Download Genetic vaccines protect against Sin Nombre hantavirus challenge

Journal
of General Virology (2002), 83, 1745–1751. Printed in Great Britain
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Genetic vaccines protect against Sin Nombre hantavirus
challenge in the deer mouse (Peromyscus maniculatus)
Mausumi Bharadwaj,1 Katy Mirowsky,1 Chunyan Ye,1 Jason Botten,1 Barbara Masten,1 Joyce Yee,1
C. Richard Lyons2 and Brian Hjelle1, 3
Center for Emerging Infectious Diseases, Departments of Pathology1, Medicine2 and Biology and Molecular Genetics and Microbiology3,
University of New Mexico School of Medicine, Albuquerque, NM 87131, USA
We used a deer mouse (Peromyscus maniculatus) infection model to test the protective efficacy of
genetic vaccine candidates for Sin Nombre (SN) virus that were known to provoke immunological
responses in BALB/c mice (Bharadwaj et al., Vaccine 17, 2836–2843, 1999). Protective epitopes
were localized in each of four overlapping cDNA fragments that encoded portions of the SN virus
G1 glycoprotein antigen ; the nucleocapsid gene also was protective. The protective efficacy of
glycoprotein gene fragments correlated with splenocyte proliferation in the presence of cognate
antigen, but none induced neutralizing antibodies. Genetic vaccines against SN virus can protect
outbred deer mice from infection even in the absence of a neutralizing antibody response.
Introduction
Hantaviruses are negative-stranded RNA viruses that
constitute a genus of the family Bunyaviridae (Schmaljohn et al.,
1985). The genomes of hantaviruses are divided into three
RNA segments : a large (L) segment encoding the viral
transcriptase, a middle (M) segment encoding the envelope
glycoproteins G1 and G2, and a small (S) segment encoding
the nucleocapsid core protein, N. Hantaviruses have long been
associated with epidemics of haemorrhagic fever with renal
syndrome in Korea, China, Russia and Europe (Lee & van
der Groen, 1989 ; Plyusnin et al., 1996). Sin Nombre (SN)
hantavirus, which is endemic in the western United States, is
the prototypical aetiological agent for hantavirus cardiopulmonary syndrome (HCPS) (Nichol et al., 1993). Approximately
1000 cases of HCPS have been recorded in nine countries since
its discovery, with an overall case-fatality ratio of about
40–45 % (Schmaljohn & Hjelle, 1997). All cases have occurred
in the western hemisphere. While at least six distinct
hantaviruses have been implicated as aetiological agents for
HCPS, SN virus remains the most important in the northern
hemisphere.
SN virus, like all pathogenic hantaviruses, is carried by a
rodent reservoir host. The reservoir for SN virus is the deer
mouse Peromyscus maniculatus (Childs et al., 1994). Although it
Author for correspondence : Brian Hjelle (at Department of
Pathology). Fax j1 505 272 9912.
e-mail bhjelle!salud.unm.edu
0001-8290 # 2002 SGM
is not uncommon to find populations of wild deer mice with
30–50 % prevalence for SN virus, and seropositive deer mice
are persistently infected, deer mice are not adversely affected
by the infection. Humans contract the infection through
inhalation of virus-contaminated excreta.
Hantavirus outbreaks can involve significant numbers of
people, and for at least one species, interpersonal transmission
of infection has been recognized (Enria et al., 1996). About
50–100 000 cases of HFRS occur every year in China (Lee &
van der Groen, 1989). In the western hemisphere, HCPS has a
much greater economic importance than would be suggested
by the sheer number of cases. HCPS outbreaks in New Mexico
in 1993 and in Bariloche, Argentina in 1996 caused considerable economic damage and stigma to affected communities. Despite years of effort, to date there are no vaccines
proven to be highly efficacious against hantavirus diseases.
Potential recipients for a vaccine against SN virus could
number in the millions, since virtually anyone living in a rural
area in the western United States and Canadian provinces is at
risk for infection (Khan et al., 1996).
The envelope glycoproteins of SN virus are expressed as a
precursor molecule of 1140 amino acids. At least for the
prototypical hantavirus Hantaan, the precursor polypeptide is
cotranslationally cleaved into G1 and G2 by a cellular protease
immediately after a hydrophobic region that ends in the
conserved sequence WAASA (Schmaljohn et al., 1987 ;
Arikawa et al., 1990 ; Lober et al., 2001). The two glycoproteins,
which are both transmembrane proteins, assemble into heterodimers and for most members of the family Bunyaviridae they
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M. Bharadwaj and others
are retained efficiently in the Golgi (Schmaljohn, 1996). For
unclear reasons, it has been difficult to obtain good expression
of recombinant SN virus glycoproteins in eukaryotic systems
except when the genes are fragmented into small pieces
(unpublished data of the authors).
