REVIEW ARTICLE
BioDrugs 2001; 15 (5): 303-323
1173-8804/01/0005-0303/$22.00/0
© Adis International Limited. All rights reserved.
Prophylaxis and Treatment of
Influenza Virus Infection
Ruth Kandel and Kevan L. Hartshorn
Hebrew Rehabilitation Center for Aged, Harvard University School of Medicine and Section of
Hematology/Oncology, Boston University School of Medicine, Boston, Massachusetts, USA
Contents
Abstract
1. Background and Scope of the Problem . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1 Host Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1.1 General Clinical Characteristics . . . . . . . . . . . . . . . . . . . . . . . . .
1.1.2 Immunity to Prevalent Influenza Strain . . . . . . . . . . . . . . . . . . . . .
1.2 Viral Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2. Potential Points of Attack: The Viral Life Cycle . . . . . . . . . . . . . . . . . . . . . . .
3. Prevention of Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4. Emerging Methods of Influenza Vaccination . . . . . . . . . . . . . . . . . . . . . . . .
4.1 Live, Attenuated Influenza Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2 Novel Vaccination Strategies to Increase Cell-Mediated and Heterosubtypic
Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5. Current Antiviral Therapies for Influenza . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1 The Adamantanes (Amantadine and Rimantadine) . . . . . . . . . . . . . . . .
5.2 The Neuraminidase Inhibitors (Zanamivir and Oseltamivir) . . . . . . . . . . . . .
5.2.1 Zanamivir . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2.2 Oseltamivir . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2.3 Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3 Summary of Antiviral Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6. Emerging Antiviral Therapies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.1 Antiviral Agents Directed Against Specific Aspects of the Influenza Viral Life Cycle
6.1.1 Inhibition of Haemagglutinin Activity (HA) . . . . . . . . . . . . . . . . . . .
6.1.2 Proteolytic Cleavage of HA . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.1.3 Viral Uncoating, RNA Polymerase, Budding and Release . . . . . . . . . .
6.1.4 Disruption of the Viral Envelope . . . . . . . . . . . . . . . . . . . . . . . . .
6.2 Potential Value of Recombinant Collectins in the Therapy of Influenza . . . . . .
7. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Abstract
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Influenza virus infections remain an important cause of morbidity and mortality. Furthermore, a recurrence of pandemic influenza remains a real possibility.
There are now effective ways to both prevent and treat influenza. Prevention of
infection is most effectively accomplished by vaccination. Vaccination with the
inactivated, intramuscular influenza vaccine has been clearly demonstrated to
reduce serious morbidity and mortality associated with influenza infection, especially in groups of patients at high risk (e.g. the elderly). However, the inactivated,
304
Kandel & Hartshorn
intramuscular vaccine does not strongly induce cell-mediated or mucosal immune
responses, and protection induced by the vaccine is highly strain specific. Live,
attenuated influenza vaccines administered intranasally have been studied in
clinical trials and shown to elicit stronger mucosal and cell-mediated immune
responses. Live, attenuated vaccines appear to be more effective for inducing
protective immunity in children or the elderly than inactivated, intramuscular
vaccines. Additionally, novel vaccine methodologies employing conserved components of influenza virus or viral DNA are being developed. Preclinical studies
suggest that these approaches may lead to methods of vaccination that could
induce immunity against diverse strains or subtypes of influenza.
Because of the limitations of vaccination, antiviral therapy continues to play
an important role in the control of influenza. Two major classes of antivirals have
demonstrated ability to prevent or treat influenza in clinical trials: the adamantanes and the neuraminidase inhibitors. The adamantanes (amantadine and rimantadine) have been in use for many years. They inhibit viral uncoating by blocking
the proton channel activity of the influenza A viral M2 protein. Limitations of the
adamantanes include lack of activity against influenza B, toxicity (especially in
the elderly), and the rapid development of resistance. The neuraminidase inhibitors were designed to interfere with the conserved sialic acid binding site of the
viral neuraminidase and act against both influenza A and B with a high degree of
specificity when administered by the oral (oseltamivir) or inhaled (zanamivir)
route. The neuraminidase inhibitors have relatively low toxicity, and viral resistance to these inhibitors appears to be uncommon. Additional novel antivirals that
target other phases of the life cycle of influenza are in preclinical development.
For example, recombinant collectins inhibit replication of influenza by binding
to the viral haemagglutinin as well as altering phagocyte responses to the virus.
Recombinant techniques have been used for generation of antiviral proteins (e.g.
modified collectins) or oligonucleotides.
Greater understanding of the biology of influenza viruses has already resulted
in significant advances in the management of this important pathogen. Further
advances in vaccination and antiviral therapy of influenza should remain a high
priority.
1. Background and Scope
of the Problem
Influenza viruses are a major cause of morbidity
and mortality worldwide. In the US alone, influenza epidemics account for an average of approximately 20 000 deaths per year.[1] Although influenza generally causes a self-limited illness, serious
morbidity and mortality can result under some circumstances. The likelihood of a severe outcome
from influenza is determined both by host factors
and by differences between viral strains. Table I
provides a summary of these factors. Important
host factors include not only general clinical characteristics such as age and presence of other med Adis International Limited. All rights reserved.
ical illnesses, but also the extent to which the host
has acquired immunity to the prevalent viral strain
(either through natural exposure or vaccination).
1.1 Host Factors
1.1.1 General Clinical Characteristics
Certain populations are at increased risk of complications of influenza, including the elderly, individuals with cardiac disease, pulmonary disease,
renal insufficiency or diabetes mellitus, and those
who are severely immunocompromised (e.g. bone
marrow transplant recipients or patients with human immunodeficiency virus infection).[1] Excess
deaths associated with influenza infection have
been documented in pregnant women. The rate of
BioDrugs 2001; 15 (5)
Prophylaxis and Treatment of Influenza Virus Infection
Table I. Risk factors for increased morbidity and mortality from
influenza infection
Host factors
General clinical characteristics (indications for influenza
vaccination)
medical illnesses − cardiac disease, pulmonary disease
(asthma, chronic obstructive pulmonary disease, cystic
fibrosis, etc.), diabetes mellitus, renal insufficiency,
haemoglobinopathies
age ≥50 yearsa
immunocompromised states − advanced HIV infection,
transplantation, etc.
residents of nursing homes and chronic care facilities
pregnant women
Lack of immunity to prevalent viral strain
not vaccinated
vaccine did not include current strain
host failed to mount effective adaptive immune response to
vaccine
Viral factors
Differences in virulence among strains
Evolutionary changes in viruses that result in failure of previous
adaptive immune responses (antibody or cell-mediated) to inhibit
viral replication
antigenic drift − new strains developed as a result of stepwise
mutations in viral envelope proteins
antigenic shift − major change resulting from incorporation of
segments of genomic RNA from animal (e.g. avian or
porcine) strains; associated with pandemics
a
In the US, the recommended age for vaccination was recently
amended from ≥65 to ≥50 years in an effort to capture more
persons at risk because of chronic medical illnesses.
hospitalisation for women in the third trimester of
pregnancy is comparable to that of nonpregnant
women who have high risk medical conditions.[1]
Influenza viral pneumonia, a complication of
influenza infection, is uncommon but carries a
poor prognosis. Influenza also predisposes highrisk individuals to bacterial superinfections (see
Hartshorn[2] for review); bacterial pneumonias are
the most common form. A specific association has
been demonstrated between influenza infection
and Staphylococcus aureus pneumonia. However,
the most common cause of bacterial pneumonia
occurring in patients with influenza is Streptococcus pneumoniae. The predisposition of influenzainfected patients to develop bacterial pneumonia
has been documented repeatedly during epidemics
throughout the past century. Other bacterial infec Adis International Limited. All rights reserved.
305
tions closely associated with prior exposure to influenza include otitis media, and, rarely, meningococcal
infections.[3,4] Hence, bacterial superinfections are
a major contributor to morbidity and mortality associated with influenza epidemics.
