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s
Medical Intervention and
Technological Solutions
OVERVIEW
The post-eradication era is a period of history for which there has been
no precedent whatsoever in terms of a zero base of immunity. Cessation of
immunization will eventually create a population susceptible to widespread
infection in the event of accidental or intentional reintroduction or re-
emergence of the eradicated virus. Thus, even after immunization ceases,
vaccine production must continue.
However, many currently available vaccines may not be appropriate
for continued post-eradication vaccine production or reinstatement. Vac-
cines must be continually improved and ongoing vaccination research main-
tained. Other potentially useful antiviral strategies antivirals, prophylaxis,
and probiotics must also be considered as means to strengthen the im-
mune system and serve as adjuvant or prophylactic therapies.
In the case of polio, for example, it remains to be determined which
vaccine (oral polio vaccine [OPV] or inactivated polio vaccine [IPV]), or
variant thereof, should be produced in the post-eradication, post-vaccina-
tion era. A detailed plan for vaccine production will require more informa-
tion on OPV-derived viral persistence and transmission, as well as continu-
ing dialogue between public health and research communities in order to
ensure that appropriate vaccination research continues.
The immune system may face unforeseeable challenges when immunity
in the community at large wanes in the post-immunization era, and even
immunized individuals may be at risk. Molecular biology technology has
121
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122
CONSIDERATIONS FOR VIRAL DISEASE ERADICATION
advanced to the point where antiviral drugs couici be developed to target
specific viruses. With the exception of HIV and influenza, however, ctis-
eases for which antiviral therapy has been consiclered are not usually con-
sidered epidemic. The research community and pharmaceutical industry
must make a concerted commitment to developing antiviral therapies for
use as potential adjuvants for vaccine-preventable diseases.
Immunoprophylaxis includes both nonspecific approaches to stimula-
tion of innate antiviral defenses and specific prophylaxis clirected at par-
ticular pathogens. Currently, the best understood nonspecific prophylaxis
is interferon (IFN) A/. However, viruses display tremendous variability in
their reponses to the effects of IFN alp, and many viruses have evolved
ways around IFN a/,B's antiviral pathways. As is the case for antivirals,
technology has advanced to a point where specific prophylactics could be
developed for use against vaccine-preventable diseases including small-
pox, polio, and measles but this has not been done.
Finally, current studies suggest that probiotic bacteria living microbes
introduced into the body in order to improve intestinal microbial balance-
could be used to strengthen the immune system, even in immuncompromised
individuals. Novel microbial mechanisms need to be further studied for
their potential use as antigen delivery vehicles and adjuvants.
In an age of unprecedented successful vaccination initiatives, public
and private sector support has led to the rapid development of vaccines for
numerous infectious diseases. Implementation of these products has helped
encourage confidence in the biomedical research and public health commu-
nities and garnered political will for disease eradication initiatives. This
support, confidence, and political will must continue in the post-eradica-
tion era. Strong commitment is needed from both the public and private
sectors to share the costs and risks associated with developing new vaccines
and therapeutic products which may have only a very short product life
cycle. Effective and appropriate antiviral therapies are critical for the pro-
tection of future populations in a post-immunization era.
THE POLIO ERADICATION EFFORT: SHOULD VACCINE
ERADICATION BE NEXT;
Vincent R. Racaniello, Ph.D.
Higgins Professor, Department of Microbiology
Columbia University College of Physicians and Surgeons
New York, NY
The World Health Organization (WHO) goal to eradicate polio by the
year 2000 (now extended to 2005) has resulted in an extraordinary recluc-
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MEDICAL INTERVENTION AND TECHNOLOGICAL SOLUTIONS 123
tion in global incidence of the disease. According to the WHO global plan,
vaccination can stop when eradication is certified, laboratory stocks of
poliovirus are contained, and there is no evidence of persistent vaccine-
derived poliovirus circulation (WorId Health Assembly, 1988~. Although
eradication may eventually be certified, it is likely that poliovirus will never
be completely contained, and recent findings indicate that vaccine-derived
polioviruses can circulate and cause disease. Consequently, vaccination will
probably not be discontinued anytime in the foreseeable future.
Although the use of live, attenuated polio vaccine (OPV) has been
crucial to the success of the eradication program thus far, unique properties
of the vaccine complicate the decision to cease vaccination. Before we can
stop vaccination, we must answer the following questions:
1. How long will OPV persist after it is no longer administered to
humans? Will such persistence (including virus excreted by immunocom-
promised individuals) constitute a threat to the growing number of unvac-
cinated individuals?
2 What is the transmissibility of OPV strains?
3. Will it be possible to eliminate all potential sources of poliovirus in
the post-vaccine era?
4. How will we respond to an outbreak of polio in the post-vaccine
era?