To overcome the problem of poor glycoprotein expression,
we previously prepared a series of 10 overlapping clones of the
SN virus envelope glycoproteins G1 and G2, each 500 nt in
length and sharing 100 nt of overlap with the more 5h clone
(Bharadwaj et al., 1999). We immunized groups of BALB\c
mice with these individual clones, and separately with a fulllength clone of the viral N gene, and measured the immunological responses. Several clones induced strong splenocyte
proliferative response, especially those at the 5h and middle
portion of the G1 glycoprotein. Neutralizing and nonneutralizing antibody responses against the G1 and G2
antigens were present in some cases, but titres were very low.
We noted that these vaccine candidates would be worthy of
evaluation upon the development of an animal challenge
model for SN virus (Bharadwaj et al., 1999). Recently, we
developed such a model by using a novel outdoor containment
laboratory in which we could conduct the challenge experiments (Botten et al., 2000a, b). The challenge experiments may
be conducted safely in an outdoor setting but would require a
biosafety level 4 (BSL-4) facility if conducted indoors (Centers
for Disease Control & Prevention, 1994 ; Mills et al., 1995).
Methods
Deer mice and virus. Six-to-ten week-old outbred P. maniculatus
rufinus were obtained from the University of New Mexico’s colony in
Albuquerque, NM, USA. The colony was founded from wild specimens
collected in 1998 (Botten et al., 2000b, 2001). Most subjects were used at
the F1 or F2 generation. The virus, strain SN77734, was isolated from a
mouse that was collected at the same time and geographical site as were
the virus-negative founder mice. We used the virus at deer mouse
intramuscular (i.m.) passage 2 ; the isolate of SN77734 we used had never
been passaged in tissue culture (Botten et al., 2000a). The research was
conducted according to protocols approved by the UNM animal research
and biosafety committees.
Construction of recombinant plasmids. We previously described how we cloned the G1 and G2 glycoprotein genes of SN virus
strain CC107 into the CMV expression vector pCMVi (-H3) UBs
(Bharadwaj et al., 1999). The M segment fragments 3h of the first
fragment were prepared in a similar manner, such that each expression
construct shared 100 nt of sequence at the 5h end with the 3h end of the
fragment that preceded it. The coordinates of each of the ten glycoprotein
fragments, designated M-CMV-A thorough -I, are indicated in Table 1.
We also cloned the entire SN virus N gene in a single fragment in a
separate expression construct. The same viral cDNA fragments were
cloned into bacterial expression vectors to allow bacterial synthesis of the
cognate antigens as fusion proteins, as described (Bharadwaj et al.,
1997 ; Yamada et al., 1995).
Expression and purification of fusion proteins. We obtained
optimal expression of M segment fragments with the pATH 23
expression system, but since that system does not incorporate affinitypurification tags, we used elution from preparative SDS–polyacrylamide
gels for purification of each of the bacterially expressed G1 and G2
peptides. For the N gene, we used metal-chelation columns for
purification via a C-terminal polyhistidine track (Bharadwaj et al., 1997).
Yield and purity were assessed as described (Bharadwaj et al.,
1997 ; Bradford, 1976). Previously, we used ‘ cross-proliferation ’ analysis
(assessing proliferation of splenocytes induced by peptides other than
that encoded by the immunizing plasmid) to show that the immunological
responses to the same gel-purified glycoprotein peptides, when used to
evaluate vaccine responses by BALB\c mice, were specific to the viral
peptides and responses were thus not related to any contaminating
bacterial products (Bharadwaj et al., 1999).
Immunoblot tests. Immunoblot tests were used to detect antibodies to N antigen in mice vaccinated with N antigen as well as those
Table 1. Splenocyte proliferation responses in the presence of cognate antigen in deer mice that were not challenged
with SN virus
Coordinates
Gene
G1
G2
N
–
[3H]Thymidine incorporation (c.p.m.) (mean)
Construct*
Nucleotide
Amino acid
No antigen (NA)
With antigen (WA)
MeanpSD
A
B
C
D
E
F
G
H
I
N
pCMVi
49–549
448–948
847–1347
1246–1746
1507–2007
2008–2508
2392–2907
2509–3006
2902–3474
43–1330
–
1–167
132–299
265–432
399–565
486–652
1–167
134–300
266–433
400–488
1–428
–
4 708
4 480
4 220
4 715
4 922
4 088
4 447
3 480
3 900
3 980
4 855
67 892
52 406
65 732
48 644
22 984
15 665
18 632
18 274
34 162
74 010
15 128
13n5p1n2
11n1p2n8
15n2p3n5
10n0p3n7
3n0p0n6
1n6p0n4
3n6p1n1
4n3p0n6
8n1p2n1
18n1p3n8
1n9p0n5
* Construct identical to construct described in Bharadwaj et al. (1999).
