Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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TEMPERATURE-SENSITIVE AND COLD-ADAPTED HUMAN PARANFLUENZA
VIRUS TYPE 2 (HPIV-2) AND VACCINES BASED ON SUCH VIRUS
Background of the Invention
The present invention relates to isolated, attenuated viral strains of human
parainfluenza virus 2 (HPIV-2), which are useful in live vaccine preparations.
These
strains exhibit a temperature sensitive and cold adapted phenotype useful for
stimulating a protective immune response in an inoculated mammal without
producing the severe symptoms caused by the wild type virus.
The human parainfluenza viruses (HPIV), types 1, 2, and 3, are important
pathogens in infants and young children. HPIV routinely causes otitis media,
pharyngitis, and the common cold. These upper respiratory tract infections
(ITRI)
occur commonly and may be associated with lower respiratory infections (LRI)
including croup, pneumonia, and bronchiolitis. Primary infection in young
children is
associated with lower respiratory disease and often leads to hospitalization.
As a
1 ~ group, the parainfluenza viruses are the second most common cause of
hospital
admission for respiratory infection and are second only to respiratory
syncytial virus
as a significant pathogen in young children. Parainfluenza type 3 is unique
among the
parainfluenza viruses in its ability to commonly infect young infants less
than 6
months of age. Bronchiolitis and pneumonia are common in infants infected with
this
type; in this regard, HPIV-3 is similar to respiratory syncytial virus. A
number of
reviews on HPIV have recently been published (Ray and Compans, 1990;
Kingsbury,
1991; Henrickson et al., 1994) concerning the various aspects of these virus
infections.
HPIV-2 infection occurs in yearly outbreaks in the United States (Downham et
al., 1974). This pathogen has a peak incidence in the fall to early winter
with a
slightly longer "season" than HPIV- 1. Croup is the most frequent LRI caused
by this
virus, but it can also cause any of the other respiratory illnesses associated
with HPIV-
1. The peak incidence of HPIV-2 infections occurs in the second year of life
with
approximately 60% of infections taking place in children less than 5 years of
age. Of
interest is the observation in one study that more girls than boys were
symptomatic
with LRI caused by HPIV-2, than LRI caused by HPIV-1 or 3 (Downham et al.,
1974). LRI caused by HPIV-2 has been reported less frequently than with HPIV-1
and
HPIV-3. Recent reports have indicated that either geographic differences or
differences in isolation and detection techniques may play a role in under-
reporting
this virus (Downham et al., 1974; Henrickson et al., 1994). It has been
estimated that
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during the 1991 epidemic, as many as 157,000 children under the age of S were
seen
in emergency rooms, and 35,000 children were admitted to hospitals in the
United
States with HPIV-2 infection. This epidemic resulted in almost 5200 million of
direct
patient care costs for HPIV-1 and -2 combined.
S All of the human parainfluenza viruses are very similar in structural, physi-
cochemical, and biological characteristics. A prototypic HPIV virion is
composed of a
single RNA strand of negative polarity surrounded by a lipid envelope of host
cell
origin. These are pleiomorphic, or mufti-formed, viruses which have an average
diameter of 1 S0-250 nm. The typical HPIV genome contains approximately I
5,000
nucleotides of genetic information (Storey et al., 1984) and encodes at least
six viral
proteins (3"-Iv'p-P(+C)M-F-HN-L-S') (Storey et al., 198=l). In addition, HPIV-
1, 2,
and 3 encode an additional nonstructural protein, "C," and HPIV-2 a protein
"V."
These proteins are produced from overlapping reading frames within the P gene
and
may require editing of the mRNA (Matsuoka et al., 1991). The complete
nucleotide
1S sequence of the HPIV-2 genome has not been published.
The human parainfluenza viruses are classified within the genus
Paramyxoviridae. There are five major serotypes within this genus: the HIPV's
1-4,
and mumps. The HPIV serotypes can be grouped antigenically into two divisions:
(1)
HPIV-1 and HPIV-3, within the genus Paramyxovirus, and (2) HPIV-2 and HPIV-4,
within the genus Rubulavirus (Coffins et al., 1996). HPIVs all share common
antigens
and variable levels of heterotypic antibody are often detected during
infection. Thus, it
is difficult to determine whether the heterotypic responses are reflective of
past
infections, or simply are cross reactions to similar antigens during serologic
testing.
However, specific hyperimmune animal serum in the past and, more recently,
2S monoclonal antibodies have been employed to differentiate these viruses in
standard
assays (Sarkkinen et al., 1981).