3t
Poliovirus infections, which are transmitted by fecal-oral contamina-
tion, begin in the pharyngeal and intestinal mucosa before spreading to the
blood and invading the central nervous system. Paralytic disease, which
occurs in about 1 in 100 infections, results from the destruction of motor
neurons. Poliomyelitis can be prevented by the use of either an injected,
formalin-inactivated vaccine (inactivated polio vaccine, IPV), or a live, at-
tenuated vaccine which is taken orally and replicates in the intestine (oral
polio vaccine, OPV). Both vaccines generate humoral immunity, but only
OPV produces local antibodies in the intestine. As a result, wild poliovirus
can replicate in the gut of individuals immunized with IPV, but not in the
gut of those immunized with OPV.
The OPV strains used in the WHO eradication effort were developed
by Albert Sabin, who identified variants of the three poliovirus serotypes
that were immunogenic but did not cause disease. Since then, molecular
biological tools have been used to identify the mutations responsible for the
attenuation phenotypes of the vaccine strains. In the 1980s, scientists dis-
covered that these mutations revert to pathogenicity during replication in
the human gut, which explains why OPV-shed virus is more neurovirulent
than the administered parent virus.
Will virulent, OPV-derived viruses shed by vaccinees be a threat in the
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124
CONSIDERATIONS FOR VIRAL DISEASE ERADICATION
post-vaccination era? To answer this question, we must first consider how
long these viruses persist in the environment. In a recent stucly carried out in
Japan, neurovirulent, OPV-derived viruses were isolated from sewage and
river water up to three months after routine immunization (Yoshida et al.,
2000~. The authors concluded that there is an environmental risk of vac-
cine-associated polio as long as live vaccine is not replaced by inactivated
vaccine. Similar studies in Cuba suggest that OPV may persist in the popu-
{ation for several months after vaccination. During the type 3 polio epi-
demic in Finland in 1984, OPV was detected up to six months after mass
immunization. All of these studies were conducted in communities with a
high proportion of immune individuals; it is not known if the level of
immunity to poliovirus affects the duration of persistence.
The problem of OPV persistence is further complicated by the observa-
tion that immunocompromised individuals who receive OPV may excrete
virus for extended periods. For example, in one study, a patient who re-
ceived monotypic Sabin type 3 in 1962 excreted neurovirulent type 3 virus
for 637 days with no symptoms of polio (Martin et al., 20001. Individuals
with B cell deficiencies often go undiagnosed and may excrete enteroviruses
for long periods. The extent to which immunodeficient individuals are
infected with polio is unknown and needs to be determined.
After the cessation of polio immunization, OPV will likely continue to
circulate in most populations for at least a few months, perhaps up to a
year. At the same time, the number of susceptible individuals will increase.
This raises the questions: will OPV-derived viruses pose a threat to unvac-
cinated indivicluals, and can OPV-derived viruses be transmitted and cause
disease in humans;
As long as OPV has been in use, scientists have recognized its transmis-
sibility among humans. Numerous studies have documented the develop-
ment of anti-poliovirus antibodies in nonimmunized persons in communi-
ties undergoing vaccination. For example, in one study of a U.S. Amish
community where many individuals refuse vaccination, 89% of unvacci-
nated children developed antibodies to type 2 poliovirus, presumably from
circulation of the vaccine virus from neighboring areas where the vaccine
was usecI. This ability to immunize non-vaccinated individuals is considered
to be an advantage of OPV, especially in Third World countries where
immunization levels are low and poor sanitation promotes extensive virus
spread. However, in the post-eraclication era, live vaccine strain transmissi-
bility will be a liability. It will be ironic if it becomes necessary to continue
vaccination as protection against vaccine-derived polioviruses.
Several recent studies confirm that OPV-like strains excreted after im-
munization can be transmitted and cause poliomyelitis among humans. In
2000, a neurovirulent derivative of the Sabin type 2 OPV strain was iso-
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MEDICAL INTERVENTION AND TECHNOLOGICAL SOLUTIONS 125
lated from sewage in Israel (Shulman et al., 2000~. The extent of sequence
divergence of this strain from Sabin type 2 indicates that it had probably
been replicating in one or more people for at least six years. These observa-
tions indicate that OPV-like virus can be transmitted "silently," i.e., in the
absence of disease, in an immunized population. In Egypt, 32 polio cases
that occurred from 1988-1993 have been attributed to a type 2 vaccine-
derived poliovirus strain (Centers for Disease Control and Prevention
[CDCI, 2001~. Analysis of the virus isolate sequences indicates that they
were probably derived from a single infection in 1982, the progeny of
which circulated in Egypt for the next 10 years. During Tuly and November
2000, an outbreak of poliomyelitis occurred in Hispaniola (CDC, 20003.
The virus responsible for this outbreak was derives! from the Sabin type 1
strain. Sequence analysis indicates that it had been circulating in the region
for approximately two years. All of these findings demonstrate that
neurovirulent revertants of OPV can be transmitted among humans and
cause poliomyelitis. In light of this information, it is impossible at this time
. . , . . . . .
to plan cessation ot 1mmumzatlon against po 1O.