BHEG
Stimulation index
(WAkNA)/NA
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Genetic vaccines against Sin Nombre virus
challenged with SN virus. We prepared strip immunoblot assays (SIA) as
previously described (Bharadwaj et al., 2000 ; Botten et al., 2000a). Briefly,
we suctioned the following materials onto separate lanes of an 8i10 cm
nitrocellulose membrane using a ‘ slot blot ’ maker, from top to bottom :
(1) a Coomassie blue solution to allow orientation of the strips ; (2) a
dilution of deer mouse serum that contained sufficient IgG antibodies to
produce a dark grey (‘ 3j ’) reactivity with the alkaline phosphataseconjugated anti-Peromyscus leucopus conjugate upon development ; (3)
1n8 µg of affinity-purified recombinant SN virus N antigen ; and (4) a
more dilute deer mouse serum solution to show a light grey (‘ 1j ’)
reaction with the conjugate antibody upon development (Botten et al.,
2000a). We then cut the nitrocellulose membrane lengthwise into 1n6 mm
wide strips with a paper shredder. We incubated each strip with a deer
mouse serum (1 : 200 dilution) in a Western blot tray overnight, and then
detected the bound antibody with the anti-P. leucopus IgG conjugate,
followed by an alkaline phosphatase substrate. In a few cases where
antibody reactivity was slight, we used a Western blot test to verify the
results of the SIA (Yamada et al., 1995).
Genetic immunization and virus challenge. We purified
plasmid DNA with an endotoxin-free kit (EndoFree, Qiagen), and
dissolved DNA to a concentration of 1 mg\ml in 0n9 % NaCl. Five to
twelve deer mice were immunized with each construct three times at
4 week intervals, using 50 µg of plasmid into each set of quadriceps
muscles for a total of 100 µg. No adjuvants were used.
To compare immunological responses to vaccination as well as
protective efficacy of each vaccine construct, we used two experimental
groups : an immunology replicate and a challenge replicate. Both of the
experimental groups were subjected to immunization with glycoprotein
gene fragments, the intact N gene or vector alone, followed by two
identical boosts. In the immunology replicate, we did not subsequently
challenge with SN virus, but we instead sacrificed the mice for
examination of splenocyte proliferative responses to each bacterially
expressed antigen and neutralizing antibody responses to the vaccine. In
the challenge replicate, we subsequently subjected the mice to virus
challenge. Mice in the immunology replicate were killed 2 weeks after the
second booster vaccination and spleens and blood samples were collected.
All of the blood samples from the immunology replicate were tested for
neutralizing antibodies, whereas splenocytes were used for assays for
proliferation in the presence of bacterially expressed cognate antigen.
After moving the immunized mice to the outdoor biocontainment
laboratory, we challenged the mice in the challenge replicate with 5 ID
&!
of SN77734 by the i.m. route 2 weeks after the third vaccination, a dose
that corresponds roughly to 50–200 focus-forming units (Botten et al.,
2000a, b). We use animal ID to measure virus infectivity rather than
&!
plaque-forming units because it is the most relevant unit for infectivity for
the studies in question, and because we have established that animalpassaged SN virus is qualitatively different from virus that has been
passed in tissue culture (unpublished data). This regimen led to infection
of 100 % of unimmunized deer mice. Four weeks after challenge, we killed
the mice with tribromoethanol. We tested blood samples for antibodies
using the SIA (Bharadwaj et al., 2000). In addition, we prepared RNA
from 10–50 mg of heart tissue for nested RT–PCR assays using primers
either in the N gene (for mice immunized with M segment fragments or
control mice) or in the G1 gene for mice immunized with the N gene
(Botten et al., 2000a ; Hjelle et al., 1994). The purified RNAs were diluted
with 2 µl water for each mg of tissue that was used in its preparation, and
5 µl of RNA was used in nested RT–PCR reactions. The nested PCR
assay we used has been an extremely sensitive measure of infection in our
hands, with no false positive assays in more than 5 years. The sensitivity
is under 10 copies of RNA template per reaction (unpublished data).
Antibody testing. SIA and FRNT tests were as described
(Bharadwaj et al., 1999 ; Botten et al., 2000a). The SIA was conducted at
a 1 : 200 serum dilution and the FRNT screen at 1 : 40. The SIA was
considered positive if the N antigen band became darkened after
development, whereas a serum was considered to be positive in the
FRNT if it demonstrated a reduction of 80 % or more of foci.