Mucous membranes of the nose and throat are the initial site of parainfluenza
virus infection. Patients with mild disease may have limited involvement of
the bron-
chi as well. The larynx and upper trachea are involved in more extensive
infections
with HPIV-1 and HPN-2, and result in the croup syndrome. Infections may also
extend to the lower trachea and bronchi, with accumulation of thickened mucus
and
resultant atelectasis (incomplete lung expansion) and pneumonia. The possible
contribution of the immune response to the pathogenesis of this illness is
suggested by
the observation that infants and children who develop parainfluenza virus
croup
produce local, virus-specific IgE antibodies earlier and in larger amounts
than patients
of comparable age who develop infections restricted to the upper respiratory
tract
!;
i
;.
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(Welliver et al., 1982). Cell-mediated immune responses to parainfluenza viral
antigens, as well as parainfluenza virus-specific IgE antibody responses, have
been
reported to be greater among infants with parainfluenza virus bronchiolitis
than
among infected infants who develop only upper respiratory illnesses. A
prolonged
carrier state of HPIV-3 is observed in patients with chronic bronchitis and
emphysema
(Gross et al., 1973). It has been suggested that healthy adults may shed
infectious
viruses intermittently and infect susceptible individuals; furthermore,
investigators
have also suggested that persistent infection might occur (Parkinson et al.,
1980).
The hamster provides a recognized animal model for HPIV infection. Infected
animals develop recognizable pathologic changes in the lung which are not
altered by
passive administration of antibodies (Glezen and Femald, 1976). Infected
hamsters do
not show visible signs of respiratory illness or a significant weight loss
during
infection. In addition, monkeys may be used as an animal model of infection,
as
demonstrated in the Examples 4-6, below.
1 p A variety of vaccines have been developed over the years to prevent
various
viral infections in animals and humans. Two principal types of vaccines have
been
used: killed viruses and attenuated live virus. A killed virus is typically
inactivated by
chemical or physical treatment, but is generally less effective in stimulating
a lasting
immune response than an attenuated live virus. Attenuated live viruses are
typically
more effective, but may revert back to their virulent state while in the body.
The time
and cost involved in developing either killed or live vaccines is significant.
Live, attenuated vaccines may be obtained directly from progeny viruses
isolated from infected animals. For example, U.S. Patent No. 3,927,209 to
Straub
discloses a parainfluenza type-3 vaccine isolated as a virus strain from a
bovine
2~ respiratory tract. Live attenuated vaccines may also be obtained by
repeatedly cold
passaging a wild-type strain through suitable cultures until the virus has
lost its
original pathogenic properties. A "cold passage" is the growth of a virus
through an
entire life cycle (infection of the host cell, proliferation in the host cell,
and escape
from the host cell) at a temperature lower than that in which the virus
normally
replicates. For example, cp45, a cold-adapted, temperature sensitive strain
was
obtained by passing the wild-type virus (JS strain) of HPIV-3 45 times at
reduced
temperatures. (Belshe and Hissom, 1982). The temperature sensitive cp4~ strain
is
currently under evaluation for use as a candidate vaccine for HPIV-3 in
humans.
(Karron et al. 1995; Hall et al. 1993; Belshe et al. 1992; Clements et al.
1991;
3~ Crookshanks-Newman and Belshe 1986). Recent evaluation in children has
revealed
the cp45 strain to be highly attenuated and effective in stimulating an
immunogenic
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response. (Karron et al. 199; Belshe et al. 1992). Although cold passaging
techniques have also been used to produce Influenza A and B vaccine strains,
no
similar successful cold passaging of an HPIV-2 virus has been described.
Attenuation in a particular vaccine strain is commonly evaluated with respect
to three phenotypes of the strain: cold adaptation, temperature sensitivity
and plaque
size or yield in tissue culture. Cold adaptation (ca) relates to the ability
of the virus to
grow at reduced temperatures around 2~ °C and temperature sensitivity
(ts) relates to
whether such growth is inhibited at temperatures around 40 °C. Plaque
titers are an
assay for quantitatively evaluating the extent of virus growth, and are
commonly used
to evaluate the extent of cold-adaptive and/or temperature sensitive
phenotypes.
Other methods for determining whether vaccine is attenuated involve
administering
the vaccine to primates. For example, the attenuation of new polio vaccine
lots is
typically tested in monkeys before being approved for sale by the FDA.
Given the propensity of HPIV-2 disease to cause severe respiratory distress in
1 ~ infants and young children, a vaccine which would prevent severe
infection, and the
resulting necessity for hospitalization and treatment, is very desirable.