In order to prevent reintroduction of the virus in the post-vaccination
era, a crucial component of the eradication effort is the identification and
destruction of poliovirus stocks. It will be an enormous task to track clown
every poliovirus stock, particularly in light of the absence of an enforce-
ment authority. We cannot simply depend on the good will of nations, as
suggested by WHO. An even greater challenge is identifying clinical labora-
tories that unknowingly harbor poliovirus. Finally, how do we deal with a
situation in which, for example, a tube labelecl "Coxsackievirus B3" actu-
ally contains poliovirus type 2? Since this has actually occurred, it is not a
hypothetical threat but a real possibility.
A paradox that arises in the post-OPV era is that it will be critically
important to continue producing vaccine stocks for use in the event of a
disease outbreak. In populations that have lost immunity to the virus, a
poliovirus vaccine production facility will be a hazard equivalent to a
bioweapons plant. With smallpox, this problem was avoided because of the
strain differences between the vaccine and wild viruses, but poliovirus vac-
cines do not offer such an easy solution.
Which poliovirus vaccine will be produced in the post-OPV era? Be-
cause the inactivated polio vaccine (IPV) is produced from wild-type strains
of poliovirus, its production would require a high containment facility.
Alternatively, IPV might be produced from the Sabin poliovirus strains,
although some research would be required to demonstrate the feasibility of
this approach. However, immunization with IPV would not prevent intesti-
nal carriage of the virus, increasing the likelihood of spread of the virus in
the population. Vaccination with OPV would probably be more effective in
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CONSIDERATIONS FOR VIRAL DISEASE ERADICATION
curtailing epidemics of poliomyelitis, but excretion of vaccine-derived OPV
would be problematic for reasons discussed above.
There are no easy answers to these questions, but it is disturbing that a
cletailed plan for poliovirus vaccine production in the post-OPV era has not
been formulated. Failure to present a coherent plan for the production of
vaccine stocks in the post-vaccination world is another reason why we
. .
cannot stop vaccinating.
The plan to eradicate polio has had an unfortunate effect on poliovirus
research. As noted recently in an article entitled "Don't Underestimate the
Enemy" in Nature, January 18, 2001, "When an infectious disease appears
to be in decline the agent that causes it tends to disappear from the biomedi-
cal research agenda." In the late 1990s, WHO and CDC began informing
polio research laboratories that they would soon be required to cease polio-
virus research and destroy virus and infectious DNA stocks. Although the
exact date was somewhat vague, the resulting uncertainty inhibited poliovi-
rus research. Graduate students and postdoctoral fellows no longer viewed
working on poliovirus as a wise career option, and funcling agencies and
their peer review groups began to question the wisdom of long-term (five-
year) investment in research programs on the virus. This effect was unfortu-
nate, because many projects relevant to the eradication effort—work on
new vaccines, animal models for virus transmission, and anti-viral com-
pounds (which might be useful in a post-vaccination-era outbreak of po-
lio) did not proceed. WHO decided not to continue poliovirus research in
1988 because the virus would be eradicated by 2000!
Today it is quite clear to many virologists that it might not be possible
to eliminate poliovirus from the world. It therefore seems unfortunate that
the poliovirus research establishment has been substantially depleted, espe-
cially since questions relevant to the eradication effort have not been ad-
equately addressed. One of the lessons we have learned from the polio
eradication effort is that there continues to be a large gap between basic
research and public health. For example, the research community has
doubted whether it will be possible to eliminate poliovirus ever since the
eradication goal was first announced in 1988. Nevertheless, the force of
public health policy has overriden these concerns, resulting in the disman-
tling of research programs that could otherwise have contributed to the
eradication effort. Future eradication campaigns should benefit from this
experience. Although it is important to convince governments and health
authorities that a disease can be eradicated, it is also important to maintain
communication with the research community so that crucial research con-
t~nues.
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MEDICA ~ INTER VENTION AND TE CHNOL O GICAL SOL UTIONS 1 2 7
ANTIVIRAL THERAPY IN THE MANAGEMENT OF POST-
ERADICATION INFECTIOUS DISEASE OUTBREAKS
Richard f. Whitiey, M.D.
Loeb Eminent Scholar Chair and Professor, Department of Pediatrics
University of Alabama at Birmingham, Birmingham, AL
Prevention must take precedence over treatment of infectious diseases.
In an age of unparalleled successful vaccination, particularly when the
eradication of smallpox has been documented and the eradication of polio-
virus is anticipated, one must question the necessity of developing antiviral
drugs targeting infectious diseases slated for global eradication. Successful
immunization against measles, mumps, rubella, diphtheria, and many other
pathogens has been demonstrated worldwide, though with varying degrees
of success.
There are several different circumstances under which re-emergence of
an infectious agent might be anticipated:
· Bioterrorism (e.g., deployment of smalIpox),
· Resurgence of an infection thought to be eradicated (e.g., poliomy-
elitis in Santo Domingo), and
· Clinical reactivation of a vaccine-preventable latent virus (e.g., va-
ricella) transmitted to a high-risk susceptible (seronegative) individual.