For the FRNT, we screened sera from the immunology replicate
animals at a 1 : 40 dilution, as described previously (Bharadwaj et al.,
1999). Briefly, we mixed 5 µl of deer mouse serum that had been diluted
with 95 µl of medium with an equal volume of medium containing 45–80
focus-forming units of SN virus strain CC 107 (a generous gift of C.
Schmaljohn) and incubated for 1 h at 37 mC, and then used the mixture to
infect a confluent monolayer of Vero E6 cells in duplicate wells of a 48well dish, using a 1n2 % methylcellulose overlay in the medium to confine
the virus to foci. After 1 week, we detected the viral foci with polyclonal
rabbit anti-N antibody followed by peroxidase-conjugated goat antirabbit IgG.
Cellular proliferation assay. Spleen cell preparation and splenocyte proliferation assays were performed by analysis of [$H]thymidine
uptake as described (Bharadwaj et al., 1999). We used 10 µg\ml of
cognate antigen for in vitro stimulation, based upon our previous dose
optimization data from the BALB\c model. The proliferative capacity of
splenocytes used in our experiments was verified by stimulation with
10 µg\ml of concanavalin A. When we developed this assay for BALB\c
mice, we demonstrated that splenocytes derived from a mouse that was
immunized with a particular construct did not proliferate in the presence
of the cognate antigen expressed from another non-overlapping vaccine
construct, indicating that the proliferation was specific for a particular
peptide that was indeed expressed in the immunized deer mouse. We
stimulated splenocytes from control mice immunized with vector alone
with the amino-terminal glycoprotein peptide ‘ A ’.
Statistical analyses. We compared the splenocyte proliferation
indices of vaccinated mice with those of mice that received the CMV
vector alone by two-way ANOVA, and the protective efficacy of the
various constructs using Fisher’s exact test.
Results
Two experimental groups of rodents were used as described
in Methods. One group was used solely to determine the
immunological responses to vaccination, another to determine
the degree of protection afforded by a particular vaccine
construct.
Immunology replicate
Ten groups of five deer mice were examined for in vitro
immunological responses to inoculation with SN virus gene
fragments, in part to allow comparison with previous studies
with the inbred Mus musculus strain BALB\c (Bharadwaj et al.,
1999). The vaccination regimen was identical to that used in
the BALB\c model : after we immunized the deer mice, we
collected spleens and serum from each animal 2 weeks after the
last boost.
Splenocyte proliferative responses induced by cognate
antigen differed markedly from construct to construct. Both N
antigen and G1 or G2 peptides induced a similar degree of
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M. Bharadwaj and others
Table 2. Fraction of deer mice infected by SN virus
challenge
Gene
G1
G2
N
None
Construct
A
B
C
D
E
F
G
H
I
N
pCMVi
Fraction
infected*
P value
(Fisher’s exact)
1\9 (11 %)
3\12 (25 %)
0\6 (0 %)
1\6 (17 %)
7\10 (70 %)
3\5 (60 %)
9\9 (100 %)
9\10 (90 %)
6\7 (66 %)
1\5 (20 %)†
9\9 (100 %)
0n0004
0n0011
0n0002
0n0020
0n2105
0n1099
1
1
0n4400
0n04995
–
* Infectious status determined by strip immunoblot assay (SIA) and
reverse transcription–PCR.
† Four out of five positive by SIA, but negative by PCR.
splenocyte proliferation from genetically immunized deer mice
in comparison to BALB\c mice (Table 1). The comparisons
between the results of Bharadwaj et al. (1999) and the present
study indicate that inbred BALB\c M. musculus and outbred
P. maniculatus do not differ dramatically in splenocyte proliferative responses to SN virus antigens delivered through
genetic immunizations.
Four of the subjects from group N were examined for
antibodies to SN virus N antigen by SIA (data not shown). Like
the results of Bharadwaj et al. (1999), some (four) deer mice
produced an antibody response against N antigen in response
to the genetic vaccine, with an endpoint titre of at least
1 : 10 000. No neutralizing antibody response to immunization
with glycoprotein segments was evident by FRNT, probably
reflecting the inability of the short peptide products to fold
into conformations that mimic the intact glycoproteins of the
virus.
We were unable to examine the antibody responses
produced by the immunized deer mice against the partially
purified G1 and G2 fragments, as described in our previous
work, because we encountered much higher background and
lower signal with the immunized deer mice than with the
BALB\c mice (Bharadwaj et al., 1999).
