Jpn. J. Infect. Dis., 53, 219‑228, 2000

Review

Identification of Effective Constituents of Influenza Vaccine bylmmunization with Plasmid DNAs Encoding Ⅵral Proteins

Ze Chen, Takeshi Kuratal and Shin‑ichi TamuraI *College ofLlfe Science, Hunan Normal Universio!,
Changsha 410081, Hunan, PeopleをRepublic of China andJDepartmen t of Pathology, National Institute oflnfectious Diseases,

Toyama 1‑23‑1, Shinjluku‑ku, TobJo 162‑8640, Japan(Received November 29, 2000. Accepted January 1 8, 2001 )CONTENTS:
1. Introduction

2･ Protection agalnSt influenza virus infection by immunization of mice with plasmid DNAsencoding
various viral proteins3 ･ Protective effect of a mixture of plasmid DNAsencoding hemagglutinin (HA) and neuraminidaLSe (NA)molecules

4･ Cross‑protection agalnSt innuenza virus infection provided by DNA vaccine to NA

5･ Differences between mouse strains in development of immunityagainst the plasmid DNAs
6･ Effectiveness of current inactivated HA vaccine supplemented with NA
7. Perspectives

SUMMARY: The use of plasmid DNA encoding influenza viral proteinsto vaccinate animals constitutes apromlSlng approach to the development of effective subunit vaccines. This review describes the results obtainedby the immunization or mice with such plasmid DNAs. (i) Both hemagglutinin (HA)‑ and neuraminidase (NA)‑expressing DNAsfor the surfaceglycoproteinsfrom AnⅣ8/34 (H IN 1 ) or B/Ibaraki/2/85virus can provide themost effective protection against influenza A‑type or B‑type virus infection among the various viral protein‑
expressing DNAs tested in BALB/c mice･ (ii) A mixture or plasmid DNAs encoding HA and NA can provide
more effective protection agalnSt Virus challenge than plasmid DNA encoding HA or NA alone in BALB/c mice･(iii) NA‑DNA can provide protection against infection not only by homologous virus but also by driR viruses.
(iv) HAIDNAfrom A仲RJ8/34 (H IN 1 ) virus provides signiflCant Protection only in BALB/C (H‑2b) mice, whereas

HA‑DNA &om B/rbaraki/2/85 virus affords significant protectlOn in BALB/C, B10 (H‑2d), and C3H (H‑2k)
mice･ NA‑DNA from both A‑type and B‑type viruses provides signiflCant Protection in the three strains of mice.
These results suggest that both HA and NA molecules should beused as vaccine components to provide effective

protection against influenza A‑type and B‑type virus infection in genetically heterogeneoushumans.

type yiTus NA‑like molecules) protein･ NS2 is also found in

1. IAtrOductiotI

the vlnOnS. On the other hand, NSl is found in abundant

Influenza is a highly contag10uS acute reSPlratOry disease,caused bytinfection of the host resplratory tract by the influ‑

quantities in innuenza virus‑infected cells.
Infection is initiated by the binding of HA protein to aenza virus (1 , 2)･ The infectious virions contain eight gene

receptor on the host cells (1‑3). The HA protein is a trimericsegments of negative‑strand RNA coding for ten viral protelnS,
which are nucleoprotein (NP), three polymerase proteins (PA,glycoprotein embedded in the viral membrane, With each

PB 1 , PB2), two matrix proteins (Ml and M2), hemagglutinin

protein subunits, designated HAl and HA2, formed throughmonomeric glycoprotein composed of two disulfide‑linked(HA), neuraminidase (NA), and two nonstmctural proteins(NS 1 and NS2)･ The RNA segments, comple又ed with NP andproteolytic cleavage of a single chain precursor (HAO)･ HA I
lS responsible for the binding of the innuenza virus to thea trimeric RNA‑dependent RNA polymerase (PA, PB I , PB2),

sialic acid receptor on the host cell surface. Followlng Virus

make up theviral RNP (nucleoprotein‑RNA‑polymerase) coreattachment, virions are endocytosed by the host cell. Inthe(Fig･ 1)･ The core is su汀Ounded by a layer of matrix proteln

acidic environment of the endosome, the hydrophobic fusion(Ml), which is encircled by a viral envelope, derived frompeptide of HA2 is translocated towards the target membrane

the plasma membrane of the infected cells after budding･ In

this viral envelope, two glycoprotein spikes, HA and NA, areso as to mediate the fusion between the endosomal membrane
and the viral membrane. The viral M2 protein mediates viralembedded and the ratio of HA and NA in the virions averages

uncoatlng by functionlng aS a hydrogen ion channel for the

5 to l･ The envelope of type A or type B innuenza viruses
also contains one minor component, M2 protein or NB (B‑required for the dissociation of the MI protein from RNP*Corresponding author: Tel: +81‑3‑5285‑I 1 1 1 , Fax: +8 I‑3‑5285‑

the endosome, they mlgrate tO the nucleus where viral tran‑

acidification of virions within the endosome. A low pH iscomplexes･ Followlng the release orthe RNP complexes丘om