This overview focuses on the potential utility of specific antiviral and
more generalized broad-spectrum antiviral agents in a post-eradication vac-
cine era.
Available Therapeutic Resources
The armamentarium of the public health physician with regard to anti-
viral agents is limited, at best. Successful antiviral therapy has only been
demonstrated in four general infectious areas:
1. The management of influenza virus infections with tricyclic amines
and neuraminidase inhibitors,
2. The treatment of HIV infection with reverse transcriptase inhibi-
tors, protease inhibitors, and other novel therapeutics,
3. The therapy of several herpes virus infections, including herpes
simplex virus, cytomegalovirus, and varicella zoster virus, with nucleoside
and nucleotide analogs, and
4. Therapeutic interventions for hepatitis B and hepatitis C with
nucleoside analogs and interferons.
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CONSIDERATIONS FOR VIRAL DISEASE ERADICATION
While therapy for each of these broact infectious disease agents has
been shown to be clinically efficacious, resulting in decreased morbidity
and mortality, no therapeutic intervention should supplant disease preven-
tion by vaccination. Toward this end, some therapeutic agents have been
cleveloped for pre-emptive antiviral therapy to be administered before overt
disease but in the presence of viral antigenemia. This approach has proven
very successful in the management of cytomegalovirus disease in organ
· . .
transplant recipients.
Currently, none of the immunological interventions or modulators (e.g.,
interferon or interferon-like compounds) have proven valuable in the pre-
vention of viral disease. With the exception of limited monoclonal antibod-
ies (e.g., palivizumab for respiratory syncytial viruses), disease prevention
has not been achieved by this modality.
Public Health Implications
With the exception of influenza and HIV infections, those diseases for
which antiviral therapy exists are not usually considered epidemic. Diseases
that could take on epidemic proportions namely, smallpox, measles, ru-
bella, polio, dengue, ant! Ebola have never been considered candidates for
antiviral drug development. This is alarming in light of the fact that extrem-
ist governments or individuals will likely consider using these agents as
bioterrorist weapons in the post-vaccine eradication era when seroprotec-
tion will have waned in the community at large.
Scientists have identified molecular targets amenable to the develop-
ment of selective and specific antiviral agents. The knowledge of viral-host
interactions should lead to the development of specific and more general-
ized modulators of host response, such as induction of intracellular inter-
feron pathways.
The unique properties of each virus need to be considered when devel-
oping selective, specific inhibitors to viral replication. For example, several
viruses primarily the herpes viruses but also hepatitis B and C- have a
propensity to establish latency. Recognizing that reactivation can occur,
even with an effective vaccine, exposure of susceptible (seronegative) or
non-vaccinated individuals could result in exaggerated disease. An example
of this is the reactivation of varicelIa poster virus which results in shingles,
or chickenpox. Shingles is contagious for seronegative individuals and is
always more severe in adults than in children.
Changes in the antigenic nature of an organism may also render it more
pathogenic for the population at large. This phenomenon has Greatly been
documented by the detection of the.HSN1 influenza A strain in Hong
Kong. It is anticipated that a major antigenic shift in the near future will
result in a worldwide influenza pandemic. The lack of adequate vaccine
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MEDICAL INTERVENTION AND TECHNO LOGICAL SOLUTIONS 129
stores and vaccines containing the appropriate antigens, combined with the
inability to generate sufficient quantities of antiviral drugs, would leave the
worId's population at significant risk for disease caused by pandemic influ-
enza.
Conclusion
In an era of rapid vaccine deployment and committed attempts at
worldwide eradication of diseases other than smallpox, questions regarding
the need to develop additional antiviral agents are very serious. With the
lingering threat of bioterrorism, the availability of therapeutics to treat
vaccine-preventable diseases, such as smallpox, should be considered a high
. .
prlorlty.
It is impossible to envision a universal vaccine program for the preven-
tion of such diseases as rabies, Ebola, dengue, and others. However, all of
these viruses are amenable to the development of specific antiviral agents.
Molecular biology tools are now available for the development of anti-
viral agents. Plus, the knowledge derived from developing therapeutics for
one virus can be applied to other viruses. For example, therapeutics di-
rected against polio can be applied to other members of the Picornavirus
family, including hepatitis A virus, rhinoviruses, enteroviruses, and
coxsackieviruses, all of which cause significant morbidity in the worId's
population. Toward this end, the pharmaceutical industry must make a
commitment to the development of antiviral interventions.
POTENTIAL USE OF CYTOEC[NES AND ANTIBODY FOR POST-
EXPOS~E PROPHYLAXIS IN THE POST-ERADICATION ERA
Diane E. Griffin, M.D., Ph.D.