Although the aetiology of bacterial superinfections complicating influenza is complex, influenzainduced depression of phagocyte function may be
an important contributor. Influenza A virus depresses the ability of neutrophils and monocytes to kill
bacteria in vitro and in vivo.[2,5,6] This inhibiting
effect correlates in vivo with increased susceptibility to bacterial superinfection.[7] Other factors
that may contribute to bacterial superinfection include damage to the airway epithelium, cleavage
of the viral haemagglutinin (HA) by bacterial proteases,[8] and alterations in quantity or functional
activity of surfactant collectins (see section 6).
Infants and young children may experience
complications of influenza, including respiratory
illnesses and otitis media, sometimes requiring
hospitalisation.[9,10] However, most negative outcomes of influenza occur in elderly populations,
especially those living in long term care facilities.
The reasons why very young or elderly individuals
or those with certain medical illnesses are more susceptible to serious morbidity and mortality from
influenza have not been fully elucidated. Some of
these patients clearly have an impaired ability to
mount a fully effective adaptive immune response
(see section 1.1.2). However, other host aspects, including deficiencies in innate immune functions or
respiratory physiology, probably explain the enhanced susceptibility of some patients. Hopefully,
these aspects of the host response to influenza virus
will be better characterised in the near future.
1.1.2 Immunity to Prevalent Influenza Strain
Adaptive immunity can be acquired by exposure to a given strain through natural infection or
vaccination. A given host may be vulnerable to influenza infection because a vaccine was never received or it did not include the infecting strain of
influenza, or because of failure of the vaccine to
induce a protective immune response. The most
prevalent method of vaccination currently in use
BioDrugs 2001; 15 (5)
306
(intramuscular injection with inactivated, trivalent vaccine) is not as effective as natural infection,
or infection with live attenuated virus, at inducing
mucosal immune responses [e.g. immunoglobulin
(Ig) A production], or cell-mediated immunity.[11,12]
In addition, the inactivated vaccine does not induce
protective antibody or T cell responses as frequently
in the elderly as it does in healthy young adults.[13,14]
Severely immunocompromised patients may also
have a reduced immune response to vaccination.
1.2 Viral Factors
One of the most difficult challenges for those
involved in the control of influenza is the propensity of the virus to undergo rapid evolution.[15] This
results in part from the inherent propensity of RNA
viruses to undergo mutation. Of particular importance are mutations in the viral coat proteins, HA
and neuraminidase. As a result of changes in the
viral coat proteins, most frequently the HA, immunity acquired to prior strains is no longer protective
against infection. The ability of influenza viruses
to evolve into new strains as a result of small mutations in viral coat proteins is known as antigenic
drift.
Another more drastic source of variation among
influenza A virus strains results from exchange of
components of the RNA viral genome of a human
strain with those of a closely related animal strain
through a process called reassortment.[15] Major
antigenic changes resulting from reassortment are
termed antigenic shift and result in viral strains to
which most people have little pre-existing immunity. Influenza A viruses have RNA genomes composed of 8 segments, 1 of which encodes the viral
HA. Replacement of the HA RNA of a circulating
human strain with that of an avian strain accounted
for the introduction of the H3 HA subtype into the
human population, and led to the influenza pandemic of 1968.[16] A similar reassortment between
human and avian strains of influenza is likely to
have been responsible for the 1918 pandemic.[17]
The H1N1 strain responsible for the 1918 pandemic
killed more than 20 million people, readily infect Adis International Limited. All rights reserved.
Kandel & Hartshorn
ing healthy young adults and causing a very high
incidence of viral pneumonia and death.
A recent outbreak of avian influenza in Hong
Kong resulted in 6 fatalities in 18 patients with this
strain.[18] This outbreak was the result of direct infection of humans by the avian strain and was limited by lack of human to human transmission, as
well as prompt slaughter of poultry. Had reassortment occurred between this strain and a human
strain, rapid spread among humans could have resulted in a pandemic. The host factors outlined
above could well be of less importance in the event
of such a pandemic. Surveillance programmes are
critical for preventing such developments. Preparedness for rapid development of vaccine against
new pandemic strains is also essential.
2. Potential Points of Attack:
The Viral Life Cycle
As depicted in figure 1, influenza virus infection
is initiated by attachment of the viral HA to nasopharyngeal and respiratory tract epithelial cell surface proteins and lipids that contain carbohydrates
terminating in sialic acid. A variety of protective
mechanisms may act to inhibit infection at this
stage. Some innate immune mechanisms have been
defined, and others are likely to be identified in the
future. Collectins are collagenous lectin molecules,
2 of which, surfactant proteins A and D (SP-A and
SP-D), are present in the airway. These proteins are
capable of binding to the viral HA and inhibiting
the virus from establishing infection.[20,21] Mice
lacking SP-D or SP-A due to gene deletion have
increased weight loss, viral titres and pulmonary
inflammatory responses after infection with a collectin-sensitive strain of influenza (A.M. LeVine and
K.L. Hartshorn, unpublished observations).
Another recently identified SP-D binding protein, gp340, present in both saliva and pulmonary
secretions,[22] inhibits influenza viral infectivity by
itself and through cooperative interactions with
SP-D (K.L. Hartshorn and U. Holmskov, unpublished observations). Adaptive immune mechanisms can prevent infection at this early stage as
well if the host has previously been exposed to the
BioDrugs 2001; 15 (5)
Prophylaxis and Treatment of Influenza Virus Infection
307
Live, attenuated
vaccines
K
A
Neutralising
antibodies,
HA-binding
agents
Cytotoxic
T cells
L
J
I
B
C
H
Adamantanes
Neuraminidase
inhibitors
D
E
G
F
Respiratory epithelial cell
Fig. 1. Influenza virus life cycle, showing infection and propagation in respiratory epithelial cells and points of action of anti-influenza
agents. Free viral particles (A) enter the upper respiratory tract by large droplet transmission. Infection could be prevented at this
stage by antibodies [especially immunoglobulin (Ig) A], mucins, innate immune proteins (e.g. collectins), gp340 or other substances
that can bind to virus particles and prevent establishment of infection of epithelial cells. Since viral attachment to cells is mediated
mainly by the viral surface haemagglutinin (HA), infection is most effectively inhibited at this stage by antibodies or other substances
that bind to the HA. Viral particles that are not neutralised in this manner can infect epithelial cells by attachment of the HA to cell
surface proteins or lipids that contain carbohydrates terminating in sialic acid (B). The viral particle is then internalised by endocytosis
(C). The endosome enters the cytoplasm and the pH within the endosome lowers. The viral core is then released from the endosome
when the viral HA protein undergoes a pH-dependent conformational change, which exposes a domain that induces fusion between
the viral and endosomal membranes (D). The viral core contains viral ribonucleoprotein (RNP), which is composed of 8 distinct viral
genomic RNA segments complexed with the viral polymerase proteins, the viral nucleoprotein and the viral M1 protein. The lowered
pH of the interior of the endosome allows dissociation of the RNPs from the M1 protein and release of the RNPs from the endosome.