1 1 89, E‑mail: stamura@nih.go.jp

scrlPtlOn ensueS･ The initial step of influenza virus replica‑219

In瓜uenza vims

HA

isolation of viral RNA

PBl &2I
cDNA synthesis

100 nmlcloning into plasmid PCAGGS‑P7 (5.20 kb)$811

CMV‑Ⅰ班n ; CMV Imediate‑carly enhancer
β一aCtin ; β‑actin promotor

rbg Pe ;.rabbit P‑globulin polyadenylation site
PCAGGS IP7作IA

Ori ; slmam Vims 40 Origin of replication
P7 ; polylinker

PCAGGS‑P7/NA (1.41 kb)
PCAGGS‑PγMl (0.78 kb)

PCAGGS‑P7nW (1.57 kb)
PCAGGS‑P7/NSl (0.74 kb)(1.74kb)Fig. 1. Structure ofinnuenza virus and plasmid DNA encoding HA, NA, Ml, NP, Or NS 1 fiom A/PR/8/34 virus in a chicken
β ‑actin‑expressed vector (PCAGGS), constructed by Niwa, etal. (50). The diagT･amOfa virion illustratesthe main features of

A‑type influenza virus. TTle Surface of the virion containsthree kinds of spike proteins,the hemagglutimin (HA), neuraminidase(NA),and matrix protein (M2), embedded in a lipid bilayer derivedfromthe host cell and covers the matrix protein (M 1 )that
surrounds theviral core. The core is made up of theribonucleoprotein complex which is comprised of eight single‑Stranded
RNA segments associated withthe nucleoprotein (NP) and three polymerase proteins (PA, PB 1 , PB2).

tion involves the transcrlptlOn Of its anti‑sense viral RNA

Wide pandemics of resplratOry diseases every year as a result(VRNA) to sense messenger RNA (mRNA). Each viral genesegment then replicates. Newly assembled vims particles

B‑type viruses have been iTPlicated in local outbreaks every

bud from the plasma membrane of infected cells by destroying

year as a result of undergolng antigenic drift.

of their undergolng antlgenic shift and drift, while influenza

the receptor by the action of the NA protein.

To control influenza, current inactivated vaccines, which are

Influenza virus replicates throughout the resplratOry tract

composed of entire virions ("whole vims" vaccines), Virions

and is recovered from the upper and lower resplratOry tracts

subjected to treatment with ether (…split‑productM va.Cones),of people naturally or experimentally infected with the virus

or purified glycoproteins ("subunit vacc.lneS"), are Injected(2). A comlation is obseⅣed between the level ofvims shed‑parenterally (2). These inactivated vacclneS mainly induce

ding and the magnitude of the clinical response. Since theserum antibodies (Abs) against HA, which directly neutralize

resplratOry Symptoms Caused by infection develop aRer a shortvirus infectivity. However, the influenza virus alters the anti‑

incubation period, the immunitydeveloped within several days

genic propertiesl of its HA and NA molecules, to circumventaRer infection cannot prevent the onset of these symptoms.

pre‑existing immunity (2, 3). Thus, the inactivated vaccinesThus, to prevent influenza, a protective immunlty must be

only protect agalnSt Viral strains having HA molecules
comprised of identical or cross‑reactive epltOPeS. Accordingly,

induced in advance by vaccination.

Influenza viruses are divided into types A, B, and C based

the protective efficacy of the vaccines is high agalnSt anon the antigenic differences between their NP and M protein

epidemic or homologous vims but low agalnSt an epidemic

antigens ( 1 , 2). Influenza A‑type viruses are血此her subdividedof heterologous vims (4‑6).

into subtype viruses (H INl , H3N2, etc･), which are generated
resultingfrom replacement of gene se甲IentS (antigenic shift)･

On the other hand, Kilboume et al. proposed that purified
NA molecules should be added to the conventional vaccines
for more effective prophylaxis of influenza (7‑9). Naturally

The subtype viruses of the A‑type vlruS Cause minor anti一or experimentally acquired Abs to NA have been reported togenie changes in the HA and NA molecules (antigenic drift).
Thus, influenza A‑type viruses have been implicated in world‑

confer resistance to experimental or natural influenza virus

by marked antlgenic changes in the HA and NA molecules

infection in humansand mice (10‑16). Abs to NA limit viral220

replication in a multiple‑cycle infection and can reduce therecombinant expressing the HA or NA gene of the influenza

level of viral replication below the pathogenic threshold ( I 71

virus were protected agalnSt Challenge infection by the20)･ Although NA, a minor compone.nt iraccines.?r on thehomologous virus, while mice immunized with a vaccinia

intact virion, routinely tmdergoes antlgenlC COmpetlt10n With

recombinant expressing the Ml, M2, NP, NSl, NS2, 0r

immuno‑dominant HA, this antigenic competition between

polymerase (PA, PBl or PB2) gene, or a mixture ofall eight

HA and NA canbe eliminated by adding NA to the vaccinesof the latter vectors, Were not protected against challenge

as a separate purified protein (7‑9, 21‑25). In addition, thefact that NA evolves more slowly than HA (26) suggests that

infection (47). On the other hand, plasmid DNA encoding
the NP of influenza A viruses, administered by intramuscularenhanced protection agalnSt infection can be achieved by

Injection, provided cross‑subtype CTL‑mediated protection

irrmunization with an anti‑influenza vaccine containlng NA(28, 36). ln addition, a fusion protein, Combining the core

antigen of a hepatitis B virus with a highly conserved portion

as an additive.