Although the
need for an HPIV-2 vaccine has been recognized for over two decades, and
despite
successes in isolating vaccine strains for HPIV-3 in the early 1980's, there
is currently
no vaccine available to immunize children against HPIV-2. Prior to the
discovery of
the applicants, HPIV-2 had not been successfully cold passaged. The difficulty
in
isolating attenuated strains of the HPIV-2 virus, as compared to the HPIV-3
virus, can
be explained by the considerable morphological and phenotypic differences
bet<veen
the tvvo viruses. Although they are antigenically similar, HPN-2 is much more
difficult to adapt to in vitro growth conditions and reduced temperatures than
HPIV-3.
2~ Summary of the Invention
Therefore, it is an object of the invention to provide vaccine strains of HPIV-
2
which may be used to immunize mammals, including humans, against wild-type
HPIV-2 infection. It is a further object of the invention to provide vaccine
strains
which are greatly reduced in symptoms produced by the vaccine strain
infection, as
compared to infection with a wild-type HPIV-2 virus strain. It is a further
object of
the invention to provide a vaccine strain of HPN-2 which will generate a
protective
immune response in the patient to whom it is administered.
Applicants have developed and isolated cold adapted vaccine strains of HPIV-
2 from the Saint Louis University wild type strain of HPIV-2 designated SLU
72».
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Several attenuated strains have now been isolated which have
the desirable phenotype of cold adaptation and temperature
sensitivity.
Thus, the present invention is drawn to isolated,
5 attenuated strains of HPIV-2 virus which exhibit the
phenotypic properties of cold adaptation and temperature
sensitivity. Preferred isolated strains with these
characteristics are those designated C3396, C3464, C3490,
C3440, and C3444. More preferred isolated strains are those
designated C3464, C3490, and C3440, In addition, the
present invention is drawn to isolated, attenuated strains
of HPIV-2 which exhibit the cold adapted and temperature
sensitive phenotypes which are progeny or sub-clones of the
isolated strains designated C3396, C3464, C3490, C3440, and
C3444.
In addition, the present invention is drawn to
vaccine compositions for use as live, attenuated vaccines
which comprise any of the HPIV-2 viral strains described
above and a pharmaceutically acceptable carrier. These
compositions may also include any pharmaceutically
acceptable excipients, diluents, and/or adjuvants. The
present invention is also drawn to a method of producing a
protective immune response in a mammal by inoculating the
mammal with a live, attenuated viral strain of the present
invention.
In one aspect, there is described an isolated
viral strain selected from the group consisting of those
deposited with ATCC under Accession Nos: PTA-1471, PTA-1472,
PTA-1473, PTA-1474, and subclones or progeny of any of the
aforementioned strains, and their use for inducing a
protective immune response in a mammal.
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Brief Description of the Figures
FIGURE l: A cold passaging diagram showing the
lineage of isolated viral strains C3440 and C3490, described
in the specification.
FIGURE 2: This graph shows the active viral titers
of nasal washes collected from hamsters which have been
inoculated with strain C3490 (~), C3440 (a), C3464 (~), or
the wild type strain, pool 453 (0).
FIGURE 3: This graph shows the active viral titers
of bronchial/lung washes collected from hamsters which have
been inoculated with strain C3490 (t), C3440 (a), C3464 (~),
or the wild type strain, pool 453 (O).
Detailed Description of the Invention
Unlike HPIV-3, wild-type strains of HPIV-2 which
can be successfully cultured and maintained in vitro have
proven difficult to isolate. Applicants tested over fifty
strains of wild-type virus before discovering a wild type
strain which could be sucessfully maintained in in vitro
culture. As shown in the disclosure below, applicants have
developed isolated temperature sensitive (ts) and
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cold adapted (ca) viral strains from non-temperature sensitive
and non-cold adapted wild type (wt) HPIV-2 viral strains. As
shown in FIGURE 1, applicant successfully modified the wild
type strain of HPIV-2 to grow at reduced temperatures, creating
strains preferably adapted to less than 30°C, more preferably
to less than 26°C, and most preferably to less than about 24°C.
These cold adapted strains are then assayed to confirm that
they are appropriately temperature sensitive. Applicants have
discovered that a fraction of the cold adapted strains will
exhibit temperature sensitivity to the degree necessary to
prevent viral growth and proliferation in the lower respiratory
tract, and the accompanying severe symptoms of HPIV-2 illness.
The combination ca and is phenotypes of these developed strains
make them excellent attenuated strains for use as live vaccine
against HPIV-2 infection, as they are able to grow without
restriction at cooler production temperatures, and are
attenuated in the patient to whom they are administered.
Although a particular HPIV-2 wt strain was used for
developing the strains disclosed below, it is believed that any
wt strain which can be maintained as an in vitro culture may be
used to develop a is and ca attenuated strain of HPIV-2 using
the methods demonstrated by the applicant. Fetal Rhesus monkey
lung (FRhL-2) cells are preferred as hosts for cold-passaging,
as they are well characterized cells used in vaccine studies.