Professor and Chair, W. Harry Feinstone Department of Molecular
Microbiology and Immunology
Johns Hopkins School of Hygiene and Public Health, Baltimore, MD
An important benefit of viral eradication, in addition to elimination of
morbidity and mortality due to infection, is the elimination of the need for
continued immunization of large numbers of people. Discontinuation of
universal immunization will result in considerable cost savings. However, it
will also eventually create a population susceptible to widespread infection
in the event of reintroduction or re-emergence of the eradicated virus.
Because reintroduction will always be a possibility, even in the best-con-
trollecl circumstances, it is necessary to have a planned response if and
when it should occur. There are several possible responses:
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CONSIDERATIONS FOR VIRAL DISEASE ERADICATION
· Resumption of widespread immunization, assuming that the vac-
cine and vaccine-manufacturing capacity are available.
· The use of antiviral drugs for prophylaxis or treatment (see previ-
ous section of this chapter).
· The use of immunoprophylaxis for protection, including both non-
specific approaches to stimulation of innate antiviral defenses and specific
prophylaxis directed at particular pathogens.
Nonspecific Inhibition of Virus Replication
The first line of defense against viral infection is the innate immune
response. Innate defenses not only act to control virus replication early after
infection, but they also shape and influence the nature of subsequent spe-
cific immune responses. This continuum between the innate and acquired
immune responses to pathogens is increasingly being recognized. Many
Or
important components of the innate immune response contribute to the
early control of viral replication. The best understood is interferon (IFN), a
cytokine which is produced by many types of cells and was first recognized
for its ability to make previously susceptible cells resistant to infection by a
wide variety of viruses. In addition to the first recognized IFN, now known
as type I or IFN odd, there are several other cytokines with important
antiviral properties. Cytokines produced early after infection include type II
IFN or IFN-^y, which is produced by natural killer (NK) cells, and tumor
necrosis factor (TNF)y, which is produced by phagocytic cells such as mac-
rophages.
Current knowledge and therapeutic experience is most extensive for
type I IFN, which induces an antiviral cellular state by interacting with the
IFN alp receptor, IFNAR. IFNAR signals through a pathway involving
transcription factors STAT-1 and STAT-2 to induce transcription of a large
number of IFN-responsive genes and subsequent production of antiviral
proteins. The best studied of these proteins and pathways are those involv-
ing the dsRNA-activated protein kinase, PKR, which inhibits protein syn-
thesis; the dsRNA-activated oligoaclenylate system, which degrades RNA;
and the MX GTPases, which inhibit RNA synthesis. In addition to these
direct antiviral responses, IFN also upregulates expression of major histo-
compatibility complex (MHC) molecules on the cell surface, thereby en-
hancing recognition from cells involved in inducing an immunologically
specific immune response. From extensive study of these IFN-regulated
pathways, several facts are clear:
1. The pathways described to date involve only a small proportion of
the messages known to be induced by IFN (i.e., IFN ac/p probably induces
about 90 different pathways).
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MEDICAL INTERVENTION AND TECHNOLOGICAL SOLUTIONS 131
2. Each pathway affects replication to a different degree, depending
on the particular virus. A pathway that interferes with the replication of
one virus may have absolutely no effect on the replication of another virus.
3. Our understanding of how IFN inhibits viral replication is incom-
plete.
4. Viruses have evolved a large number of mechanisms to counteract
the effects of IFN. These mechanisms may or may not be preserved in the
tissue culture-adapted virus strains most often used for study.
Several recombinant forms of both IFN-a and IFN-p are currently
available and licensed for treatment of a variety of diseases, including mul-
tiple sclerosis, lymphoid tumors, and chronic viral infections (particularly
hepatitis B and hepatitis C). Therefore, we have knowledge of dosing and
side effects for prophylaxis against chronic infections in humans. However,
our experience with prophylaxis against acute infections is very limited.
IFN has been used locally for prophylaxis against upper respiratory infec-
tions, and, although effective, open causes side effects resembling symp-
toms of the disease being prevented. Many people would rather have a cold
than suffer these side effects. Experience with preventing systemic infec-
tions is limited to animal models, where efficacy can be demonstrated as
long as the IFN or IFN-inducer (e.g., poly IC) is administered before or
shortly after exposure to the virus. Therefore, although our experience with
this approach is limited, prophylactic use of IFN is certainly a rational
approach to protection from infection early after exposure. However, its
effectiveness against the specific wild-type virus of interest would need to be
confirmed.
As mentioned above, viruses have evolved many ways to circumvent
host cell antiviral activities (Alcami and Koszinowski, 2000~. For example,
viruses from many different families (e.g., picornaviruses, rhabdoviruses,
reoviruses, retroviruses, orthomyxoviruses, adenoviruses, herpesviruses,
poxviruses) block the activation of the PKR pathway by either producing
decoy RNAs, binding dsRNA, or degracling PKR protein. These mecha-
nisms are especially prevalent in wi13-type viruses, whose ability to escape
the effects of IFN is likely to be important for virulence and transmission.