In influenza A, the viral M2 protein enables RNPs to dissociate from M1 by acting as a pH-sensitive ion channel through the viral
membrane. The adamantanes inhibit viral replication principally by blocking the ion channel activity of the M2 protein. The RNPs
translocate to the nucleus, where viral genomic RNA and mRNA are transcribed (E). Viral mRNA enters the cytoplasm and viral
protein synthesis takes place (F). The viral polymerase proteins and nucleoprotein return to the nucleus where they are assembled
into new RNPs (G), which are then transported out of the nucleus for assembly of new viral particles (H). Viral envelope proteins
(HA, neuraminidase and either M2 or NB, respectively, in influenza A or B) are produced in the endoplasmic reticulum and Golgi
apparatus and expressed on the luminal surface of the epithelial cell membrane (I). Live, attenuated vaccines can act at this point
by activating cell-mediated immunity against the exposed viral epitopes. Newly formed viral particles then bud off the cell surface,
using cell surface lipids to form the new viral envelope (J). The viral surface neuraminidase protein must enzymatically cleave terminal
sialic acids off viral and cell surface carbohydrates at this stage to allow the viral particle to bud freely off the cell (K). The neuraminidase inhibitors act principally at this stage. Inhibiting neuraminidase activity results in self-aggregation of viral particles and/or
reattachment of viral particles to the cell surface (L), and failure of the virus to propagate to other cells in the same host or to travel
out of the airway to infect other hosts. For human influenza strains, extracellular proteases in the airway cleave the viral HA protein
into HA1 and HA2 subunits. This cleavage is critical for the ability of the newly formed viral particles to infect other cells. In some
avian influenza strains, intracellular proteases can cleave the neuraminidase. For an excellent description of the influenza viral life
cycle see Lamb & Krug.[19]
viral strain either through natural infection or vaccination. Intramuscular injection of inactivated influenza vaccine leads to generation of neutralising
antibodies (mainly IgG) that can prevent infection
 Adis International Limited. All rights reserved.
by binding to the virus in the airway. The mechanisms through which such antibodies inhibit infection may involve inhibition of viral binding to
cells, or inhibition of viral infection after uptake
BioDrugs 2001; 15 (5)
308
into the cell (e.g. via inhibition of HA-mediated
fusion of viral and endocytotic membranes).[23]
Live attenuated vaccines administered through
intranasal inoculation are more effective at inducing antiviral IgA production in the airway, and result in greater protection from infection when
given together with inactivated vaccine to certain
patient groups.[11,24] Neutralising antibodies against
influenza are directed against the viral HA.
As a prerequisite for infection of epithelial cells,
the viral HA must also undergo cleavage into HA1
and HA2 subunits. Cleavage is mediated by host
proteases present in the airway. Recent studies
have indicated that the ability of a given influenza
viral strain to bind to host proteases is an important
virulence factor.[25]
Susceptibility of the viral HA to cleavage by
lung proteases may account for the confinement of
influenza infection to the respiratory tract.[26] The
HAs of human influenza strains are principally
cleaved by extracellular proteases in the airway,
whereas HAs of some virulent avian strains are
cleaved by ubiquitous intracellular proteases.
If the virus does succeed in attaching to the cell,
it generally enters via endocytosis and is processed
via endocytotic pathways. Another remarkable
property of the HA molecule is critical in allowing
the virus to exit the endocytotic vesicle, allowing
release of viral particles into the cytoplasm. As the
pH of the endocytotic vesicle decreases, the HA
molecule undergoes a conformational change that
exposes a domain of the protein that can mediate
fusion of the viral membrane with the membrane
of the endocytotic membrane. As a result of this
fusion, the viral core containing viral ribonucleoprotein (RNP) is released into the cytoplasm and
can travel to the cell nucleus where viral RNA transcription occurs. The RNP is composed of 8 distinct viral genomic RNA segments, each of which
is complexed with the viral polymerase proteins
(PB1, PB2 and PA) as well as the viral nucleoprotein.
Influenza A viral strains have an envelope protein, called the M2 protein, which functions as a
pH-dependent ion channel. During the process of
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Kandel & Hartshorn
acidification of the virus-containing endosome, the
M2 protein allows protons to enter the viral particle.
Acidification of the interior of the viral particle facilitates dissociation of the viral RNP from the M1
protein, which is necessary for release of RNPs into
the cytoplasm and their transition to the nucleus. The
antiviral drugs amantadine and rimantadine inhibit
replication of influenza A viral strains by binding
to, and inhibiting proton channelling through, the
M2 protein.[27]
After entering the cytoplasm, the viral transcriptional apparatus travels to the nucleus of the cell,
where viral genomic RNA and mRNA are formed.
The viral polymerase and nucleoproteins, formed
from viral mRNA in the cytoplasm, return to the
nucleus of the cell where they are packaged along
with new viral genomic RNA segments into new
RNPs. The new RNPs are then transported back to
the cytoplasm for inclusion in new viral particles.
Newly synthesised viral envelope proteins (i.e.
HA, neuraminidase and either M2 or NB, respectively, in influenza A or B viruses) are produced in
the endoplasmic reticulum and Golgi apparatus
and directed to, and expressed on, the mucosal aspect of the cell surface. Cell-mediated immunity
plays a critical role in ultimate clearance of influenza infection,[28] principally through acting at this
stage of the virus life cycle. Prior to budding of
viral particles from the cell surface, expression of
viral proteins render the infected cell susceptible to
lysis by cytotoxic T cells. Viral nucleoproteins are
largely conserved in strains of influenza A or influenza B and are the major target for cross-reactive
antiviral T cells. Live attenuated vaccines, or other
vaccination techniques, may be more effective at
inducing cell-mediated immune responses than inactivated vaccines.[12,29] In addition, cell-mediated
immune responses can be directed against more
conserved viral proteins (e.g. the nucleoprotein or
the M2 protein[18,29]), leading to greater heterosubtypic immunity than is induced by inactivated
vaccines. Heterosubtypic immunity refers to protective immunity against influenza A viral strains
of serotypes differing from the strain used for initial immunisation.
BioDrugs 2001; 15 (5)
Prophylaxis and Treatment of Influenza Virus Infection
New virions bud off from the cell, encased in an
envelope composed of lipid derived from the cell’s
membrane and viral envelope proteins. After release of viral particles, infection ultimately results
in apoptosis or lysis of the epithelial cell. A critical
step in the budding, release and subsequent dissemination of new viral particles is mediated by the
viral neuraminidase. Mammalian cells add sialic
acid as the terminal sugar on carbohydrate attachments on many cell surface proteins and lipids.
Since the viral envelope proteins are heavily
glycosylated and are formed in the cell’s protein
synthesis pathway, they too are sialylated. However, mature viral particles lack sialic acid by virtue of the enzymatic activity of the neuraminidase.
Removal of these terminal sialic acids on viral or
epithelial cell surfaces is critical in allowing the
viruses to avoid reattachment to the same cell or to
other virus particles. The neuraminidase inhibitors,
zanamivir and oseltamivir, were synthesised based
on knowledge of the sialic binding site of the neuraminidase derived from x-ray crystallography.[30,31]
These drugs are synthetic derivatives of sialic acid,
which bind irreversibly to the sialic acid binding
site of neuraminidase and inhibit its function.
When the neuraminidase is impaired, clusters of
viral particles can be seen to accumulate at the cell
surface on electron microscopy.[32] For an excellent description of the influenza viral life cycle and
background references see Lamb & Krug.[19]
3. Prevention of Infection
Vaccination is currently the most effective and
economical method of controlling influenza virus
infection. Vaccination may prevent infection by
inducing production of specific antibodies that complex with the virus prior to infection of the respiratory epithelium or through cell-mediated destruction of infected cells early in the cycle of
infection. Several significant challenges are involved in vaccination against influenza. As summarised in section 1.2, continual evolution of novel
viral strains necessitates development of new vaccine preparations each year. Extensive surveillance
and careful analysis are required to successfully
 Adis International Limited. All rights reserved.
309
identify the strains that will be prevalent each year
so that sufficient quantities of vaccine can be produced in advance of the influenza season.