The development of a more effective vaccine remains anof M2, provided protection agalnSt a lethal influenza virus

important goal in the control of influenza. The first step in thechallenge when administered intraperitoneally or intranasallydevelopment of an effective innuenza vaccine is to identify

to mice (48)･ Based on these.results, we designed experiments

the viral proteins, the immune responses to which provide

to identifythe rpost protectlVe DNAsamong influenza viralprotein‑expressing DNAs･

the highest protection against infection. Immunization withplasmid DNAs expresslng Viral proteins has recently proved

We prepared plasmid DNA encoding HA, NA, Ml , NP, orto be a promisingヮew approach in this respect (27)･ The

NSl from the A/PR/8/34 (PR8) (HINl) virus in a chicken

plasmid DNA vaccclne mlmlCS natural viral infection in that
antlgenS are Produced in their native conformation and are

β ‑actin‑expressing vector (PCAGGS) (Fig. 1 ) and compared

the ability of each plasmid DNA to protect agalnSt influenza
in BALB/c mice (49, 50). Each DNA was inoculated twice,presented in the context of major hitocompatibilitycomplex(MHC) class i and class II molecules to elicit cytotoxic T

3 Weeks apart, at a dose of I LLg per mouse by particle‑

lymphocyte (CTL) responses and other immune responses,

mediated DNA transfer to the epidermi(gene gunPethod)･

including Ab productlOn, respectively. Several publishedstudies have demonstrated that plasmid DNA encoding thechallenged with homologous virus, and the abilityofDNA to

HA or the NP of influenza A‑type viruses, administered byprotect mice from influenza was evidenced by decreased lungeither a gene gun method or by intramuscular Injection,vims titers 3 days and increased suⅣival rate 3 weeks a鮎relicited specific immune responses and provided protection

the challenge infection. The results showed that mice admimiS‑
tered HA‑ or NA‑expresslng DNA were well protected agalnSt
the challenge infection (Table I). Thus, the lung virus titer 3
days aRer infection in mice administered HA‑ or NA‑express‑
lng DNA was slgnificantly lower than that in control mice,

Seven days after the second immunization, the mice Wereagainst influe甲a in mice, ferrets, and chickens (28‑36)･ NP‑

DNA vaccinat10n by intramuscular Injection provided cross‑subtype CTL‑mediated protection (28, 36)･ How.ever･ the mostprotective DNAs among influenza viral proteln‑eXPreSSlng
DNAs remain to be identified.

accompanied by a lOO% survival rate 3 weeks after the

In addition, althoughinfluenza A‑type virus DNA vaccines

challengeinfection. Under these experimentalConditions, thesignificant reduction of lung viruStiters, less than 1 O4･9 EID50/

have been studied extensively, influenza B‑type virus DNA
infection requlrlng hospitalization is about one‑quarter thatml, correlated with long‑termsurvival (5 I). The protection
was accompanied by a high level of specific Ab response.

of influenza A‑type virus infection, but B‑type virus infec‑

The decreased lung virus titers 3 days after the challenge

vaccines have not. The frequency of serious B‑type virus

tion sometimes causes severe illness such as myocarditis andinfection in mice, administered HA‑ or NA‑expresslng DNA

Reye's syndrome (37‑46). Therefore, a study to identifythe
B‑type viral proteins, the immune responses to which give themay be explained by the induction of the specific Ab response

greatest protection against infection, remains to be conductedbeen reported that the influenza virus‑Specific CD8'effector

rather than the induction of a CTL response, because it has

to prevent the disease onset.

or memory T cells fわr CTL responses appear in the

Asdescribed above, a systematic examination for identifi‑

bronchoalveolar lavage of mice from 5 days a洗er pnmary or

cation of the viral proteins, the immune responses to which

secondary infection (52). Onthe other hand, mice administeredprovide the highest protection against innuenza A‑type or B‑
plasmid DNAsencoding viral proteins. The objective of thisMl‑, NPl 0r NSl‑DNAwere not protected (Table 1), although
Ml ‑ and NP‑DNAs induced detectable Ab responses. It has
been reported that Ab responses agalnSt Ml and NP are notrevie† is to identifythe most protective viral proteins withinvolved in protection against innuenza (4, 28).

the alm Of developlng a more effective vaccine. The main

In addition, We detemined the level of protection agalnSt
a lethal‑dose influenza B virus infection in BALB/c mice

type vims, has not yet been made by immunization withpart or this review is composed of our data obtained byimmunizationwith plasmid DNAsencoding influenza A‑

imunized with plasmid DNAs encoding HA, NA, NB, andtype or B‑type viral proteins.