As another example, the virulent myxomatosis in the poxvirus family pro-
duces proteins that bind host I-NF, IFN, and a broad range of chemokines.
Because viral defenses against host innate immune responses are not neces-
sary for viral replication in vitro, they may be lost, not expressed, or mu-
tatect in tissue culture-adapted strains of virus. However, they are very
important for in viva virulence.
In addition to IFN alp, other less well-studied antiviral cytokines in-
clude IFN-y and TNFa. In vitro, both exhibit antiviral activity against some
viruses in some cells, but their effects are much more variable that those of
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CONSIDERATIONS FOR VIRAL DISEASE ERADICATION
IFN a/b. "Upstream" inducers of these effector cytokines, such as IL-12 or
immunostimulatory oligonucleoticles, could potentially be developed as
broadly active prophylactic agents. However, considerable research on tox-
icity and effectiveness would need to be performed before any of these
agents could be considered for widespread prophylactic use.
Specific Inhibition of Virus Replication
Acquired immune responses provide specific protection against re-in-
fection by many viral pathogens and, as such, serve as the basis for protec-
tion by immunization. In the pre-immunization era, immune globulin con-
taining polyclonal antibodies to specific viruses was used for prevention
and treatment for a number of infections (Ordman et al., 1944~. For both
polio and measles, data from excellent controlled studies show that passive
prophylaxis can prevent disease in outbreak situations; protection can last
~ ~ ~ · 1 1 ~ r . 1 1 1 1 1 1
tor weeks alter a single nose. come or these data nave been used to deter-
mine what levels of antiviral antibody need to be inducer! by vaccines in
order to provide protection from infection.
However, there are a couple of serious problems with passive transfer
of immune globulin. First, the amount of antibody against the virus may be
relatively low but the volume needed relatively large. This problem will be
exacerbated as the population's immunity to the virus wanes following
eradication and cessation of immunization. Second, using large pools of
donors to generate the immune globulin carries the risk of transmitting
other infectious agents.
Fortunately, there has been considerable progress on this front since the
early days when immune globulin was used for passive protection against
polio and measles. This is illustrated by the current products available for
prophylaxis against respiratory syncytial virus (RSV), a cause of serious
lower respiratory disease in young infants, particularly those with cardiac
and pulmonary abnormalities.
There is no vaccine for RSV. Passive transfer of immune globulin is
protective, but not all infants can tolerate the volume loads required to
achieve protective antibody levels (PREVENT, 1997~. More effective and
potent prophylactic products have been developed and licensed. In particu-
lar, animal studies have shown that a mouse monoclonal antibody (MAb)
provides protection against RSV by binding to the F protein. Determinants
of antibody specificity lie in the variable complementary determining re-
gions (CDRs) of MAb's Fab H and L chains. But the rest of the mouse MAb
molecule induces an immune response in humans. Through genetic engi-
neering, mouse MAb has been "humanized" so that every region of the
molecule, except for those portions of the CDRs that determine specificity
for binding to the RSV F protein, are now human. Humanized MAb pro-
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MEDICAL INTER VENTION AND TE CHNO ~ O GICAL SO ~ UTIONS 1 33
vices effective prophylaxis against severe RSV-inducec! disease (The Im-
pact-RSV Study Group, 1998~.
With recent technological improvements, variable regions of human
antibodies with desired specificity can be cloned directly from antibody-
secreting cells in the blood or bone marrow (Hoogenboom and Chames,
2000; Little et al., 2000~. These clones can then be cloned into another
vector and converted directly into whole human IgG molecules Manna et
al., 1999~. By knowing which antibody specificities are protective against
eradicated viruses, clinically useful immunoprophylactic reagents could be
generated relatively easily. These antibodies would then be available for
production and use in the event of reintroduction or re-emergence of an
eradicated virus.
Conclusion
Immunomodulators that would be broadly protective against viral in-
fections—such as IFN alp, TNFa, and immunostimulatory DNA are in
the early stages of development. IFN oc/p is the best characterized but has
been used primarily for treatment of chronic viral infections, not prophy-
laxis against acute viral infections. Humanized MAbs have proven success-
ful at preventing acute viral infections and are currently being used as a
prophylaxis against RSV. Technology has advanced to a point where spe-
cific prophylactic MAbs could be developed for use against other viral
pathogens besides RSV, but this has not been done for polio, measles, or
smallpox.
Given the diversity of viruses, it seems unlikely that a universal prophy-
lactic agent will be identified. Rather, studies will need to focus on develop-
ing prophylaxis for those infections deemed to pose the greatest risks.
Prophylactic agents must be developed before they are needed in the post-
. .
erac Cation era.
THE POTENTIAL ROLE OF PROBIOTICS AND MICROBIAL
ECOLOGY IN HOST DEFENSE
Susanna Cunningham-R2`ndles, Ph.D.