Currently, trivalent intramuscular, killed virus
vaccines are used most commonly. The vaccine includes the most prevalent influenza A strains (generally one each of H1N1 and H3N2 subtypes) and
an influenza B strain. Current recommendations in
the US call for vaccination of all individuals who
are at increased risk of morbidity and mortality
from influenza and those in contact with such individuals (see table I, ‘Host factors’), although vaccination is beneficial for healthy adults as well (see
below). Use of this vaccine has been shown to significantly reduce the rate of hospitalisation from
pneumonia and other serious complications of influenza infection in patients at high risk (e.g. the
elderly or patients with diabetes).[33-35]
One approach to improving the effectiveness of
vaccination programmes involves identification of
new groups for vaccination. Vaccination of schoolage children may be an effective means of limiting
the spread of influenza in the community since
transmission within schools and then to adult family members is an important means of viral dissemination.[36] Vaccination of children also has benefits for the children themselves (e.g. reduced asthma
exacerbations).[37] Vaccination of employees of
healthcare institutions, particularly those involved
with care of vulnerable populations, is strongly
recommended. Vaccination of healthcare workers
in long term care facilities has been shown to reduce mortality among elderly residents.[38,39]
Although hospitalisation for pneumonia or mortality is rare among healthy working adults infected
with influenza, vaccination does reduce morbidity
and days lost from work by this group.[40] It should
be emphasised as well that evolution of a new pandemic strain of influenza due to antigenic shift could
result in serious morbidity or mortality in healthy
adults (as in the 1918 and 1968 epidemics). Under
such circumstances, vaccination (if available) would
be universally indicated. However, with current
technology it appears unlikely that sufficient vaccine could be produced to meet this need.
BioDrugs 2001; 15 (5)
310
Inactivated influenza vaccines currently in use
are generally well tolerated. Since the vaccines are
produced in hens’ eggs, they are contraindicated in
individuals with egg allergy. If a person has an active febrile illness it is recommended that vaccination be withheld until symptoms have resolved.
The vaccine raised against the swine influenza
strain of 1976 was associated with an increased risk
of Guillain-Barré syndrome (approximately 10 additional cases/million vaccinated). Such an association is less clear for subsequent influenza vaccines. If there is an added risk it appears to be very
slight (i.e. in the range of 1 additional case/million
vaccinated).[41] It is also unclear if influenza vaccination could induce relapse of Guillain-Barré syndrome. Two patients who received the 1976 vaccine did suffer such a relapse; however, there have
not been subsequent reports of this association.[42]
A recent study found no risk of relapse of multiple
sclerosis associated with influenza vaccination.[43]
In contrast, naturally occurring influenza infection
has been associated with relapse of multiple sclerosis.[43] There is conflicting data regarding possible increased replication of HIV in HIV-infected
patients vaccinated for influenza.[44] It is currently
recommended that HIV-infected patients receive
influenza vaccination since they are probably at risk
for more prolonged or more severe influenza infection.[1,45]
Unfortunately, some of the groups most vulnerable to complications of influenza may not mount
an effective immune response to the intramuscular
inactivated influenza vaccine. This includes elderly and immunocompromised individuals. Recent studies also demonstrate that chronic stress
resulting from the need to care for a spouse with
dementia is associated with a reduced immune response to influenza vaccination.[46,47] Although
many individuals at high risk may not be fully protected against infection, the vaccine lessens the risk
of complications in these patients[1] and should still
be used.
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Kandel & Hartshorn
4. Emerging Methods of
Influenza Vaccination
There are 2 major limitations to the intramuscular, inactivated influenza vaccine, which have
spurred attempts to develop alternative vaccines.
First, the current vaccine has reduced effectiveness
for inducing protective immunity in high risk
groups (e.g. the elderly). Second, immunity induced by the current vaccine is specific for the viral
strains included in the vaccine. Both of these problems may relate in part to the fact that the intramuscular, inactivated vaccine predominantly induces a
serum antibody response, and is relatively less effective for inducing mucosal IgA production, or
cell-mediated immune responses. Alternative vaccine preparations and routes of administration are
under study in an attempt to overcome these problems (see table II).
4.1 Live, Attenuated Influenza Vaccines
Live, attenuated influenza vaccines, administered through the intranasal route, have been extensively studied in human trials. Generally, these attenuated vaccines are cold-adapted variants of
circulating viral strains developed through repeated passage at suboptimal temperature. Such
vaccines have been shown to be well tolerated in
many high risk patient groups, including those
with chronic lung disease[11] or HIV infection,[48]
and in children[24] and the elderly.[12] The intranasal
vaccine is associated with mild nasal symptoms but
is otherwise well tolerated. One possible advantage
of intranasal administration is increased production of respiratory IgA.[11,24] In addition, combined
use of live, attenuated, intranasal vaccine and intramuscular, inactivated vaccine has been found to
cause greater induction of cell-mediated immune
responses to influenza in the elderly than inactivated, intramuscular vaccine alone.[12,49] Live,
intranasal, attenuated vaccine has also been shown
to protect children against natural or experimental
influenza infection.[24] Further trials will be needed
to determine whether the live vaccine provides
greater protection than inactivated vaccine.
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Table II. Novel vaccination strategies
Limitations of the current inactivated, intramuscular vaccine
Failure to induce immunity to unrelated strains; need for yearly
reformulation
Limited induction of cell-mediated immune response
Limited induction of mucosal immunoglobulin (Ig) A response
Elderly and immunocompromised individuals are less likely to
develop protective immune response
Novel vaccination strategies
Vaccines in clinical trials
live attenuated, intranasal vaccines: provide increased
mucosal IgA and cell-mediated immune response
Vaccines tested in animals
vaccination with conserved viral components (e.g.
nucleoprotein, M2 protein): provides immunisation against
divergent viral strains
DNA vaccines: provide increased cell-mediated immunity
sequential administration of DNA vaccine followed by viral
protein vaccine, or recombinant vaccinia vaccine: potentiate
response to DNA vaccine, effective protection against
simian immunodeficiency virus in macaques
adminstration of vaccine with cytokines: increase
cell-mediated response using type 1 cytokines (e.g.
interleukin-12)
Recent studies have determined the molecular
basis of temperature sensitivity in some strains.
These discoveries have allowed development of attenuated strains through planned mutagenesis.[50]
A new method of efficiently generating mutant influenza strains entirely from cloned viral cDNAs
could facilitate production of attenuated strains.[51]
Viral strains with mutated neuraminidase have attenuated growth characteristics and induce immunity against wild-type virus in mice.[52]
4.2 Novel Vaccination Strategies to
Increase Cell-Mediated and
Heterosubtypic Immunity
One of the important long term goals of influenza (or other virus) research has been to develop
vaccines that could induce heterosubtypic immunity, or immunity that would protect the host
against variant strains of influenza virus. Such an
achievement would be of enormous significance
not only because it would obviate the need for
yearly vaccination, but also because it could provide protection against reassorted, pandemic viral
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strains. Efforts to engender heterosubtypic immunity have focused on vaccination using more highly
conserved viral components (e.g. the viral nucleoprotein or the M2 protein) or methods of vaccination that more strongly induce cell-mediated immune responses.
Recent advances in immunology have provided
better understanding of the mechanisms of immune
response to vaccination, which appear to allow more
precise tailoring of these immune responses. The
adaptive immune response to infection or vaccination can be broken down into 2 major categories,
type 1 and type 2, depending on the nature of cytokines and helper T cells involved. Type 1 responses
involve strong cell-mediated immunity, with relatively weak antibody production, whereas type 2
responses favour antibody production with weak
cell-mediated immunity. The failure of neonates to
mount an effective immune response to standard influenza vaccination has been ascribed to their tendency to preferentially develop type 2-like immune responses.[53] The factors that influence the
development of type 1 or type 2 immune responses
to vaccination or infection include the local cytokine milieu, presence of immunologically active
hormones, dosage or route of antigen administration, or the type of cell involved in antigen presentation.[54] Effective generation of heterosubtypic
immunity, and more effective vaccination of individuals at high risk, will probably involve techniques
that lead to stronger type 1 immune responses than
are afforded by inactivated, intramuscular or viral
protein-based vaccines alone.
