocytes from mice immunized with F cDNA (G2 aa 1±167). As with the non-cryopreserved splenocytes, cryopreserved cells from mice immunized with fragment D cDNA (G1 aa 399±565), three mice had little or no proliferative responses (mean 5893.532 482.9) but two other showed 19,000 CPM and 47,921 CPM ( p>0.3 for overall stimulation compared to controls). Two antigen preparations elicited small, apparently nonspeci®c responses: splenocytes from mice immunized with gene segment H proliferated in response to E antigen, and I-segment-immunized splenocytes proliferated in response to A antigen (Table 1). 3.2. Western blot analysis Antibody responses of the cDNA-immunized mice were evaluated by western blot analysis. Of the 50 immunized mice tested nineteen mice produced detectable antibody responses against A, C, D, E, F, or G peptides as well as against N protein (Table 1, Fig. 1). Fig. 1(b) showed the partially puri®ed Coomassiestained fusion proteins (fragments A through I, and N protein). Note that the 428-aa N antigen migrates at the same position as do the glycoprotein antigens because the N antigen has only approximately 3 kDa of ¯anking fusion protein compared to 37 kDa for the glycoprotein antigens. Fig. 2(c) showed selected western blots using mouse sera that showed positive antibody responses. All responses against M-segment antigens were dicult to detect, but the four mice that produced antibodies to N protein had easily detectable responses even at 1:1500 serum dilutions (data not shown). The trpE protein alone did not react with any of the immunized mouse sera (data not shown). 3.3. Focus reduction neutralization test The 50 mouse serum samples were tested individually for their neutralization activities against SN virus (strain CC 107). Sera from most mice immunized against peptides A, C, D, E, F and G cDNAs showed neutralization activity at a 10-fold serum dilution. Serum from only two mice of ®ve immunized with fragment C cDNA immunized showed signi®cant neutralization activity against SN virus. Three mice genetically immunized against peptide A and two mice immunized against peptide G had 50% neutralizing activity at a 20-fold dilution Table 1. Two mice immunized with the D glycoprotein gene construct showed both exceptionally high lymphoproliferation and higher (20-fold dilution) neutralization titer than did
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other mice immunized with the same construct Table 1. Immunization with the N gene did not produce any neutralizing antibodies. 4. Discussion Vaccination of BALB/c mice with Sin Nombre viral DNA resulted in speci®c immune responses in some mice. G1, G2 and N cDNA fragments induced speci®c lymphoproliferative responses in the presence of their cognate antigens. Fragments C and D elicited the highest speci®c proliferative responses of the envelope gene fragments, although three mice inoculated with the D gene fragment apparently did not generate any immune response. The N gene cDNA elicited stronger proliferative and antibody responses than did any glycoprotein fragment but as has been noted previously, such antibodies were not neutralizing [14,15]. Several envelope fragments induced antibody responses against the cognate antigens, albeit inconsistently. As might be expected with a strategy that uses small portions of genes, the neutralization responses to the glycoprotein antigens were of modest titer. We believe that selected high-risk groups could bene®t by development of a vaccine against SN virus. These include certain Native American groups, forestry and agricultural workers, and ®eld biologists. Currently there is no infection or disease model available to evaluate the ecacy of vaccine preparations for SN virus. Pending the development of such animal models, it is helpful to determine whether BALB/c (Mus musculus ) mice could be used to screen genetic vaccine candidates for SN virus. We showed here that most of the glycoprotein fragments studied induced antibodies that can neutralize SN virus at modest dilution. We choose to introduce no non-native secretory signal upstream of our gene fragments, because viral peptides produced by the transfected tissues may be expressed as antigens at least in the class I pathway even when the peptides lack such signals [29,33]. Choi et al. showed that in case of rotavirus capsid protein VP7-containing plasmid gene immunized mice, had no dierence in immune responses regardless of the presence of a secretory signal peptide [30]. Several recent reports indicate immunization that resulted in both cellular proliferation and antibody production with or without neutralizing antibody can be associated with protection from virus challenge [31,32]. In Aujeszky's disease, the glycoprotein construct gC elicited very low virus-neutralizing activity, but gave the greatest protection against virus challenge. In contrast, a gD gene construct gave no protection, even though neutralizing activity was demonstrable [31]. Our tests did not discern which population(s) of lymphocytes were actually
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stimulated to proliferate. Nevertheless, glycoprotein cDNA fragments A, C, D, F and G, which induced speci®c splenocyte proliferation and neutralizing activity may be candidates for further study. Nucleocapsid gene immunization induced good lymphoproliferative responses to the N antigen as well as a strong non-neutralizing antibody response. Such responses may nevertheless result in protection from challenge, at least when administered as exogenous peptide [15]. Although Mus musculus mice such as BALB/c are not the natural carriers of SN virus, these inbred mice help de®ne possibly important epitopes of the viral antigens and allow us to preliminarily characterize cellular and humoral responses where no eective animal models exist. At present, little is known about the nature of protective immunity to hantaviruses, and the relative roles of cell-mediated immunity and antibody responses in such protection. We believe that the demonstration of detectable immune responses elicited by naked DNA vaccines in the BALB/c system oers hope that such vaccine candidates may also elicit protective responses in an infection model. These data thus should encourage the development of such models using the deer mouse (P. maniculatus ). We recently established a deer mouse colony for that purpose.
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Acknowledgements We thank S. Johnston for providing valuable technical advice, the pCMVi (±H3) UBs vector and unpublished data; C. Schmaljohn for providing the CC107 virus and helpful discussions; H. Artsob for providing unpublished data, and the UNM protein chemistry laboratory for technical assistance. This work was supported by the Department of Defense Advanced Projects Agency (DARPA) to CRL and S. Johnston and Public Health Services grants RO1 AI 41892 and RO1 AI 36336 to BH (National Institute of Allergy and Infectious Diseases). CRL is a Charles E. Culpeper Foundation Medical Scholar and the work was supported in part by the Charles E. Culpeper Foundation.
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