Professor of Immunology, Department of Pediatrics
Weill Medical College of Cornell University, New York, NY
The human immune system provides host defense against sudden inva-
sion from exogenous pathogenic microorganisms and viruses, while simul-
taneously maintaining continual surveillance against incursion from endog-
enous microbes. Immunization will create a specific pathogen-free
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134
CONSIDERATIONS FOR VIRAL DISEASE ERADICATION
environment only if there is a continuous normal immune response within
an immunized majority of the community. An apparently healthy person's
response to vaccine is often taken for granted, even though detailed knowI-
edge of the normal immune response is lacking. There has been little con-
sideration given to the possibility that the human immune system may be
affected by selective pressure from changing world conditions. As immunity
wanes, even immunized individuals may be highly vulnerable in the absence
of total eradication. Immunity in the community at large is not determined
by the poor response to some vaccines by young children and immuno-
compromised persons if the proportionate representation of these groups is
small. However, the increasing size of this poor response population for
example in parts of the world with a high incidence of HIV infection—may
significantly affect whether standard immunization practices can lead to the
eradication of infectious pathogens.
The strength of the immune system is both challenged and maintained
3:
,
through continual interaction with an internal microbial milieu. Under-
standing this fundamental interaction will provide new insights into what
makes an immune response functional and will likely lead to novel ap-
proaches to restoring or enhancing immune function.
In healthy people, microflora are normally present on all external sur-
faces and the internal surfaces of the upper respiratory tract, gastrointesti-
nal tract, perineum, vagina and distal urethra. They are usually absent from
the internal surfaces of the bronchi, alveolar spaces, urinary tract, and
uterus, as well as the blood, deep tissues, organs, and brain.
Within the gut, there are distinct, closely regulated differences in the
relative density of bacteria. Mechanisms that mediate and maintain these
regional differences include physical structures, such as the glottis; physi-
ological barriers, such as gastric pH; and the continual action of both the
innate and adaptive immune systems. The normal human gut is persistently
colonized. Since there is no fixed boundary between colonization and infec-
tion, response to persistent colonization likely involves repeated waves of
immune activation. Thus, gastrointestinal colonization conditions the acti-
vation potential of the immune system.
The gut immune system operates independently of the systemic im-
mune system, and the gut's resident T cells have developed specialized
functional capacities independent of thymic influence. Recent studies (Gill
et al., 2000; Macpherson et al., 2000; Walker, 2000) have shown that the
gastrointestinal-immune interface is a frontier zone, and the gut's local
innate response to antigenic or pathogenic challenge has a surprisingly
strong influence on the systemic immune response. A key mediator for this
response is the natural killer, or NK, cell. NK cells are characterized by
their spontaneous ability to kill tumor or virally infected cells. They also
produce cytokines, which regulate host defense against bacteria and influ-
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MEDICAL INTERVENTION AND TECHNOLOGICAL SOLUTIONS 135
ence the development of the adaptive immune system. NK cells begin func-
tioning at birth, when microbial colonization of the gut occurs. In the early
post-birth period, neonatal NK cells show absent or decreased cytoly~cic
activity against the reference erythroleukemia tumor cell target K562. How-
ever, as demonstrated in Figure 5-1, certain bacteria can directly activate
the neonatal NK cell system. This functional response is accompanied by he
nova induction of gamma interferon procluction. The preparations of bac-
teria used in these studies ImuVert (ribosomal vesicles from Serratia
marscese?~s) and OK432 (whole inactivated Streptococcus pyogenes) have
broad immunoadjuvant properties. These experiments in vitro mirror what
happens in viva in response to conventional environmental microbes. This
30
25
Ox 20
a
a
~ 15
a,
10
~ O- .
Endogenous
~ S. marscesens
· S. pyogenes
~ , ~ , rim ~
1
50:1
25:1
Effecter: Target Ratio
12:1
FIGURE 5-1 Stimulation of neonatal natural killer cell activity by bacteria. This
shows the effect of S. marscesens and S. pyogenes on NK activity of peripheral
blood mononuclear cells in the short-term Cr5i release assay against K562. Data
are given as percent specific release at three effecter target ratios. (Cunningham-
Rundles and Nesin in "Persistent Bacterial Infections," 2000, reprinted with per-
mission from ASM Press)
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136
CONSIDERATIONS FOR VIRAL DISEASE ERADICATION
unique response to bacteria probably evolved to provide a transition be-
tween the pre-natal suppression of fetal immune effecter activity, which is
necessary for the maintenance of maternal fetal tolerance, and the post-
natal requirement for rapid response toward potential microbial pathogens.
Microflora directly alter the architecture and physiology of the mucosa
by inducing an immune response, which they probably continue to influ-
ence and regulate throughout life. Emerging studies (Word and Adlerberth,
2000) have suggested that the specific composition of microflora is highly
varied among different cultures and that it tenets to remain constant for the
individual once established after birth. Normal flora do not directly harm
the normal host; plus they contain commensals which produce nutrients,
absorbable peptides, and vitamins, all of which benefit the host. It is now
possible to stucly the potential significance of this lifelong interaction, thanks
to the advent of genetic typing, which spurred investigation of flora com-
prised of species resistant to current culturing methods. One study (Ahrne
et al., 1998) showed that the we11-characterized beneficent commensals,
such as lactobacilli, form a small and rather fragile part of the overall flora.