NP from the B/Ibaraki/2/85 virus (53‑55). The influenza
B vimS NB protein is speculated to be a counterpart of theinnuenza A virus M2 protein which exhibits ion channel

2. Protection against in爪uenza virus infection by

activity(2). Each DNA was administered twice, 3 Weeks apart,

immunization of micewith plasmid DNAs

at a dose of 1 LLg per mouse by particle‑mediated DNA transfer
to the epidermis (gene gun.method) or at a dose of30 LLg per

encoding various viral proteins

lt has already been shown that Abs against HA and NA

mouse by electroporation lntO the muscle. Three weeks a洗er

provide protection agalnSt infection by preventlng infection

the second immunization, the mice were challenged with aand by reducing the level of vims replication to below thelethal dose ofhomologousviruS. The results showed that HA‑

pathogeneic threshold, respectively, while Abs against NPandand NA‑DNAsConferred protection against the lethal‑dose

Ml proteins do not provide prワtection (1 ‑4･ 1 7‑20)･ Epsteinviruschallenge (Table 2). The protection was accompanied by

et al. have demonstrated that mlCe immunized with a vaccinia

a high level of specific Ab response. The degree of protection

221

Table I. Protection agalnSt a lethal PR8 virus infection andantibody responses in mice immunized bythe genegun methodwith plasmid DNAsencoding various PR8 viral proteins且Serum antibody titers (10 days after 2nd immunization)

Protection agalnSt PR8 vims challenge

Plasmid DNA

Lung virus liters No. of survivors/

ELISA NI assay lmmunoblotting(log.. EID5./ml)

no. tested (3 weeks)(LLg/ml)

HA

3.1±1.2*5/5*NA

3.1±0.2*

引 α α α W α5 5 5 5 ‑ 1

MI

6.3±0.521±1

NP

6.4±0.210±0

NSl

6.1±0.4

Contro1

6.0±0.3

Infection alone

6. 1 ±0.2

(2

n)

(2

n)

18±0

5.6±1.0
7.5±0.7

9±1

<1
<0.

0 0

1

<3

<1

aMice were immunized with 1 LLg Ofplasmid DNAsencoding variousviral proteins from PR8viruS on day 0 and 21 andchallengedwitha lcthaldose ofPR8virus on day 28 (7 days afterthe second immunization)I Lung virus titers 3 daysand
the survival rate 3 weeks after the challenge infection were determined as an index of protection. Serum lgG antibody
titers 1 0 days aRer the second immunization were determined as an index of antibory responses. Valuse represent mean± SD
of

each

group

of

five

mice.

*Significant

difference

(P

<

0･05)

(ref･

49)

Table 2. Protection agalnSt a lethalB/Ibaraki virus infection and antibody responses in mice immunized bythc gene gun

method (G.G) orthe electroporation method (Electro.)withplasmid DNAsencoding various B/Ibaraki viralproteinsA
Protection agalnSt B/IbarakiviruS infection

Method Plasmid DNA

Lung virus liters No. of survivors/(PFU/ml, Ion)

G.G.

HANANBN P他

no. tested (3 weeks)2.7±0.6*

5 5 0 0 0/ / / / 5/ 5 5 5 5**Serum.antibody titers (24 days after 2Td
lmmun ization)

ELISA NI assay lmunoblotting
(2m)

(2m)

8.5±2.5

3.2±0.4*

2.5±0.7

5.8±0.1

5.7±0.2●一8.5±3.1

5.9±0.2‑Or

E1 らC r1 0

HANANBNP他<1

2.5±0.5書

5 5 1 0 0/ / / / 5 / 5 5 5 5

*

*

<3

11.8±0.5

5.0±0

6.6±0.1†■<1

10.5±1.0

2.7±0.3書a(2n)

8.6±1.2

6.7±0.2

16.2±1.7

6.4±0.1

<1

<3

<1

&Mice were irrununized bythe gene gune method (G.G.) with I LLg ofplasmid DNAsor the electroporation method (Electro･)
with 30 LLg ofplasmid DNAsencoding various viral proteinsfrom B/Ibaraki virus on day Oand 2 1 ･ Three weeks after thesecond immunization,the mice were challengedwith a lethal dose ofB/Ibaraki virus. Serum samples were obtained 24 days
afterthesecondimmunization.

For

details,seelegends

to

Table

1.