As noted, live virus vaccination appears to engender greater cell-mediated immunity than killed
vaccine. A recent study demonstrated that immunisation of mice with a live virus that shared
internal viral protein genes, but not the HA gene,
with the highly lethal A/HongKong/156/97 avian
virus, protected mice against subsequent challenge
with the A/HongKong/156/97 strain.[18] In this
model, the protection was T cell mediated, since
vaccination was also effective in mice deficient in
antibody production.
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Modifying the route of vaccine delivery, or the
use of DNA vaccines, may also favour development of cell-mediated responses. Delivery of either
DNA or inactivated influenza vaccines through
‘gene gun’ injection into the skin can lead to protective immunity that is less strain-specific than
that obtained with current vaccination methods.[55]
This appears to result from greater access of viral
antigen to antigen-processing cells, resulting in potent stimulation of an HLA Class I restricted T cellmediated (i.e. type 1) immune response.[56-58]
Immunisation using plasmid DNA encoding the
influenza viral HA has been shown to induce strong
cell-mediated immunity to influenza in mice,
which was protective against respiratory challenge
with live virus despite low levels of anti-HA antibody production.[58] Plasmid DNA encoding the
influenza nucleoprotein also induced protective
cell-mediated immunity in mice.[29] The effectiveness of DNA vaccination in protecting against infection may be increased by concurrent inoculation
with genes encoding viral and host defence proteins [e.g. granulocyte-macrophage colony-stimulating factor (GM-CSF) or the co-stimulatory molecule B7.2], or by following DNA vaccination with
use of a protein vaccine or live virus vaccine.[57]
For instance, use of DNA vaccine followed by live
boosting with a fowl pox virus incorporating genes
for common human immunodeficiency virus proteins resulted in protection of rhesus macaques
from simian immunodeficiency virus infection, despite engendering low-to-undetectable levels of
neutralising antibodies.[59]
Another method of increasing cell-mediated immune response to influenza vaccination could involve co-administration of vaccine along with cytokines that favour development of a type 1 immune
response. Administration of interleukin-12 concurrently with inactivated influenza vaccination to
neonatal mice has been shown to have such an effect.[53]
Although the bulk of evidence suggests that enhancement of cell-mediated immunity is critical to
the development of heterosubtypic immunity to influenza, there is also evidence that antibody pro Adis International Limited. All rights reserved.
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duction and B cells may mediate broad-based immunity in murine studies.[60] A recent study demonstrated that a subunit vaccine incorporating the
conserved extracellular domain of the M2 protein
of influenza A virus elicited long-lasting production of immunoglobulin against the M2 protein that
protected mice against divergent strains of influenza.[61] The evolving research into the basis for
heterosubtypic immunity to influenza is extremely
important as a strategy for reducing mortality in
future pandemics.
5. Current Antiviral Therapies
for Influenza
Approaches to antiviral therapy for influenza,
both in current use and in development, are listed
in table III. Four antiviral agents are currently
available for the treatment of influenza, 2 adamantane derivatives (amantadine and rimantadine) and
2 neuraminidase inhibitors (zanamivir and oseltamivir). The antiviral activity of the adamantanes
is restricted to influenza A viruses, whereas the
neuraminidase inhibitors have activity against both
influenza A and influenza B. For optimal effect, all
of these drugs must be started as soon as possible
after recognition of clinical illness. It is recommended that treatment begin within 2 days of onset
Table III. Approaches to antiviral therapy for influenza
Inhibit binding of haemagglutinin to respiratory epithelium
carbohydrate inhibitors of haemagglutinin
collectins
Inhibit proteolytic cleavage of haemagglutinin
Inhibit fusion activity of haemagglutinin
Inhibit M2 proton channel
adamantanes (approved for use)
novel compounds
Inhibit viral RNA polymerase
antisense oligonucleotides
Inhibit neuraminidase
zanamivir and oseltamivir (approved for use)
novel compounds: aromatic inhibitors
Disrupt viral envelope
defensins
Enhance host defence function of phagocytes, lymphocytes
collectins
defensins
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of illness. The duration of drug treatment is 5 days
for all the anti-influenza drugs. Amantadine and
rimantadine are available for use in chemoprophylaxis of influenza A infection. Oseltamivir was recently approved for prophylaxis against influenza
A and B infection in the US. Initial studies (see
section 5.2.1) have indicated that zanamivir is effective for prophylaxis as well.
5.1 The Adamantanes (Amantadine
and Rimantadine)
As noted in section 2, these agents inhibit viral
replication principally by blocking the ion channel
activity of the influenza A M2 protein. Both
amantadine and rimantadine are approved for prophylaxis of influenza A in individuals aged >1 year.
These medications provide an important adjunct to
vaccination in the management of influenza outbreaks in institutional settings. Centers for Disease
Control (CDC) recommendations call for drug administration for ≥14 days after recognition of an
outbreak (or for approximately 1 week after the
end of the outbreak).[1,62] The dosages recommended for prophylaxis are the same as for treatment.
A recent review evaluated the potential for prophylactic use of these agents in the event of pandemic
influenza.[63] One advantage of these compounds
is their extraordinary stability in storage at room
temperature.[64]
Both amantadine and rimantadine are effective
for the treatment of established influenza A infection, reducing viral shedding and symptom duration if begun within 48 hours of the onset of symptoms of influenza.[65,66] Rimantadine is approved
for the treatment of influenza in people aged ≥14
years, while amantadine is approved for treatment
of those aged ≥1 year. The adamantanes have not
been compared directly with neuraminidase inhibitors in randomised trials. Reduction of serious
complications of influenza (e.g. hospitalisation,
pneumonia, death) has not been clearly documented for any of the adamantanes. A recent trial
of rimantadine treatment for healthy individuals
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ding and symptoms, but failed to show improvement of otological abnormalities resulting from infection.[67]
There are several limitations to the clinical usefulness of the adamantanes. These drugs have activity only against influenza A strains. Furthermore,
resistance to the adamantanes develops frequently
and rapidly. Resistant viral isolates have been obtained from treated patients within 2 to 7 days of
treatment. Resistance appears to be mediated by
point mutations in the M2 viral protein.[68] Rimantadine-resistant strains are completely cross-resistant to amantadine.[65] It has been estimated that 10
to 30% of immunocompetent adults will develop
resistant viral isolates after treatment.[65] The incidence of resistance is probably higher than this in
severely immunocompromised patients (e.g. bone
marrow transplant recipients) who shed virus for
longer periods than immunocompetent individuals.[69] Rimantadine-resistant strains can be transmitted to household contacts causing typical influenza infection.[70] However, there is no evidence
that rimantadine-resistant strains are more or less
virulent than wild-type strains, and rimantadine resistance remains low in field isolates of influenza
A viruses.[71]
There are some important differences between
amantadine and rimantadine. Although the cost of
rimantadine is somewhat greater than that of amantadine, there are some clinically important advantages with rimantadine. The occurrence of CNS adverse effects was significantly less for rimantadine
than for amantadine in a randomised trial of sequential prophylaxis in elderly nursing home
residents.[72] Despite adjustment of dosage for creatinine clearance, amantadine caused a 18.6% incidence of CNS toxicity overall compared with
1.9% for rimantadine. Although male gender and
reduced creatinine clearance were independent
risk factors for CNS toxicity, amantadine use was
a considerably more powerful risk factor. The most
common CNS toxicity with amantadine was confusion (10.3%); however, amantadine can increase
the incidence of seizures in patients with seizure
disorders as well. Seizures have also been reported
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in patients receiving rimantadine, but appear to occur less frequently than with amantadine.[65] Adverse effects of confusion or gait difficulties resulting from the use of amantadine are of particular
concern in the nursing home setting because of the
risk of falls or aspiration. There is a roughly similar
occurrence of gastrointestinal adverse effects for
amantadine and rimantadine. Nausea has been reported in approximately 3% of patients receiving
rimantadine.[65]
There are also significant differences in the
management of dosage adjustments between amantadine and rimantadine. Amantadine is excreted essentially unchanged in the urine, and it is necessary
to make dosage adjustments for relatively minor
changes in renal clearance. In practice, it is necessary to determine creatinine clearance prior to administration of amantadine, especially among elderly patients in whom renal insufficiency and risk
of serious toxicity are more likely. Because of this
problem, reduced dosage is recommended for
standard therapy in patients over 65 years of age;
however, even at this reduced dosage, excess rates
of adverse effects can occur. Rimantadine is metabolised by both renal and hepatic routes; however,
dose adjustment is required only for severe renal or
hepatic insufficiency.