However, other cultured mammalian host cells are contemplated
for use in producing the attenuated viral strains of the
present invention. Likewise, one of skill in the art may
choose to modify the cold passaging technique, using different
temperatures or numbers of cold passages at each temperature.
However, such modification would preferably maintain gradually
stepped temperatures, similar to those described by the
applicants below.
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7
Strain SLU 7255 of parainfluenza virus type 2 (HPIV-
2) was isolated from a 6 month old child hospitalized with
croup and pneumonia (deposited with ATCC on March 9, 2000,
Accession No. PTA-1474). Although it was originally isolated
in primary Rhesus monkey kidney (RMK) cells, SLU 7255 was
adapted to grow in fetal Rhesus lung (FRhL-2) cells, a diploid
cell line used for vaccine studies. Following adaptation to
the FRhL cells, SLU 7255 was serially passaged in the cold
(__<30°C) to produce vaccine candidates in a similar fashion to
the JS strain of HPIV-3, described in Belshe and Hissom, 1982.
The wt strain was first passaged 6 times at 30°C, then 6 times
at 28°C, then 8 times at 26°C, then 13 times at 24°C. See
Figure 1 for a diagram of the cold passaging process.
Applicants were surprised to find that the cold-passaging
temperature had to be stepped down gradually in order to
successfully adapt HPIV-2 virus, unlike HPIV-3, which can be
immediately cold passaged at 22°C. After cold adaptation,
clones were selected by passing a Pasteur pipet through an
agarose overlay using a standard plaque assay technique in
primary African green monkey kidney (AGMK) cells, aspirating
the agar plug, and inoculating the clone into a tissue culture
tube containing primary AGMK cells. After a primary screening
of clones, clones 02450 and 02768 were further cold passaged
about 18-30 times at 23-24°C to yield the isolated clones
03464, 03440 and 03490 (later subcloned to produce 03605). No
successful cold passaging of the HPIV-2 virus had previously
been disclosed. Clone 03490 and 03605 have been deposited with
ATCC on March 9, 2000. Clone 03490 has Accession No. PTA-1473.
Clone 03605 has Accession No. PTA-1472.
To determine if the HPIV-2 clones were temperature
sensitive, the titers of each clone at 32°C and 39°C were
compared using the hemadsorption plaque assay. A clone is
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considered to be "temperature sensitive" when it exhibits a
>_ 100-fold decrease in titer at 39°C compared with its titer at
32°C, and, conversely, is considered to be a wt virus if it
exhibits < 100-fold decrease in titer at 39°C compared with
32°C. More preferably, a clone has a titer of <1.0 pfu/ml at
39°C. The results of the is phenotyping using the
hemadsorption screening assay showed that the majority of the
clones tested exhibited the is phenotype.
To determine if clones possessed the cold-adapted
property, their growth at 23°C was compared with their growth
at 32°C. See Table 1. Each of the clones was inoculated onto
tissue culture tube monolayers of either Vero cells or primary
AGMK cells (data not shown) and incubated at either 23°C or at
32°C. Tube cultures were harvested from each of the clones on
day 7 and day 14 post inoculation when incubated at 23°C and on
day 7 post inoculation when they were incubated at 32°C. The
titer of virus in culture supernatants was determined by plaque
assay plaque assayed at 32°C on Vero cells. Plates were
visualized by staining the cells with hematoxylin and eosin
after 5 days. A clone which had a titer at 23°C that was
within one hundred fold of its titer at 32°C was considered to
be cold adapted (ca).
Six of the clones tested were cold adapted, however,
one of them, C3252, did not grow at either temperature. In
contrast to the cold adapted clones (C3396, C3464, C3490,
C3457, C3440, and C3444), the wild type parent control, Pool
453, did not grow in Vero cells at 23°C.
Efficiency of plaquing (EOP) assays were performed to
determine the cut-off temperature of each clone. Each vaccine
candidate was analyzed for its ability to produce plaques in
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7b
Vero cells at 32°C, 36°C, 37°C, 38°C, and
39°C. See Table 2.
C3464, C3490, C3457, C3440 and C3252 exhibited a cut-off
temperature of 38°C
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while two of the clones (C3396 and C3444) were 1000-fold restricted in growth
at
39°C compared with their growth at 32°C.
Clones other than C3396, C3464, C3490, C3457, C3440, and C3444 which
are developed and isolated from wild type HPIV-2 virus in a manner similar to
that
disclosed by the applicant and which also exhibit the is and ca phenotype are
within
the scope of the present invention. Following the examples and teachings set
forth in
this specification, one of ordinary skill in the art would be able to develop
and isolate
is and ca clones from wild type HPIV-2 virus using routine methods.