(refl

53)

relatively low doses of antigens is essential for the develop‑

provided by gene gun immunization roughly corresponded
to that provided by electroporation immunization, althoughment of a more effective influenza vaccine. Thus, our results
suggest that both HA and NA molecules can be used as
vaccine components to provide effective protection agalnSt
influenza A and B virus infection.

the titers of anti‑HA and anti‑NA Abs, the pro血lCtion of which

was induced by the gene gun method, Were slightly lower

than those of the Abs, the production of which was inducedby the electroporation method (53). This finding suggests thateven a relatively low level of anti‑HA or anti‑NAAb is suffi‑

3. Protective effect of amiⅩture or plasmid DNAs

cient to prevent vims replication. On the other hand, NB‑

encoding HA and NA molecules

and NP‑DNAsfailed to provide protection agalnSt infection

The above results show that viral surface glycoproteins,(Table 2), although NB‑ and NP‑DNAs induced detectable
Ab responses. Thus, Ab responses against B type vims NB
and NP seem not to be involved in protection, as described

HA and NA, are the most protective antlgenS among Several
viral proteins from A and B viruses in mice. The next step ln

forAb responses againstA type virus Ml and NP (4, 28, 49).

the development of an effective vaccine agalnSt influen2:a A
and B virus infection is to prepare a mixture of effective viral

Our results, described above, are consistent with theresults obtained uslng a VaCCinia virus vector expresslng

proteins rather than uslng a Slngle protein.

influenza viral proteins (47)･ On the other hand, oT results
are different from those that showed intramuscular Injectionof plasmid DNA encoding NP of innuenza A viruses to

We compared the abilityof plasmid DNA encoding HA,
NA, Or Ml from influenza virus A/PR/8/34 (HINl), andmixtures of these plasmid DNAs(HA+NA and HA+NA+M I )provide cross‑subtype CTL一mediated protection (28). The

to protect against homologous virus infection in BALB/c mice

discrepancy between the results can be explained by the fact(56). Mice were inoculated with each DNA, twice 3 Weeks

thatinour procedures,the mice were immumized lessfrequently
with relatively lower doses of DNA vaccine than in otherapart, at a dose of I LLg per mouse by the gene gun method

studies (27, 28, 49, 53). Less frequent immunization witha洗er the second immunization. The ability ofDNA to protect

and challenged with a lethal dose ofhomologous virus 7 days222

or in conjunction with other antigens(25).

mice from influenza was evidenced by decreased lung virus
titers and increased survival rates. The results showed thatadministration of a plasmid DNA mixture of either (HA+NA)

4. Cross‑protection against innuenzavirus infectionor (HA+NA+M I ) provided almost complete protection inprovided by DNA vaccine to NA

both lung vims titer and suⅣival rate against the PR8 vims

challenge (Table 3), and that this protection wast accompanied

To prevent influenza, inactivated influenza vaccines, inby high levels of speci丘c antibody responses to the respective

which HA is a major component, are Injected subcutaneously.

components. The degree of protection afforded in these groups

The protective efficacy of these vaccines is highagalnSt

is slgnificantly higher than that in mice administered either

homologousvirus but low against heterologousviruSinfecdon

HA‑ or NA‑expresslng DNA alone, which provided signifl‑(4‑6)･ This is because the influenza virus alters the antigepic

cantly lower lung vims titer and a lO0% suvival rate agalnSt
PR8 virus challenge, or that in mice administered M I ‑express‑

properties of its HA molecules to escape attack by pre‑existlng

The results described above are explained by assumlng thatAbs (1‑3). On the other hand, Kilboume et al. demonstrated
the relatively slower antlgenic evolution of NA than that or
HA (26). The results reviewed in the preceding sections
confirm that the NA molecule should be considered one of
the most effective components for use in the development ofa more protectiveinfluerLZa Vaccine. Thus,the ability ofplasmidDNAs encoding NA molecules, which alter their antlgenic
structure more slowly thando HA molecules, to provide cross‑the HA and NA molecules, derived from the respective

protection agalnSt lethal‑dose heterologous virus infection

plasmid DNAs, can be recognized independently by the hostremains to be detemined.

lng DNA, which failed to improve both lung virus titer andsuvival rate. Thus, ln mice immunized by the gene gun

method, a mixture of plasmid DNAs encoding HA and NA

provided the most effective protection agalnSt the virus
challenge. The addition of M 1 ‑expresslng plasmid DNA tothis mixture did not enhance the degree of protection afforded.

immune system and that the immune responses induced by

We investigated whether NA‑expresslng DNA fromthe respective molecules are highly protective agalnSt thedifferent A viruses, used for the immunization of BALB/cvirus infection. This explanation may be consistent with thatmice, could provide cross‑protection agalnSt lethal‑dose

provided by Kilboume et al.; that is, immunization with aheterologousinfluenza viruSinfection (57). NA‑DNA, preparedfrom A/Guizhou/54/89 (A/Guizhou, H3N2), A/Aichi/2/68 (A/Aichi, H3N2) or A/PR/8/34 (PR8, HINl) virus, was immu‑

mixture of purified HA and NA molecules eliminates the
antlgenic competition which is observed when a large amountof HA and a small amount ofNA are present together on
intact innuenza virus particles (7‑9, 2 1 ‑25). JohanssoP and

nized twice, 3 Weeks apart, at a dose of I LLg Per mouse by

Kilboume have also shown that no additional protection iszation, mice were challenged with a lethal dose ofA/Gui血ou

conferred on mice immunized with Ml and NP either alone

vims and the ability of each NA‑DNA to protect the mice

the gene gun method. Three weeks aRer the second irrununi‑

Table 3. Protection agalnSt a lethal PR8 virus infection andantibody responses in mice immunized bythe gene gun

method withplasmid DNAsencoding various PR8 viralproteinsa
Protection agalnSt PR8virus challenge Serum antibody titers ( 10 days after 2nd immunization)
Plasmid