5.2 The Neuraminidase Inhibitors (Zanamivir
and Oseltamivir)
The neuraminidase inhibitors zanamivir and oseltamivir block the propagation of influenza virus
by complexing with the sialic acid binding site of
the viral neuraminidase. Numerous studies have
now shown that these agents reduce the duration of
viral shedding and provide significant clinical benefits in treatment of naturally occurring influenza.
Overall, the results with zanamivir and oseltamivir
are fairly similar. Both have been approved for
treatment of influenza A and B on the basis of large
treatment trials of naturally occurring infection. In
the US, oseltamivir is now also available for the
prophylaxis of influenza.
Neuraminidase inhibitors are distinguished
from the adamantanes in that they have activity
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against both influenza A and influenza B viruses.
Influenza A has predominated in the studies of
treatment of naturally occurring influenza with
neuraminidase inhibitors. Although some trials of
zanamivir have included >30% patients with influenza B,[73,74] very few patients with influenza B
have been included in trials of oseltamivir. However,
all evidence to date suggests that both agents have
similar activity against influenza A and B.
When used in treatment of naturally occurring
influenza, the neuraminidase inhibitors reduce the
duration of viral shedding and duration of symptoms of influenza by approximately 1 to 3 days. In
general, treatment has been started <48 hours after
onset of symptoms in these studies. There is some
evidence that starting treatment sooner than 48
hours may provide more benefit.[75] In contrast,
there is no evidence that starting treatment later
than 48 hours is beneficial. Given the need to start
treatment early, trials in naturally occurring influenza have included all patients with influenza-like
symptoms in the context of an apparent influenza
outbreak. Results of viral culture and/or serum
samples generally come back after the initiation of
treatment. The percentage of individuals who have
been confirmed positive for influenza infection by
culture, antigen test or serum conversion has varied
between 57% and 79% in various studies.[75,76]
5.2.1 Zanamivir
Zanamivir is administered by oral inhalation using a specially designed inhalation device. Results
of zanamivir treatment of naturally occurring influenza in adults in the US, Europe, Australia, New
Zealand and South Africa (the MIST Trial), have
shown similar results overall.[73,75,76] As noted, the
duration of symptomatic influenza has been reduced by at least 1 day in these trials, with various
indicators of clinical improvement being noted.
Since similar clinical benefits were obtained with
10mg twice daily and 10mg 4 times daily drug administration regimens, 10mg twice daily has become the recommended treatment dose. A recent
trial in children aged 5 to 12 years showed similar
activity and no increased incidence of adverse effects.[74]
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Prophylaxis and Treatment of Influenza Virus Infection
The majority of patients included in these treatment trials have been healthy. However, some data
are now available regarding clinical benefits of
zanamivir in individuals at high risk for complications of influenza. In one trial approximately 90
individuals with high risk features (i.e. age >65
years or cardiac, pulmonary, endocrine or metabolic diseases) were randomised to receive zanamivir, with a similar number receiving placebo. The
subset of individuals who had high risk features
and had confirmed influenza infection appeared to
have the greatest clinical benefit from treatment
with zanamivir (e.g. symptoms reduced by 3 days
in this group). In the MIST trial, 37 high risk individuals were treated with zanamivir. A significant
reduction in complications and antibacterial use
was noted in this group compared with placebo. A
recent pooled analysis of randomised, placebocontrolled trials of treatment of influenza A and B
with zanamivir showed a 2.5-day treatment benefit
in terms of overall symptoms, and a 45% reduction
in complications requiring antibacterials among
154 high risk patients.[77] There is insufficient data
as yet regarding results of treatment of medically
unstable or immunocompromised patients with
neuraminidase inhibitors.
Although zanamivir has not been approved for
prophylaxis of influenza, a large study involving
1107 adults demonstrated that taking 10mg of
zanamivir once daily for 4 weeks during the influenza season was 67% effective in preventing illness, and 84% effective in preventing febrile illness, from influenza.[78]
In general, adverse effects of zanamivir have
been minor and have not exceeded those observed
with placebo. The one exception to this, however,
is the observation that zanamivir can exacerbate
respiratory distress in patients with chronic obstructive pulmonary disease (COPD) or asthma.[79]
In an initial study regarding safety of zanamivir in
patients with asthma (not infected with influenza),
1 of 13 patients experienced bronchospasm.[80]
Current recommendations include instructing patients with asthma or COPD to stop zanamivir if
respiratory function worsens, and for such patients
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to have a fast-acting bronchodilator on hand when
taking zanamivir. Revised labelling for zanamivir
now includes a warning that it is not generally recommended for patients with underlying airway
disease.[81] This adverse effect clearly requires
more study and is of concern because patients with
asthma and COPD are among those most at risk of
complications of influenza. Presumably, adverse
respiratory effects of zanamivir are a reflection of
the route of administration, since similar findings
have not been reported thus far with oseltamivir.
On the other hand, since zanamivir is administered
by oral inhalation, only 20% of the drug is absorbed systemically, 90% of which is excreted unchanged in the urine. As a result, no dosage adjustments appear to be necessary for patients with renal
failure undergoing a 5-day course of zanamivir.
5.2.2 Oseltamivir
There are some salient differences between
zanamivir and oseltamivir both in terms of the clinical properties of the available preparations and in
terms of clinical trial data that has been assembled
thus far. Oseltamivir is administered as an oral tablet. Large trials carried out in the US[82] and Europe[83] have suggested that a dose of oseltamivir
75mg orally twice daily has clinical benefits in naturally occurring influenza similar to those of
zanamivir. Neither trial included enough high risk
patients to analyse results separately in this group.
In addition, as noted, very few patients with influenza B were included in the trials. On the basis of
enrolment criteria of the US trial, oseltamivir is
approved for treatment of individuals aged ≥18
years.
Since oseltamivir is administered orally, it is excreted more extensively in the urine than zanamivir. Hence, unlike zanamivir, dose adjustment
of oseltamivir is recommended for patients who
have renal failure (i.e. creatinine clearance <30
ml/min). Another consequence of the differing
mode of administration is that oseltamivir has an
adverse event profile somewhat distinct from that
of zanamivir. Again, adverse effects are very mild
overall and generally do not exceed those seen with
placebo. However, oseltamivir does cause a signifBioDrugs 2001; 15 (5)
316
icantly greater incidence of nausea and vomiting
than placebo (e.g. 18% nausea and 14% vomiting
as compared with 7.4% and 3.4% for placebo[82]).
This effect may possibly be reduced by administration of the medication with food. Thus far, oseltamivir has not been reported to cause exacerbation
of respiratory insufficiency, although further study
in patients with underlying chronic respiratory
conditions is needed. It is noteworthy that there
have not been significantly increased numbers of
patients discontinuing treatment because of adverse effects of neuraminidase inhibitors (compared with placebo) in randomised trials.
Oseltamivir has been administered at 75mg
orally twice daily for 6 weeks during the peak influenza season in a prophylaxis trial.[84] This trial
demonstrated a 74% reduction in the incidence of
influenza infection as compared with placebo. The
only adverse effect that occurred more frequently
with oseltamivir than with placebo was nausea.
Oseltamivir (75mg daily for 7 days) reduced the
incidence of clinical influenza by 89% in household contacts of individuals with influenza, without any evidence of development of viral resistance
in treated individuals.[85] On the basis of these trials, oseltamivir has been approved for prophylaxis
of influenza A and B in the US.