Challenge replicate
The results of the challenge experiments are summarized in
Table 2, and typical data are depicted in Fig. 1. We assessed
infection by (1) seroconversion to N antigen by SIA, and (2)
RT–PCR detection of SN virus RNA in the heart. With the
glycoprotein constructs, there was a close correspondence
between seroconversion in the SIA and the detection of SN
virus S segment RNA in the heart of challenged mice. In a few
cases, anti-N antibody reactivities were slight by the SIA but
their presence could be confirmed in a Western blot test (data
not shown) using N antigen as target (Table 2, groups G and
I). SN virus S segment RNA could be detected in the hearts of
those specimens, confirming that they had become infected.
Ultimately, there were no discrepancies in the comparisons
between the SIA test and RT–PCR analysis.
Constructs designed to express glycoprotein antigens
differed markedly in their ability to protect deer mice from
subsequent viral challenge, with protective efficacies ranging
from 0 to 100 %. The most active constructs were A through
D (Table 2), which protected 75 % to 100 % of deer mice from
infection, as well as the N gene. For the glycoprotein vaccines
and the N vaccine, there was a strong correlation between the
splenocyte proliferation in the presence of cognate antigen and
the fraction of deer mice that were protected from subsequent
SN virus challenge (Fig. 2).
Protective efficacy of N gene
Since some mice immunized with the N gene construct
produced an antibody response to N antigen, the SIA was not
Fig. 1. Representative data from
challenge group showing partial
protection against challenge with SN virus
by genetic vaccine construct E
(glycoprotein G1 gene residues
847–1347). After immunization and two
boosts with 100 µg of construct E, deer
mice were challenged with 5 ID50 of SN
virus SN77734 by the intramuscular
route. (A) The presence of serum
antibodies to the viral N antigen was
assessed at a 1 : 200 dilution by strip
immunoblot assay. (B) The presence of
viral RNA in the heart of each mouse was
assessed by nested RT–PCR. CB,
Coomassie blue ; 3j and 1j, intensity
control bands. Note correlation between
detection of viral RNA in tissues and the
presence of serum antibodies.
BHEI
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Genetic vaccines against Sin Nombre virus
experiments, to difficulties establishing animal models, and\or
to difficulties in obtaining high-level expression of the
envelope glycoproteins of SN virus and other agents of HCPS.
Efficacious hantavirus vaccines can be prepared from
short genomic segments
Fig. 2. For each glycoprotein fragments (
) and N gene construct ($)
listed in Table 1, the splenocyte proliferative response elicited by the
cognate antigen (immunology replicate) is correlated with the degree of
protection from viral challenge elicited by the same construct (challenge
replicate). Proliferative responses are expressed as the stimulation index
relative to splenocytes that were not exposed to antigen, after subtraction
of the spontaneous uptake of tritium. A trend line has been drawn to
depict the relationship for the glycoprotein gene data.
useful in assessing infection. For this group, we assessed
infection by RT–PCR with primers from the G1 gene of SN
virus. (Hjelle et al., 1994). In this group, one of the five mice
was positive by RT–PCR using primers in G1, indicating
that the N construct vaccine was 80 % protective against
subsequent challenge.
Discussion
Vaccines against SN virus
A vaccine prepared against any single hantavirus might not
be able to provide protection against every member of the
antigenically diverse genus Hantavirus. Early vaccines were
produced from inactivated virus produced in rodent brains, but
immune responses have been short-lived (Cho & Howard,
1999). Recent efforts to develop hantavirus vaccines have
included genetic vaccine approaches as well as subunit vaccines
utilizing purified proteins or vaccinia delivery systems (Chu et
al., 1995 ; Hooper et al., 1999 ; Schmaljohn et al., 1992). Clinical
trials using recombinant vaccinia virus vaccines did not
consistently produce immune responses in vaccinia-immune
volunteers (McClain et al., 2000). Recently, investigations
using hamster and other rodent challenge models have shown
that genetic vaccines can be effective against the agents of
HFRS, but no similar progress has been noted with agents of
HCPS. In a hamster model, however, it has recently been
possible to demonstrate that hantaviruses that are apathogenic
in that species, including Hantaan and SN viruses, confer
protection from disease by a virus that produces pulmonary
oedema, Andes virus (Hooper et al., 2001). The slow progress
with vaccines against the agents of HCPS could be related to
the need for biosafety level 4 containment for challenge
Most studies of hantavirus vaccines have associated
protective efficacy with the induction of neutralizing antibodies. However, some investigations have shown partial or
complete protection using the viral nucleocapsid antigen as
immunogen, which does not elicit neutralizing antibodies
(Lundkvist et al., 1996 ; Schmaljohn et al., 1990 ; Ulrich et al.,
1998). By contrast, a role for T cells in protective immunity has
been little-studied, but adoptive transfer studies have suggested that T cells can confer protection to a naı$ ve host
(Yoshimatsu et al., 1993). Previously, it has been possible to
localize the protective epitopes within a peptide vaccine
through deletion analysis (Lundkvist et al., 1996). Since we
have previously experienced difficulties obtaining adequate
expression of the envelope glycoproteins of SN virus in
eukaryotic systems, we chose to scan the G1 and G2 genes for
protective epitopes by introducing the genes in small fragments that were known to be competent for expression
(Bharadwaj et al., 1999). That the glycoprotein gene segments
were in fact being expressed was demonstrated by splenocyte
proliferation in the presence of peptide after vaccination, an
effect that was specific for those mice that had received the
genetic vaccine encoding that cognate antigen.