DNA

Lung

virus

(log..

titers

EID5｡/ml)

Not

of

no.

testedsurvivors/(3weeks)

ELISA

NI(〝g/ml)assay

Immunoblotting

(2n)

HA

4.5±0.3*

10/lo♯

NA

2.9±1.1*

10/lo♯

MI

5.8±0.3

1/10

HA+NA

0.8±0.2書

10/lo♯

25.4±9.4

7.7±0.6

HA+NA+M 1

0.5±0.2*

10/lo♯

14.8±1.3

7.7±0.6

Control6.1±0.6

0/10

Infection alone

5.9±0.4

0/10

(2n)

10.1±4.7

7.3±0.613.3±1.2<0.

116.0±2.0<3<1

AMice were immunized twice, 3 weeks apart with l唯Ofplasmid DNAsencoding HA, NA and Ml &om PR8 virus andtheir mixtures (HA+NA or HA+NA+M 1) by the gene gun method. One week after the second immumization,mice werechallenged witha lethaldose ofPR8 virus. For details, see legends to Table 1 ･(ref･ 56)

Table 4. Protection against a lethal A/Guizhou virus infection and antibody responses inmice immunized bythe gene gun
mcthodwithplasmid DNAsencoding the neuraminidase (NA) protein of various innuenza A virus strainsa
Protection against Guizbou‑X vims
Serum antibody liters (24 days a氏er 2nd immunization)

cb al lenge

Plasmid DNA

NI assay against each virus strain

Lungvirus liters No. of survivors/

AノGuizhou(H3N2)

A/Aichi(H3N2)

PR8 (HINl)(PFU/ml, 10n)

no. tested (3 weeks)

AノGuizhou NA

3.4±0.4*

10/lo♯

8.3±1.5

6.0±1.4<1

A/Aichi NA

3.9±0.2*6/lo♯

6.5±0.6

9.0±0.5<1

PR8 NA

5.4±0.2

1/10

2.5±0.7

Vector

5.2±0.30/10<1

3.5±0.7<1

9.1 ± 1.5<l

AMice were immunized bythe gene gun method with l嶋Ofplasmid DNAsencodingthe NA proteins of various innuenza

virus strains on day 0 and 21. Three weeks afterthe second immunization,the mice were challengedwith a lethaldose of
A/Guizhou virus. Serum samples were obtained 24 days after the second immunization･ For details, see legends to Table 1 ･(ref. 57)

223

Table 5. Protection against a lethalPR8 virus infectionandantibody responses inmice immumizcd withplasmid
DNA encoding HA, NA and NP舟om PR8virus in different strains ofmouseA

Plasmid Mouse strain
DNA(H

Protection against PR8 virus challcngc

Lungvirus titer No. of survivors/‑2)(logl. EID50/ml)

HA

no. tested (3 weeks)′b ′▲U ′hV つJ 3 4 0 3 4 つJ つJ

immunization)

Anti‑HA Anti ‑NA Anti ‑NP
(2m)

(d)

4.2±0.4事

C3H(k)

6.0±0.5

10.4±1.8

B10(ら)

6.2±0.2

4.0±1.2

BALB/C(d)4.4±0.7*C3H(k)

5.0±0.3*BIO(b)

4.8+0.4*

BAL良/C(d)

6.1±0.2C3H(k)

6.0±0.IBIO(b)

6.1±0.2

Vector BALD/C (d)6.2±0.3

NA

NP

BALB/C

Serum antibody 一iters ( 1 0 days after 21d

C3H

(k)

5.9±0.3BIO(b)

6.4±0.3

tJ

つJ 1 1〃 e! 伯 仲 伯 伯 仲 化 花 川 乃 C! 0 0 0 0 0 0 0 0 0 0 0 0

●

■●■

(2n)

(2m)

15.2±3.2

aDifferent strains ofmousc, BALB/C, BIO and C3H, were immunized with1嶋Ofplasmid DNAscncoding HA, NA

and NP from PR8 virus on day 0 and 21 and challenged with a lethaldose ofPR8 virus on day 28 (7 days afterthe
second immunization). For details, see legends to Table 1(ref. 62)

血e lung vims titers and the suⅣival rate. The results showedsignificant protection only in BALB/c mice (Table 5). The
levels of serum Ab titers against NA or HA molecules inthat A/Guizhou virus NA‑DNA could provide complete protec‑

BALD/C, C3H, and 良lo mice were high, intemediate, andtion against homologous virus infection, while A/Aichi virus
NA‑DNA provided a slgnificant degree of cross‑protection
against infection by a variant (drift) virus of the same
subtype (H3N2) (Table 4). PR8 virus NA‑DNA failed tolow, respectively. NP‑DNA failed to provide protection inany mouse strain. These results suggest that NA‑DNA can beused as an effective vaccine component to provide protection

from A/Guizhou virus infection was evaluated by determlnlng

agalnSt infection in various mouse strains.