Additionally, it
is well within the ordinary skill of a practitioner in the art of virology to
further cold
passage sub-clones of the disclosed preferred strains, or to adapt these
strains for
culture in other host cells by utilizing established methods. Thus, subclones
and
progeny of the above preferred strains are also within the scope of the
present
invention.
As shown in the examples below, the isolated viral strains of the present
1 ~ invention are useful in vaccine compositions for inducing a protective
immune
response in mammals. An isolated, attenuated HPIV-2 viral strain of the
present
invention is preferably administered as a live vaccine in an effective amount
which
will allow some growth and proliferation of the virus, in order to produce the
desired
immune response, but which will not produce HPIV-2 disease symptoms. The
proper
amount of the virus to use in the live vaccine will depend on several factors,
including: the virulence or hardiness of the particular isolated, attenuated
HPIV-2
strain; the age of the patient to whom the vaccine will be administered; the
body mass
and general health of the patient to whom the vaccine will be administered;
and
whether the immune system of the patient to whom the vaccine will be
administered
is compromised.
The isolated, attenuated strains of HPIV-2 of the present invention may be
formulated into vaccine compositions for administration to the patient by any
usual
route (as an intraperitoneal or intravenous injection, topically applicable
formulation,
formulation of oral administration, etc.), but is most preferably formulated
as a spray
or wash for application to the mucosa of the upper respiratory tract. Such
application
will assist in stimulating local mucosal immunity, which will offer greater
protection
against later infection by the HPIV-2 wild type virus. Such vaccine
formulations
comprise the isolated, attenuated virus of the present invention and a
pharmaceutically
acceptable carrier, such as sterile saline. In addition, the vaccine
formulation may
comprise pharmaceutically acceptable excipients, diluents, and/or adjuvants
which
will aid in producing a protective immune response in the patient. Excipients
which
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may be used in vaccine formulations of the present invention include agents
which
will help the virus adhere to the mucosa and spread along the surface of the
upper
respiratory tract, such as gums or starches.
Isolated, attenuated strains of HPIV-2 of the present invention may be
S administered in vaccine formulations to mammalian patients in order to
elicit a
protective immune response. After vaccination, the immune system of the
patient will
exhibit a primed immune response to challenges with a wild-type HPIV-2 virus,
moderating the severity of HPIV-2 infection and illness. Although the vaccine
strains
of the present invention are intended for use with human patients, use with
other
mammals which exhibit deleterious symptoms with HPIV-2 infection is also
contemplated 'within the scope of the invention. The vaccine strains of the
present
invention are preferably administered to the patient at a young age in order
to prevent
more severe HPIV-2 infections, which often occur in infancy. Although it is
currently
anticipated that a single administration of the vaccine strain of the present
invention
1 ~ will be sufficient to induce a primed immune response to later challenges
with the
wild-type HPIV-2 virus, more than one administration may be indicated based on
factors similar to those for dosage, listed above. One of ordinary skill in
the art would
be able to devise the proper dosing regimen for a particular patient without
undue
experimentation.
Several examples of the use of the isolated, attenuated HPIV-2 strains of the
present invention are illustrated below. It should be appreciated that these
are offered
as illustrations of the invention, and are not meant to limit the embodiments
of the
invention in any way.
Example 1
2~ Based on their phenotypic characteristics, 3 of the is and ca clones,
C3440,
C3464, and C3490, were chosen for evaluation in hamsters. Table 3 illustrates
the is
and ca phenotype of these selected clones and the w~t parent of these HPN-2
vaccine
candidates. Weanling hamsters were deeply anesthetized and intranasally
inoculated
with either the parent wt virus or one of the vaccine candidates. The titer of
the
inoculum received by the hamsters is shown in Table 4. Each animal received a
total
inoculum of O.lml (O.O~mI/nostril) using a micropipettor with aerosol
resistant pipet
tips to avoid cross contamination. Groups of twenty hamsters were inoculated
with
one of the vaccine candidates or the wt parent virus. Four hamsters from each
group
were euthanized at five time points, day 1, 2, 3, 4, and 7 post inoculation.
Ten
3~ uninoculated animals were euthanized (2 at each of the five time points) as
a control
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W
group. Blood was collected by cardiac puncture and the lungs and nasal
turbinates
were removed from each animal on the day of harvest. Each tissue homogenate
was
tested for virus by plaque assay on Vero cells at 32°C for five days.
The Vero
monolayers were fixed with formaldehyde and stained with hematoxylin and eosin
to
5 visualize virus plaques.