The deer mouse system was favoured over the bettercharacterized M. musculus for several reasons. First, hantavirus
models using M. musculus are generally unsatisfactory, allowing only transient and generally fatal infections of the central
nervous system in neonates (Tsai et al., 1982 ; Ebihara et al.,
2000). Second, outbred deer mice are the natural reservoir host
for SN virus and thus were taken as the model that is more
reflective of the normal host–pathogen relationship. Furthermore, use of the deer mouse model allows the possibility
for later studies of the modes of transmission, persistence\
reactivation, tissue and host tropism, and clearance of the virus
in its natural reservoir. It is possible that the recent advent of
the hamster infection model for SN and Andes viruses will
overcome some of these difficulties (Hooper et al., 2001).
Our data show that SN virus glycoprotein and N gene
segments induce similar proliferative responses of splenocytes
and that the N gene segment elicits similar anti-N antibody
responses in outbred deer mice and M. musculus, although a
few of the less immunogenic segments differed slightly
between the species (Bharadwaj et al., 1999). As judged by
polyclonal splenocyte proliferation, some strongly immunogenic gene segments are capable of eliciting immune responses
in disparate species and in disparate MHC backgrounds. For
this series of experiments, we elected to establish a higher cutoff dilution (1 : 40 with an 80 % reduction of foci) for detection
of neutralizing antibody responses than we did with our
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M. Bharadwaj and others
previous experiments with BALB\c mice, because we wanted
to have greater confidence that the neutralization responses we
might detect were important in protection. Using the 1 : 40
screening dilution, no evidence for such (neutralizing) response
was detected, nor would such a response been evident using
that standard in the BALB\c model.
The N gene conveyed protection in deer mice, as did
several small peptides derived from G1 and G2. The basis for
protection by the small glycoprotein fragments has yet to be
determined. The induction of neutralizing and non-neutralizing
antibodies by these small gene segments is slight, as one might
expect given their likely poor reproduction of the native
folding of the intact G1\G2 complex (Wang et al., 1992).
Because in preliminary data (not shown) non-neutralizing
antibodies to the glycoproteins were difficult to distinguish
from nonspecific reactivity in deer mice we could not study
those antibodies in this model. There appears to be a good
correlation between the splenocyte proliferation responses and
the protective efficacy of each vaccine construct. It is possible
that the glycoprotein segments are processed by the class I
pathway and elicit CD8 cell immunity, through either
production of antiviral mediators or via cytotoxic mechanisms.
The basis of protection by the short segments utilized herein
will require additional work to clarify, and may require the
development of a cytotoxic T cell assay involving inbred
strains of deer mice, and\or reagents capable of detecting deer
mouse cytokines such as interferon-γ.
By identifying multiple apparently independent targets for
sterilizing immune responses, we may be able to develop
synergistic or additive polyvalent vaccine preparations, which
may also allow the incorporation of homologous segments
from other hantaviruses. To test such cocktails it may be
necessary to use a higher challenge dose of virus to increase
the failure rate in the protective gene segments from G1 and N.
Supported by US Public Health Service Grant RO1 AI 41692 and by
the Defence Advanced Research Projects Agency. J. B. is a fellow in the
Infectious Diseases and Inflammation Program (Public Health Services
Grant T32 AI07538-01). We thank R. Ricci, F. Gurule and R. Xiao for
excellent technical assistance.
syndrome. Journal of Infectious Diseases 182, 43–48.
Botten, J., Mirowsky, K., Kusewitt, D., Bharadwaj, M., Yee, J., Ricci, R.,
Feddersen, R. M. & Hjelle, B. (2000a). Experimental infection model for
Sin Nombre hantavirus in the deer mouse (Peromyscus maniculatus). Proceedings of the National Academy of Sciences, USA 97, 10578–
10583.
Botten, J., Nofchissey, R., Ahern, H., Rodriguez-Moran, P., Wortman,
I. A., Goade, D., Yates, T. & Hjelle, B. (2000b). Outdoor facility for
quarantine of wild rodents infected with hantavirus. Journal of Mammalogy 81, 250–259.