The ecology of microflora is strongly influenced by oxygen tolerance.
Commensal bacteria are primarily obligate anaerobes, whereas key patho-
genic bacteria are facultative anaerobes that replicate faster in the presence
of oxygen. Thus lactobacilli and bifididobacteria, which are normal gut
commensals, survive and replicate in the presence of oxygen but not as
effectively as, for example, E. coli.
If beneficial microbes have a selective advantage, their colonization
may prevent outgrowth of more pathogenic bacteria. Although Metchnikoff
proposed in 1907 (Metchnikoff, 1907) that lactic acid bacteria would have
a favorable effect on health, the concept of probiotic bacteria living mi-
crobes introduced into the body to improve intestinal microbial balance is
recent (Fuller, 1989~. Probiotic bacteria have proven effective against anti-
biotic-associated diarrhea and certain persistent and clinically significant
infections, such as C. difficile. Experimental studies (Bergogne-Berezin,
2000; Hirayama and Rafter, 1999; Hove et al., 1999; Kirjavainen et al.,
1999; Majamaa et al., 1995) have shown that probiotic lactobacilli can
neutralize carcinogens, replace microflora that produce carcinogens and
tumor promoters, and produce antitumor factors through direct actions in
the gastrointestinal tract. Essential characteristics for efficacy include resis-
tance to acid and bile and ability to colonize and adhere to the colonic
mucosa (Bengmark, 1999~. Moreover, current studies (Cunningham-
Rundles et al., 2000; Devi et al., 1999; Hessle et al., 1999) have suggested
that probiotic lactobacilli may serve as immunoadjuvants, thereby increas-
ing weak systemic immune response, even in the HIV+ host. Possible mecha-
nisms of action include competition for specific ecological niches, immuno-
~:
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MEDICAL INTERVENTION AND TECHNOLOGICAL SOLUTIONS 13 7
logical stimulation of the mucosal barrier, and induction of specific cytokine
patterns.
Thus, probiotic bacteria have strong potential to sustain natural im-
mune response towards environmental pathogens in the post-vaccine era.
Additionally, probiotic lactobacillus may prove useful in strengthening im-
mune responses in persons whose host defense capacity has been compro-
mised by chronic infection or short-term stressors. However, there are a
few key questions concerning the use of probiotic bacteria in the immuno-
deficient host, including:
.
Can the immune-deficient host develop a normal immune response
towards lactobacitIus?
Is this response qualitatively or quantitatively different from that of
immunocompetent persons?
.
Are there safety issues, such as potential for transiocation?
The most extreme example of acquired immune deficiency is HIV infec-
tion. Normal bacterial flora are altered in HIV infection, as evident by the
frequency of bacteremia associated with altered gastrointestinal function,
diarrhea, and malabsorption. Failure-to-thrive is relatively common in con-
genital HIV infection and is linked to altered gastrointestinal function and
chronic cytokine activation. Our lab studies the effect of L. piantarum
299v, a specially developed probiotic lactobacillus, on growth and specific
systemic immune response following oral supplementation in the HIV+
child. There appears to be a generally beneficial effect on immune response.
Surprisingly, the HIV+ children's level of cross-reacting immune response
to LP299, as a group prior to supplementation, is essentially independent
of CD4+ T cell percentage, which is unlike response to any other activator.
Data are shown in Figure 5-2. Children who did not respond to LP299v
before supplementation did develop response after supplementation; the
oral supplement was well tolerated, colonization was temporary, and there
were no side effects. The mechanism of action is currently under study;
preliminary data suggest that treatment promotes a T helper type 1 cytokine
response.
These studies support current interest in commensal bacteria as anti-
gen-delivery vehicles, as well as potential adjuvants. The possibility that
modulation of gastrointestinal flora might be used to strengthen immune
response is especially relevant for protection of future populations against
emerging infections in a post-immunization era where, paradoxically, the
immune system may face even greater challenges.
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138
CONSIDERATIONS FOR VIRAL DISEASE ERADICATION
3500
3000 -
2500 -
2000 -
1 500 —
1 000 —
500 -
O -
CD4+ T cell = 7%
CD4+ T cell = 34%
.
HIV+ children
FIGURE 5-2 Immune response to Lactobacillus plantarum 299v in HIV+ children
in relationship to CD4+ T cells. Peripheral blood mononuclear cells were cultured
in a microtiter plate assay and pulse labeled with 3H thymidine. Data show mean
net maximum response to LP299v antigen in children grouped by CD4+ T cell
level. (Cunningham-Rundles and Nesin in "Persistent Bacterial Infections," 2000,
reprinted with permission from ASM Press.)
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Representative terms from entire chapter:
disease eradication