5.2.3 Resistance
Resistance to neuraminidase inhibitors has been
documented in vitro and in treated patients.[80] Resistance can be mediated either by mutations in the
neuraminidase or HA proteins or both.[86,87] Mutations in the HA that result in reduced binding affinity for sialic acid appear to confer resistance by reducing the requirement for neuraminidase in viral
budding and release. Detection of resistance to
neuraminidase inhibitors is technically difficult
since standard viral neutralisation assays are unreliable for this purpose.[86] Resistance to zanamivir
has been reported but appears to be uncommon in
treated patients.[87] In one trial of zanamivir treatment, viral samples were obtained before and after
treatment in 41 individuals, and no resistance was
noted on the basis of either neuraminidase assays
or detection of mutations in neuraminidase genes.[87]
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In a clinical trial of oseltamivir, 2 of 54 (4%) individuals were found to develop resistance on the
basis of neuraminidase assay.[88] Influenza strains
resistant to neuraminidase inhibitors frequently
have reduced infectivity in mice or ferrets, although their infectivity in humans is unknown.[86]
There has been no evidence thus far of transmission
of neuraminidase inhibitor resistant strains to other
patients. More experience with these drugs is required to determine whether resistance will be a
clinically important problem.
5.3 Summary of Antiviral Studies
Although the overall antiviral activity and clinical effectiveness of the currently available anti-influenza agents appear to be similar, there have not
yet been any trials comparing either of the neuraminidase inhibitors with the adamantanes. The
neuraminidase inhibitors have clear advantages
over the adamantanes, including a lower incidence
of adverse effects, a broader spectrum of activity,
and a reduced incidence of viral resistance. However, at this time the adamantanes cost considerably less than the neuraminidase inhibitors.
Amantadine is the least costly of the anti-influenza
medications; however, it is also the most toxic.
There are important limitations to our knowledge base regarding the available anti-influenza
medicines. There is insufficient information regarding the effectiveness of these agents in reducing serious complications (viral pneumonia, bacterial superinfection, cardiovascular failure, death).
Data available so far indicate that zanamivir may
be effective in reducing some complications in
high risk individuals. Although the neuraminidase
inhibitors appear to be generally well tolerated,
more data are needed regarding adverse effects in
patients with acute medical illnesses.
The importance of early detection of influenza
is underscored by the facts that all of the currently
available antivirals must be administered shortly
after onset of symptoms to be effective, and that all
are highly specific for treatment of influenza and
unlikely to benefit patients with other respiratory
viral infections. Although viral isolation remains
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Prophylaxis and Treatment of Influenza Virus Infection
the gold standard for diagnosis of influenza, more
rapid diagnosis is essential so that antiviral treatment can be initiated in a timely manner. Rapid
methods (e.g. using immunofluorescence assays or
ELISA) to detect influenza viral antigens in respiratory secretions are now available.[89]
6. Emerging Antiviral Therapies
Figure 1 illustrates the fact that currently available antiviral agents target relatively few aspects
of the viral life cycle. It seems likely that other
phases of influenza virus replication are vulnerable
to relatively specific inhibition.
6.1 Antiviral Agents Directed
Against Specific Aspects of the
Influenza Viral Life Cycle
6.1.1 Inhibition of Haemagglutinin Activity (HA)
Inhibition of binding of HA to epithelial cells
(B in figure 1) could be an efficient means of blocking infection. The crystal structure of HA complexed with various derivatives of sialic acid has
been determined.[90] Such knowledge has led to rational design of carbohydrate-based inhibitors of
HA.[91] Further development of such compounds
for clinical use is a potential strategy for treatment
of influenza, which up to now has been eclipsed by
the development of well tolerated and effective
neuraminidase inhibitors. Collectins also bind to
the viral HA and probably prevent infection
through interfering with the cellular binding activity of HA.[92] The collectins will be separately discussed in detail in section 6.2.
6.1.2 Proteolytic Cleavage of HA
Proteolytic cleavage of the HA is another critical step in the life cycle of the influenza virus that
is probably vulnerable to antiviral intervention.[25,26] The HA of human influenza strains is
cleaved extracellularly by proteases in the airway.
A protease produced by S. aureus has been implicated as an explanation for the clinical association
between influenza infection and staphylococcal
pneumonia.[8] Proteolytic inhibitors have been
shown to have anti-influenza activity in mice.[93]
In addition, pulmonary surfactant may act as an
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317
endogenous inhibitor of proteolytic activation of
the influenza A HA.[94] The specific components of
surfactant responsible for this inhibition need to be
determined. It is conceivable that topical application of surfactant components or specific protease
inhibitors in the airway could be effective in the
treatment of influenza, although potential effects
of the latter on normal airway functions are uncertain.
6.1.3 Viral Uncoating, RNA Polymerase,
Budding and Release
Determination of the molecular mechanism of
action of the adamantanes has allowed rational development of additional inhibitors of the M2 ion
channel.[95]
As noted, the influenza HA also has a fusion
domain, which is important for viral uncoating in
the cell. Characterisation of the molecular basis of
fusion activity of the viral HA has also allowed
production of potent in vitro inhibitors of viral fusion and infectivity.[96] Of interest, zanamivir has
recently been shown to inhibit cell fusion activity
of parainfluenza and influenza viruses.[97]
Antisense oligonucleotides complementary to
influenza viral RNA polymerase components have
been administered intravenously, in liposome-encapsulated form, to mice, and shown to significantly
prolong survival after infection with influenza A
virus.[98] Additional neuraminidase inhibitors are
also under study that utilise an aromatic benzene
ring in place of the pyranose ring of zanamivir or
oseltamivir.[99]
It is conceivable that collectins could also inhibit viral proliferation after budding of virus from
the epithelial cell surface. SP-D binds to influenza
viral neuraminidase and inhibits neuraminidase activity.[100] Collectins also induce viral aggregation
(see section 6.2), which could contribute to viral
clearance by phagocytic or mucociliary mechanisms.
6.1.4 Disruption of the Viral Envelope
Defensins are low molecular weight antimicrobial peptides produced by phagocytes and in various
epithelial locations, including lung and trachea.[101]
Defensins have broad spectrum activity against a
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variety of pathogens including bacteria, fungi and
viruses. Defensins have activity against various enveloped viruses, including influenza.[102] These
peptides bind to microbial surfaces and induce formation of membrane pores.[103] Defensins could,
therefore, inhibit infectivity of influenza through
disrupting the envelope of extracellular viral
particles. Defensins present in the airway (Human
β defensins 1 and 2) are also chemotactic for dendritic cells and memory T cells, and may, therefore,
stimulate adaptive immune responses.[104] Recombinant production of defensins and other low molecular weight antimicrobial peptides is an attractive area for antiviral research because of the broad
spectrum activity of these agents and their potential
to modulate host defence functions.
Combinations of different antivirals acting
against influenza at different stages of viral replication could be an important area of research in the
future based on the success of this strategy in the
treatment of HIV infection. Such a strategy might
be particularly relevant to treatment of immunocompromised patients given their propensity for
prolonged viral shedding and development of resistance with single agents.
6.2 Potential Value of Recombinant
Collectins in the Therapy of Influenza
Collectins have the potential to beneficially affect host defence against influenza viruses through
several distinct mechanisms. Collectins have activity not only against various strains of influenza, but
also against other viruses, bacteria and fungi. Data
regarding antimicrobial activities of collectins is
thus far confined to in vitro and animal studies.
Recombinant collectins neutralise influenza viral infectivity through interaction with the viral
HA.[92] There are at least 3 collectins in humans, 2
of which were originally isolated from pulmonary
fluids, pulmonary SP-A and SP-D, and another that
is mainly present in serum, mannose-binding lectin
(MBL). Recent findings indicate that SP-D is not
only present in the lung, but also in a variety of
mucosal or other epithelial surfaces in the body.[105]
Of particular note, SP-D present in the oropharynx
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(e.g. saliva, eustachian tube) is also well situated
to provide an initial barrier against influenza.