Botten, J., Ricci, R. & Hjelle, B. (2001). Establishment of a deer mouse
(Peromyscus maniculatus rufinus) breeding colony from wild-caught
founders. Comparative Medicine 51, 291–295.
Bradford, M. (1976). A rapid and sensitive method for the quantitation
of microgram quantities of protein utilizing the principle of protein-dye
binding. Analytical Biochemistry 72, 248–254.
Centers for Disease Control & Prevention (1994). Laboratory
management of agents associated with hantavirus pulmonary syndrome :
interim biosafety guidelines. Morbidity and Mortality Weekly Report 43,
1–7.
Childs, J. E., Ksiazek, T. G., Spiropoulou, C. F., Krebs, J. W., Morzunov,
S., Maupin, G. O., Gage, K. L., Rollin, P. E., Sarisky, J. & Enscore, R. E.
(1994). Serologic and genetic identification of Peromyscus maniculatus as
the primary rodent reservoir for a new hantavirus in the southwestern
United States. Journal of Infectious Diseases 169, 1271–1280.
Cho, H. W. & Howard, C. R. (1999). Antibody responses in humans to
an inactivated hantavirus vaccine (Hantavax). Vaccine 17, 2569–2575.
Chu, Y. K., Jennings, G. B. & Schmaljohn, C. S. (1995). A vaccinia
virus-vectored Hantaan virus vaccine protects hamsters from challenge
with Hantaan and Seoul viruses but not Puumala virus. Journal of Virology
69, 6417–6423.
Ebihara, H., Yoshimatsu, K., Ogino, M., Araki, K., Ami, Y., Kariwa, H.,
Takashima, I., Li, D. & Arikawa, J. (2000). Pathogenicity of Hantaan
virus in newborn mice : genetic reassortant study demonstrating that a
single amino acid change in glycoprotein G1 is related to virulence.
Journal of Virology 74, 9245–9255.
Enria, D., Padula, P., Segura, E. L., Pini, N., Edelstein, A., Posse, C. R.
& Weissenbacher, M. C. (1996). Hantavirus pulmonary syndrome in
Argentina. Possibility of person to person transmission. Medicina (Buenos
Aires) 56, 709–711.
Hjelle, B., Chavez-Giles, F., Torrez-Martinez, N., Yamada, T., Sarisky,
J., Ascher, M. & Jenison, S. (1994). Dominant glycoprotein epitope of
Four Corners hantavirus is conserved across a wide geographical area.
Journal of General Virology 75, 2881–2888.
Hooper, J. W., Kamrud, K. I., Elgh, F., Custer, D. & Schmaljohn, C. S.
(1999). DNA vaccination with hantavirus M segment elicits neutralizing
References
Arikawa, J., Lapenotiere, H. F., Iacono-Connors, L., Wang, M. L. &
Schmaljohn, C. S. (1990). Coding properties of the S and the M genome
segments of Sapporo rat virus : comparison to other causative agents of
hemorrhagic fever with renal syndrome. Virology 176, 114–125.
Bharadwaj, M., Botten, J., Torrez-Martinez, N. & Hjelle, B. (1997). Rio
Mamore virus : genetic characterization of a newly recognized hantavirus
of the pygmy rice rat, Oligoryzomys microtis, from Bolivia. American
Journal of Tropical Medicine and Hygiene 57, 368–374.
Bharadwaj, M., Lyons, C. R., Wortman, I. A. & Hjelle, B. (1999).
Intramuscular inoculation of Sin Nombre hantavirus cDNAs induces
cellular and humoral immune responses in BALB\c mice. Vaccine 17,
2836–2843.
BHFA
Bharadwaj, M., Nofchissey, R., Goade, D., Koster, F. & Hjelle, B.
(2000). Humoral immune responses in the hantavirus cardiopulmonary
antibodies and protects against Seoul virus infection. Virology 255,
269–278.
Hooper, J. W., Larsen, T., Custer, D. M. & Schmaljohn, C. S. (2001). A
lethal disease model for hantavirus pulmonary syndrome. Virology 289,
6–14.
Khan, A. S., Ksiazek, T. G. & Peters, C. J. (1996). Hantavirus pulmonary
syndrome. Lancet 347, 739–741.
Lee, H. W. & van der Groen, G. (1989). Hemorrhagic fever with renal
syndrome. Progress in Medical Virology 36, 62–102.
Lober, C., Anheier, B., Lindow, S., Klenk, H. D. & Feldmann, H. (2001).