The wt parent HPIV-2 grew equally well in both the nasal turbinates and the
lungs of the weanling hamsters (See FIGURES 2 and 3). The duration of virus
shedding was 4 days with a peak titer of 5.~ pfu/gm of tissue (all pfu/gm
values in
these examples are in logo) in the nasal turbinates on day 3 and a mean peak
titer of
10 5.2 pfu/gm of tissue in the lungs on day 2. Clone 3490, cp5l, was shed from
day 3
through day 7 in the nasal turbinates of the hamsters. The mean peak titer of
C3490
was 4.5 pfu/gm of tissue recovered on day 7. HPIV-2 was recovered from only a
few
animals inoculated with C3440 or C3464 indicating that the clones were
minimally
infectious. Virus was not recovered from the lungs of any animals inoculated
with
l~ one of the three cold adapted clones. These three cold adapted temperature
sensitive
clones were attenuated in hamsters and may be used for additional in vivo
characterization.
Example 2
Each of the clones evaluated in hamsters were also tested for genetic
stability
in vitro. We performed a stress test on each of the clones by serially blind
passing
each of them once each week for four weeks at the permissive temperature
(32°), an
intermediately permissive temperature (3~°C), and the restrictive
temperature (39°C)
to determine if the viruses would revert to the wild type phenotype under
selective
pressure against the is phenotype. The results of the stress test are shown in
Table 5
After each passage, the virus was titered at 32° to 39°C to
detect changes in the is
phenotype. Each of the clones retained their is phenotype after serial passage
at 39°C
indicating that they are genetically stable.
In addition to the stress test we selected plaques from each of these three
cold
passaged viruses to determine if there was a mixture of virus phenotypes
within the
virus pools. (Table 6) Each of the 10 subclones selected from Pool 474 (clone
3490)
were clearly is and exhibited a complete cutoff at 39°C. Two of the 10
subclones
from Pool 477 (clone 3440) exhibited some growth at 39°C but had titers
of at least
100-fold less at 39°C compared with 32°C. All 6 of the subclones
of Pool 484 (clone
3464) had a complete cutoff at 39°C and retained their is phenotype.
These results
indicate that clone 3490 and clone 3464 have a single phenotype in contrast to
clone
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11
3440 which has a mixture of phenotypes. We selected a subclone
(C3605) from C3440 in order to have a more homogenous vaccine
candidate.
Example 3
Three clones of SLU 7255 which emerged as the most
promising vaccine candidates, C3464 (cp50), C3490 (cp63), and
C3605 (cp47, a subclone of C3440). These three were evaluated
in seronegative Rhesus monkeys. Pools of virus were prepared
in Vero cells for each of the clones and the wt virus. Titers
of the pools used in the following examples are shown in Table
7. Clone C3464 has been deposited with ATCC on March 9, 2000
and has Accession No. PTA-1471.
Example 4
The objective of this experiment was to evaluate the
ability of wild type (wt) HPIV2 to infect seronegative rhesus
monkeys. Each of the rhesus monkeys involved in this and the
two following experiments was selected based on their serum HAI
antibody status against wt HPIV2. Monkeys were considered to
be eligible for inclusion if they had an HAI antibody titer of
< 1:8 to wt HPIV2 antigen. A total of 20 rhesus monkeys have
been involved with the three experiments. Sixteen of them
received either the wt HPIV or one of two cats vaccine
candidates and the other 4 animals received placebo. Two of
the 4 monkeys participated as placebo animals in more than one
experiment. Each experiment had two placebo control animals.
Pools of the wt HPIV2 parent and the cats clones,
C3490 and C3605, were prepared in Vero cells. C3605 is a
subclone of isolated strain C3440. The staff at New Iberia
diluted the viruses at the time of inoculation for the first
two experiments but we changed the procedure when the titration
of the post inoculum of Example 5 was determined to be <_ 2.0
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lla
pfu/ml when it was supposed to be 6.0 pfu/ml. The inoculum for
Example 6, both the cats vaccine candidate C3605 and the wt
HPIV2 challenge virus, were prepared at Saint Louis University
and shipped frozen to New Iberia at the intended dose.
Nasal wash (NW) samples and bronchial lavage (BL)
samples were collected from each of the monkeys on day 0 (prior
to inoculation) and on days 3, 5, 7, 10, 12, 14, 17, 19, and 21
following intranasal and intratracheal inoculation of 105.5 pfu
of virus or placebo. Samples were mixed with transport media,
aliquoted, snap-frozen in a dry ice/alcohol bath, and stored at
-70C. Serum samples were collected from each monkey prior to
inoculation and on day 7, 14, 21, 28, 42, and 56 following
inoculation.