Deficiency of MBL has been associated with increased risk for infection in children and adults.[106]
MBL deficiency is associated with increased risk
of HIV infection,[107] and increased acute respiratory infections in childhood.[108] It is possible that
qualitative or quantitative alterations in the surfactant collectins could account for the increased susceptibility of some patients to influenza viral infection or its potential complications. This hypothesis
is supported by extensive in vitro and animal studies.[21] Murine data strongly indicate that SP-A and
SP-D play a role in initial host defence against bacterial and viral pathogens, including influenza virus.[109-111] Reading et al.[100] demonstrated that the
severity of influenza virus infection in mice
correlates strongly with in vitro sensitivity of influenza strains to SP-D. Increased replication of collectin-sensitive influenza A strains has been documented in diabetic mice and has been associated
with elevated glucose in pulmonary secretions.[112]
Finally, mice lacking SP-D or SP-A because of
gene deletion have markedly increased viral replication and pulmonary inflammation after infection
with influenza (A.M. LeVine et al., personal communication). These abnormalities can be at least
partially corrected by intranasal instillation of the
collectins (A.M. LeVine et al., personal communication).
Although inherited deficiency of either of the
surfactant collectins has not been described as yet,
lower levels, or altered multimerisation, of SP-A or
SP-D have been associated with some pulmonary
diseases.[113,114] Polymorphisms of SP-A and SP-D
have been identified[115] and appear to be clinically
significant. For instance, certain polymorphic forms
of SP-A and SP-D have been associated with a
strongly increased risk of Mycobacterium tuberculosis infection.[116] Future experiments should establish whether SP-D polymorphisms affect immunity to influenza.
Although the collectins appear to inhibit infectivity of influenza principally through binding to
viral HA and altering attachment of the virus to
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Prophylaxis and Treatment of Influenza Virus Infection
cells, the exact mechanism of viral inhibition has
not been fully established and is the subject of ongoing research in our laboratory. Furthermore, the
collectins have various other functional properties
that could be of importance for their role in host
defence.[20,21,117-119] For instance, the collectins
potently agglutinate influenza virus.[20] Viral aggregation activity of various SP-D preparations has
been shown to correlate with HA inhibition and
neutralisation activity and the ability of SP-D to
promote viral uptake by neutrophils.[120,121] SP-D
not only strongly increases uptake of influenza
virus by neutrophils,[122] but also protects neutrophils against the depressant effects of the virus on
antimicrobial functions in vitro.[20,122] SP-A and
SP-D bind to and aggregate various types of bacteria, including those commonly involved in bacterial superinfections of influenza-infected patients (e.g. S. pneumoniae and S. aureus), and
increase uptake of bacteria by neutrophils[109] and
alveolar macrophages.[123] Hence, deficiency or
impaired function of SP-D could be an important
risk factor for bacterial superinfection, and administration of SP-D could also reduce the risk of bacterial superinfection through protective effects on
neutrophils.
SP-A and SP-D have a very broad spectrum of
antimicrobial activity. In addition to their ability to
inhibit many influenza A and B strains, they have
activity against other common respiratory viruses,
including respiratory syncytial virus[111] and adenovirus.[124] Instillation of a recombinant form of
SP-D in mice reduced the severity of respiratory
syncytial virus infection.[125] SP-A and SP-D have
not only antibacterial but antifungal activity as
well.[126,127] These proteins also appear to play important roles in phospholipid metabolism in the
lung,[128] and modulation of cytokine responses,
and macrophage and lymphocyte activation.[129,130]
It is possible, therefore, that instillation of collectins in the airway could not only have antiviral
effects, but could also correct other respiratory abnormalities in individuals with quantitative or
qualitative deficiencies of SP-A or SP-D.
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319
Increased understanding of the mechanisms of
interaction of collectins with influenza virus has
allowed rational modification of collectins to increase anti-influenza activity. Chimeric collectins,
containing domains of surfactant protein D combined with domains of collectins found in serum,
have enhanced ability to neutralise the infectivity
of influenza virus compared with wild-type surfactant proteins A or D.[121,131] This enhanced neutralising activity results in part from greater affinity of
these chimeric collectins for specific carbohydrates
present on the influenza viral HA.[92] Specific modifications of other domains of SP-D (e.g. neck or
collagen domain) have also been found to increase
anti-influenza activity (K.L. Hartshorn and E.C.
Crouch, unpublished observations). Recombinant
modification has also elucidated the collectin domains responsible for aggregating activity[120,132]
and enabled production of collectins with increased aggregating and opsonic activity.[121,131]
Although these data suggest that collectins have
significant promise for therapy of influenza, there
are potential limitations to their use. Strains of influenza that are highly resistant to the antiviral activities of SP-D or the serum collectins have been
developed in egg culture or through long term passage in mice. These strains have reduced or absent
oligomannose oligosaccharide attachments on their
HA.[133] Collectin resistance has not been reported
in viral isolates obtained directly from patients. However, collectin-resistant strains developed in vitro
do replicate in mice to a greater extent than wildtype strains.[100,134] Strains of influenza resistant to
SP-D are not resistant to SP-A or gp340. This is
consistent with the finding that these proteins inhibit infectivity of influenza through a mechanism
distinct from that of SP-D. SP-D inhibits infectivity through calcium-dependent binding of SP-D to
HA-associated carbohydrates. In contrast, neutralisation of influenza by SP-A[135] and gp340
(K.L. Hartshorn et al., unpublished observations) involves non–calcium-dependent binding of the virus to carbohydrates on SP-A or gp340.
BioDrugs 2001; 15 (5)
320
7. Summary
Development of vaccines that can induce more
broad-based immunity against various influenza
strains, immunise individuals at high risk (e.g.
those who are elderly or immunocompromised)
more effectively, or induce greater production of
oral or respiratory IgA production, is of the highest
priority. However, because the influenza virus can
evolve rapidly and it is not possible to effectively
vaccinate all vulnerable individuals, antiviral therapies will continue to fill an important role in the
containment of influenza infections. The neuraminidase inhibitors are an important advance in
that they are effective against both influenza A and
B, are highly specific in their mechanism of action,
have relatively low toxicity, and do not provoke the
development of viral resistance to the same extent
as the adamantanes. In addition, clinical studies
with zanamivir indicate that administration of antivirals through inhalation can be effective for the
treatment of influenza. More data are needed on the
tolerability and effectiveness of currently available
antiviral agents in patients at high risk. However,
there is now initial evidence that treatment with
neuraminidase inhibitors can reduce complications
of influenza in some high risk individuals. There is
extensive evidence that vaccination can reduce the
incidence of severe complications of influenza
(e.g. pneumonia or death).
The reasons why some people or groups of people are more at risk of serious morbidity and mortality from influenza virus infection are only partially elucidated. It appears likely that innate
immune mechanisms play an important role in the
initial containment of influenza virus. Hence, it is
possible that relative impairment of aspects of innate immunity (e.g. collectins) accounts for the increased susceptibility of some people to complications of influenza infection. As an example, the
susceptibility of diabetic patients to complicated
influenza infection may result from the ability of
glucose to interfere with the ability of collectins to
bind virus-associated carbohydrates.[112] Better understanding of the specific pathophysiological
mechanisms of susceptibility to influenza virus
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Kandel & Hartshorn
may lead to more specific therapies, and enhanced
ability to identify appropriate individuals for treatment or prophylaxis.
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Correspondence and offprints: Dr Ruth Kandel, Hebrew Rehabilitation Center for Aged, Internal Medicine/Geriatrics,
1200 Centre Street, Boston, MA 02131-1097, USA.
BioDrugs 2001; 15 (5)