Furthemore, we compared the ability of HA‑ and NA‑

provide protection against infection by a different subtype
virus. The degree of cross‑protection agalnSt infection correlated

DNAsfrom the B/Ibaraki/2/85 virus to protect agalnSt

roughly with the titers of cross‑reactlng Abs. These results
suggest that NA‑DNA can be used as a vaccine component
to provide effective protection agalnSt homologous as welllethal‑dose influenza B‑type virus infection in three different
mouse strains, BALD/C, BIO, and C3H (53). Each DNA was

as driftviruses.

mouse by the gene gun method or at a dose of 30 〟g per

administered twice, 3 Weeks apart, at a dose of I jig Per

mouse by electroporation into the muscle (53‑55). Three
weeks a洗er the second immumization,the mice were challenged

5. Differences between mouse strains in developmentwith a lethal dose of homologous vims. The results showedof immunity against the plasmid DNAs

that both HA‑ and NA‑DNAs provided protection agalnSt

The murine imune responses to various antigens and thelethal‑dose viral challenge in mice immunized by either thesusceptibity of mice to some pathogens are known to begene gun method or the electroporation method (Table 6).
The degree of protection was accompanied by high levels ofcontrolled by H‑2 or non‑H‑2 genes (58)･ We have previously
reported that anti‑HA Ab responses in mice lmmunizedintranasally with inactivated influenza virus vaccines depend

anti‑HA and anti‑NA IgG Ab responses in all of the tested
mouse strains. These results suggest that both HA and NA

on the mouse strains: H‑2k, H‑2d and H‑2b strains were high,molecules can be used as vaccine components to provide

intermediate, and low responders, respectively (59). Thisbe obseⅣed, because plasmid DNAs encoding HA stimulate

effective protection against innuenza B virus infection.
The results described above show that plasmid DNA
encoding HA ofPR8 virus provided protection only in BALB/cmice, while plasmid DNA encoding HA of B/Ibaraki virus

the immune sytstem similarly to a viral infection but dissimi‑

provided protection in three mouse strains. The discrepancy

result does not mean that strain differences in anti‑HA Ab
responses elicited by vaccination wi血HA‑DNA will routinely

larly to exogeneous protein antigens (27, 60, 61). In thisin the results f♭r inmIenZa A and B viruses can be explained

regard, strain differencesinAb responses to other viral compo‑

by differe.nces in HA‑DNA (HA molecules) of both viruses･
T cell epltOpeS On the HA and NA molecules, which arenents elicited by DNA vaccines remain to be clari丘ed.

We compared the abilityofplasmid DNAsencoding HA,

presented on antlgen‑preSentlng Cells together with MHC
antigens unlque tO each mouse strain, are different between
the influenza A and B viruses (59).

NA, and NP from the A/PR/8/34 (HINl) virus to protect
against a homologous influenza A virus challenge in threedifferent mouse strains; BALB/C (Hl2d), B 10 (H‑2b)and C3H(H‑2k) (62). Mice were inoculated with each DNA twice, 3

6. Effectiveness of current inactivated HA vaccine
Supplemented with NA

Weeks apart, at a dose of 1 jig per mouse by the gene gun
me血od and challenged wi血a le仙al dose ofhomologous vims

7 days after the second immunization. The results showed

The current inactivated vaccines are standardized onlythat NA‑DNA could provide slgnificant protection agalnSt
infection in any mouse strain, although HA‑DNA afforded

according to the content of the HA antlgen,and HA molecules
which predominate over NA molecules on an intact live

224

Table 6･ Protection against a lethalB/Ibaraki virus infection andantibody responses in ditrerent strains of mouse

irnmunizedwithplasmid DNAsencoding HA and NA &om B/Ibaraki virus bythe gene gunmethod (G.G.) or
the electroporation method (Electro.) A
Protection agalnSt B/Ibaraki virus
Plasmid

Method

DNA

Mouse

Chat

HA

NA

VectorElectro.

HA

NA

Vectorimmunization)strain Lungvirus titcr No･ of survivors/(PFU/mI, Ion)

G.G

Serum antibody liters (24 days after 2巾d

lenge

ELISA

no. tested (3 weeks)

(2n)

NI

assay
(2m)

BALB/C3.4±0.5+

10/10書

C3H

3.0±0.1*

10/10*

10.0±1.6810

2.6±0.5*

10/10*

12.0±2.3

BAL凸/C3.7±0.2*

10/lo♯

C3H

3.4±0.6*

10/lo♯

8.5±1.9BIO

3.5±0.5*

10/lo♯

8.3±1.7

9.0±3.59.3±1.5

BALB/C

6.0±0.2

I/10

C3H

5.9±0.3

0/10

<3

BIO

5.8±0.1

0/10

<3

±0.2書

10/lo♯

BALD/c1.5

く く く l一 l 1

tJ 3 5111<3

5 0 00 +一+I+I 1 1 2 0 5 . 1

C3H

1.6±0.3事

10/10+

BIO

1.5±0.2*

10/lo♯

BALD/C

3.3 ±0.9書

10/lo♯

12.5±1.9

C3H

3.6±1.5*

10/lo♯

13.0±1.2BIO

3.1±0.7*

10/lo♯

12.8±2.2

BAL良/C6.9±0. 1

0/10

<3

C3H

6.6±0.6

2/10

<3

BIO

6.9±0.2

I/10<3

AFor details, see legends to Tables I and 2.