CA 02336574 2001-03-08
w
12
Samples were inoculated into duplicate primary rhesus monkey kidney tissue
culture tubes and incubated at 32°C. R.ViIC tubes were hemadsorbed at
days 5, 9, and
14 with Guinea pig erythrocytes. Hemadsorption positive tubes were identified
using
immunofluorescence (IF). Each sample was also quantified by plaque assay on
Vero
cell monolayers at 32°C. Serum samples were tested for antibody to wt
HPIV2
by hemagglutination inhibition (HAI). All samples were tested in the same
assay
following treatment with receptor destroying enzyme (RDE) and heat activation.
Results: Wild type HPIV2 was recovered from the NW and BL samples of
each of the four of the rhesus monkeys inoculated in the first experiment. See
Table
8. Similar titers of HPIV2 were recovered from both the NW and BL samples with
the mean peak titer of z 1.73 pfu/ml on day 7 for NW samples and z I.53 pfu/ml
on
day ~ for BL samples. An HAI antibody response to wt HPIV2 was observed in
each
of the 4 animals by day 21 following inoculation. Neither of the placebo
recipients
shed virus or had an HAI antibody response.
1 ~ Example 5
The purpose of this experiment was to determine the growth, attenuation,
genetic stability, and immunogenicity of HPIV2 vaccine candidates in
seronegative
rhesus monkeys.
Two HPIV2 cats vaccine candidates, C3490 and C360~, were tested. See
Table 9A and 9B. We isolated virus from the nasal wash (NW) samples from each
of
the 4 monkeys who received C3490 although two of them shed for only one day
each
(9~N148 and 95N139). We recovered virus from the NW of 3 of the 4 animals who
received C360~ (Pool 502). One of the animals in the C3605 group, 95NI52, shed
virus on two days although this animal was seropositive (HAI titer= 32) at the
time of
vaccination. The mean peak titer of virus recovered from the NW of animals
shedding HPIV2 was 1.5 pfu/ml on day'7 for C3490 and 1.4 pfu/ml on day 7 for
C360~. None of the 8 monkeys vaccinated with either C3490 or C3605 cats HPIV2
vaccine candidates shed virus from the lower respiratory tract, i.e., from the
bronchial
lavage samples. HPIV2 was not isolated from either of the placebo recipients.
Overall, the HAI data shows a minimal rise (<4 to 8) in two monkeys from the
C3490
group (95N140 and 95N139) and a rise in monkey number 95N148 who went from an
HAI titer of 8 on day 28 to a titer of 32 on day 42 and day 56. However, this
monkey
exhibited titers of 16 from day 0 through day 21, therefore the HAI titer
increase from
8 to 32 may not represent a strong case for an antibody response.
CA 02336574 2001-03-08
~ 1
I3
There were no HAI antibody rises in the group who received C3605 or in the
placebo recipients. Results of the back titration of an aliquot of the diluted
vaccine
indicated that the inoculum for this experiment was too low. The back
titration of the
post inoculum was determined to be approximately 1.0 pfu/ml for C3490 and 2.5
pfu/ml for C360~. From this data, one can see that the vaccine strains are
attenuated
in the lower respiratory tract.
Example 6
The purpose of this experiment was to evaluate the efficacy of the a preferred
attenuated HPIV2 vaccine candidate in seronegative rhesus monkeys. Four
seronegative rhesus monkeys received intratracheal and intranasal inoculation
of a
pre-diluted dose of C360~. All 4 of the animals shed C360~ HPIV2 from their NW
samples. See Table 10. Virus was not isolated from the BL of any animal that
received the cats vaccine candidate. The peak mean titer of vaccine shed from
the
NW samples was 1.88 pfu/ml on day 7. None of the monkeys exhibited an HAI
antibody response to C360~ HPIV2. The placebo recipients did not shed virus or
have an HAI antibody response.
Fifty-six days following their original vaccination with the cats clone C360~,
each of the four monkeys was challenged with a single inoculum of wt HPIV2.
See
Table 1 I . The placebo recipients also received the wt HPIV2 challenge virus.
Virus
was not recovered from any of the monkeys vaccinated with C360~; however, both
of
the animals that originally received the placebo and then were challenged with
the w-t
HPIV2 shed virus from both N~V and BL samples. HAI antibody responses to the
wt
HPIV2 challenge virus were very vigorous in 3 of the 4 monkeys that were
vaccinated
with C360~. HAI titers of Z64 were evident by day 7 following challenge with
the wt
2~ HPIV2 virus. The fourth monkey, 9~N024, seroconverted by day 28 following
challenge. Animals that received placebo and then the challenge virus mounted
an
HAI antibody response similar to the monkeys in Example 4 who were inoculated
with wt HPIV2, i.e., both of the monkeys seroconverted by day 21 post
challenge.