The Hantaan virus glycoprotein precursor is cleaved at the conserved
pentapeptide WAASA. Virology 289, 224–229.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 07:28:54
Genetic vaccines against Sin Nombre virus
Lundkvist, A., Kallio-Kokko, H., Sjolander, K. B., Lankinen, H.,
Niklasson, B., Vaheri, A. & Vapalahti, O. (1996). Characterization of
Schmaljohn, C. S., Chu, Y. K., Schmaljohn, A. L. & Dalrymple, J. M.
(1990). Antigenic subunits of Hantaan virus expressed by baculovirus
Puumala virus nucleocapsid protein : identification of B-cell epitopes and
domains involved in protective immunity. Virology 216, 397–406.
and vaccinia virus recombinants. Journal of Virology 64, 3162–3170.
Schmaljohn, C. S., Hasty, S. E. & Dalrymple, J. M. (1992). Preparation
of candidate vaccinia-vectored vaccines for haemorrhagic fever with renal
syndrome. Vaccine 10, 10–13.
Tsai, T. F., Bauer, S., McCormick, J. B. & Kurata, T. (1982). Intracerebral
inoculation of suckling mice with Hantaan virus [letter]. Lancet 2,
503–504.
McClain, D. J., Summers, P. L., Harrison, S. A., Schmaljohn, A. L. &
Schmaljohn, C. S. (2000). Clinical evaluation of a vaccinia-vectored
Hantaan virus vaccine. Journal of Medical Virology 60, 77–85.
Mills, J. N., Yates, T. L., Childs, J. E., Parmenter, R. R., Ksiazek, T. G.,
Rollin, P. E. & Peters, C. J. (1995). Guidelines for working with rodents
potentially infected with hantavirus. Journal of Mammalogy 76, 716–722.
Nichol, S. T., Spiropoulou, C. F., Morzunov, S., Rollin, P. E., Ksiazek,
T. G., Feldmann, H., Sanchez, A., Childs, J., Zaki, S. & Peters, C. J.
(1993). Genetic identification of a hantavirus associated with an outbreak
of acute respiratory illness [see comments]. Science 262, 914–917.
Plyusnin, A., Vapalahti, O. & Vaheri, A. (1996). Hantaviruses : genome
structure, expression and evolution. Journal of General Virology 77,
2677–2687.
Schmaljohn, C. S. (1996). Bunyaviridae : the viruses and their replication.
In Fields Virology, 3rd edn, pp. 1447–1471. Edited by B. N. Fields, D. M.
Knipe & P. M. Howley. Philadelphia : Lippincott–Raven.
Schmaljohn, C. & Hjelle, B. (1997). Hantaviruses : a global disease
problem. Emerging Infectious Diseases 3, 95–104.
Schmaljohn, C. S., Hasty, S. E., Dalrymple, J. M., Leduc, J. W., Lee,
H. W., von Bonsdorff, C. H., Brummer-Korvenkontio, M., Vaheri, A.,
Tsai, T. F. & Regnery, H. L. (1985). Antigenic and genetic properties
of viruses linked to hemorrhagic fever with renal syndrome. Science
227, 1041–1044.
Ulrich, R., Lundkvist, A., Meisel, H., Koletzki, D., Sjolander, K. B.,
Gelderblom, H. R., Borisova, G., Schnitzler, P., Darai, G. & Kruger,
D. H. (1998). Chimaeric HBV core particles carrying a defined segment
of Puumala hantavirus nucleocapsid protein evoke protective immunity
in an animal model. Vaccine 16, 272–280.
Wang, M. W., Pennock, D. G., Spik, K. W. & Schmaljohn, C. S. (1992).
Epitope mapping studies with neutralizing and non-neutralizing monoclonal antibodies to the G1 and G2 envelope glycoproteins of Hantaan
virus. Virology 192, 757–766.
Yamada, T., Hjelle, B., Lanzi, R., Morris, C., Anderson, B. & Jenison, S.
(1995). Antibody responses to Four Corners hantavirus infections in the
deer mouse (Peromyscus maniculatus) : identification of an immunodominant region of the viral nucleocapsid protein. Journal of Virology 69,
1939–1943.
Yoshimatsu, K., Yoo, Y. C., Yoshida, R., Ishihara, C., Azuma, I. &
Arikawa, J. (1993). Protective immunity of Hantaan virus nucleocapsid
and envelope protein studied using baculovirus-expressed proteins.
Archives of Virology 130, 365–376.
Schmaljohn, C. S., Schmaljohn, A. L. & Dalrymple, J. M. (1987).
Hantaan virus M RNA : coding strategy, nucleotide sequence, and gene
order. Virology 157, 31–39.
Received 4 December 2001 ; Accepted 12 March 2002
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 07:28:54
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