(ref.53)

influenza virus are competitive in T‑ and B‑cell priming (2 1‑

production would improve the protective e氏cacy of the current

25). Therefore, the supplementation of conventional innuenza

inactivated vaccines. In this regard, intranasal immunization

A virus vaccines with purified viral NA induces balanced andwith vaccines has been advocated as a means of inducing

broad immTne responses (7‑9)･ Based on our previous
results showlng that Abs to NA molecules afford the highest
degree of protection agalnSt innuenza A virus infection inlgA and sys.temic IgG agaipst influenza virus (64)･ However,mice血at are low responders to HA molecule administration,

together with cholera toxin (CT) or Escherichia coli heat‑administratlOn Of the vacclne alone does not easily generate
S‑IgA. Therefわre, We attempted to administer the vaccine

it seems that cu汀ent inactivated vaccines supplemented withlabile enterotoxin (LT) as an adjuvant (65, 66).

NA molecules could provide effective protection agalnSt
influenza A virus infection in genetically heterogeneous

together with CTB* or LTB* burified CTB or LTB supple‑

humans (62). In addition, the supplementation of cu汀ently

mented with 0. 1% ofCT or 0.5% ofLT ) provides effective

available inactivated vaccineswiththe purified NA of influenzacross‑protection in the upper resplratOry tract agalnSt Variants

A virus, as well as that of influenza B virus, could provide
the most effective protection agalnSt influenza A and B virus
infection in genetically heterogeneoushumans (53, 56, 62).

(drift viruses) within the subtype of the influenza A virus?s

Intranasal immunization with current inactivated vaccines

or variants of the B vims?S (65‑69)･ The cross‑protection ln

the upper resplratOry tract lS Provided mainly by cross‑reacting
anti‑HA IgA Abs, whereas the crossIPrOteCtion in the lower

resplratOry tract is provided by the IgG Abs. We further

7. Perspectives

investigated the vaccination conditions necessary for use of

Current triValent inactivated vaccines, which containcompo‑
nents ofA and B type viruses circulating in humans, arethe adjuvant‑combined vaccine in humans. More effective

administered subcutaneously. These vaccines are shown toby imunization with two doses of vaccine, than with a slngle

induce high levels of antiviral lgG Abs in semm, and have adose of vaccine (5 1, 70). In addition, two doses oftrivalent

protective effect against homologous viral infection. However,vaccines conferred cross‑protection agalnSt Various virus

cross‑protection against lethal‑dose infection was provided

the vaccines are less effective agalnSt heterologous virus

strains. From these studies, We theorized that administration

infection withinthe save subtype, which explains theirineffec‑
tiveness when the vacclne Strain is different from the epidemic

ofadjuvant‑combined vaccine to humans could be performed
effectively and safely via two successive intranasal immuni‑

strain. On the other hand, natural innuenza viral infection

zations, at 4‑week inteⅣals, with a small volume of trivalent

has been shown to be superior to cu汀ent inactivated vaccines

vaccine, pr.epared &om HINl , H3N2, and B type virus strains

for inducing cross‑protection against Variant viral infections(recently clrCulated in humans), mixed with CTB* (or LTB*)(4). The cross‑protection induced by natural infection seems(66, 70).

to be largely due to the induction of cross‑reactlng IgA Abs

In addition, as described in this review, NA molecules, as

to the viral HA molecules in the respiratory tract (63)･ The

well as HA molecules, from influenza A and B viruses should
be considered as some of the most effective components forcross‑reactivityis derived from the polymerlC nature Of S‑
IgA, which generates a greater avidity for the influenza virus
than does serum IgG. This fact suggests that the developmentuse in the development ofaneffective influenza vaccine. This
Rnding, together with the effectiveness of a mucosal influenzaof an immunization procedure to stimulate mucosal lgA

vaccine, suggests that intranasal administration of inactivated225influenza vaccines, supplemented with purified NA, can3013.

provide the most effective protection agalnSt all epidemic

15･ Johansson, B. E. andKiIboume, E. D. (1990): Compara‑

strains of influenza virus. Further studies are needed in orderto realize the development of the most effective inactivatedtive long‑termeffects in a mouse model system ofinnu‑

influenza vaccine in the near future.

followed by sequential infections. J. Infect. Dis., 162,

enza whole virus and purified neuraminidase vaccines
800‑809.

16. Powers, D. C., Kilboume, E. D. and Johansson, B. E.

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