The foregoing descriptions of the preferred embodiments of the present
invention have been presented for purposes of illustration and description.
They are
not intended to be exhaustive or to limit the invention to the precise form
disclosed,
and many modifications and variations are possible in light of the above
teaching.
Such modifications and variations which may be apparent to a person skilled in
the art
are intended to be within the scope of this invention.
CA 02336574 2001-03-08
14
Table 1
Characterization of the wt parent and selected clones of PIV-2 (SLU 72~~) for
the
cold adapted property.
Virus Titer (log pfu/ml)
Clone #
23°C D7 23°C D14 3~°C D7
C3252 <? <?
C3396 . 4.8 6.7 6.4
C3464 3.1 s.3 6.8
C3490 3.8 6.0 . s.7
C34s7 4.0 6,4
C344o 3.4 s.s 6.s
C3444 3.8 5.7 s.3
Pool 4s3 <2 <2 6.1
(Wild Type PIV-2)
CA 02336574 2001-03-08
1J
Table 2
Efficiency of plaquing (EOP) assay of the
wt parent (Pool 453) and selected clones of PIV-2 (SLU 7255)
Clone cp Virus Titer
(pfu/mL)
# Level 32C 36C 37C 38C 39C
C3252 38 2.8 3.0 2.~ 2.7 <2
C3396 50 6.3 6.4 6.0 5.9 2.8
C3464 50 6.1 5.8 5.3 4.6 <?
C3490 63 ~ 5.2 4.9 4.5 4.4 <?
C3457 56 5.4 5.3 4.9 5.2 <2
C3440 47 4.8 4.9 4.6 4.~ <?
C3444 63 ~.8 5.7 ~.l 5.3 2.8
Poo1453 0 7.0 7.2 7.1 7.0 6.9
CA 02336574 2001-03-08
16
Table 3
Cold adapted and temperature sensitive phenotype of selected clones of PIV-2
Virus Interpretation
Titer
(log
pfu/mL)
CLONE cp 32C 39C 23C D14 32C D7 is ca
# LEVEL
C3464 50 6.1 <2.0 5.3 6.8 Yes Yes
C3490 63 5.2 <2.0 6.0 ~.7 Yes Yes
C3440 47 ~ 4.8 <2.0 5.8 6.5 Yes Yes
Pool 0 7.0 6.9 <2.0 6.1 No No
4~3 (wt)
Table 4
Titer of wt and ca PIV-? pools used to inoculate hamsters
Pool Number Clone Number cp Level Titer of inoculum
(log pfu/mL)
474 3490 63 6.0
477 3440 47 6.4
484 3464 50 6.4
453 wt 6.8
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CA 02336574 2001-03-08
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Table 6
Phenotype of PIV-2 subclones
Parent Sub- Virus Titer (Ion
Clone # Clone # pfu/mL)
Pool # 32C 39C
C3490 C359 l 474 4.4 < 1
C3490 C3592 474 5.4 <1
C3490 C3593 474 5.3 <1
C3490 C3594 474 7.0 <1
C3490 C3595 474 G.2 <1
C3490 ' C359G 474 G.7 <1
C3490 C3597 474 6.3 <1
C3490 C3~ 98 474 7.4 < 1
C3490 C3599 474 G.8 <1
C3490 C3600 474 5.8 <1
C3440 C3G01 477 7.2 3.1
C3440 C3602 477 6.9 <1
C3440 C3G03 477 7.3 <1
C3440 C3G04 477 G.6 <1
C3440 C3G0~ 477 7.3 < 1
C3440 C3GOG 477 G.6 <1
C3440 C3607 477 7.1 < 1
C3440 C3608 477 7. l < 1
C3440 C3G09 477 4.7 . <1
C3440 C3610 477 6.7 4.4
C3464 C3621 484 5.5 ~ <1
C34G4 C3G22 484 G.0 <1
C3464 C3623 484 S.S <1
C34G4 C3625 4g4 G.~ < 1
C3464 C3627 484 6.9 <1
C3464 C3628 484 7.0 ~ <1
control 491 6.4 6
SUBSTITUTE SHEET (RULE 26)
CA 02336574 2001-03-08
19
Table 7
Titers of wt and attenuated pools of HPIV-2, SLU 72~~, to be used for
the inoculation of seronegative Rhesus monkeys.
Pool # Clone # Cold passage Virus Titer (log pfu/ml)
level
32C 39C
499 C3464 50 5.4 2.0
500 C3490 63 7.2 1.3
502 C360~* 47 7.8 <1.0
504 wt 0 6.4 6.6
*C360~ is a subclone of C3440
CA 02336574 2001-03-08
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