A BRIEF INTRODUCTION TO INFLUENZA AND SWINE FLU. HOW IMMUNE STORM CAUSED BY H1N1..

1. ABSTRACT
Swine flu is an infection which caused by Influenza A viruses. The change in
ecology and evolutionary steps for influenza A viruses is very important, and
for this pig play an crucial role in it. Evolution steps for swine may include for
host adaption and mixing of genetic information. The epithelium tissue of pigs
tracheal that contain SA alpha 2,6 Gal and SA alpha 2,3 Gal . Which are acts
as receptors and that may be infected with human, avian viruses and swine.
Therefore, pigs considered as an intermediate host for the adaptation to humans.
There are three type of subtypes of influenza viruses are present currently i.e.
H1N1, H1N2, H3N2. Several studies found that there are approx. 45000 people
were died due to H1N1 stain over worldwide. Researchers have found that this
type of flu will cause cytokine storm. This will cause overproduction of
cytokine. Due to overproduction there will be inflammation in lungs and
building up fluid, which will surely have respiratory distress. The Scripps
Research Institute (TSRI) have used gene knock-out techniques to observed
response to infection by influenza virus for that they breed mice which lack 1
or more molecular sensors of influenza virus infection. From this technique
researcher have found, the drugs that we use not working through one pathways
but they are been working on broadly. So we need to understand all the
pathways which lead to overproduction of cytokines. Though vaccine is not
much useful for the complete treatment for the H1N1 flu, but a study have
found that, 60 people who are suffered from H1N1 flu, and they treated with
extracorporeal membrane oxygenation, than at the end 40 patients cured from
H1N1 flu, but still influenza virus is pandemic to world. Better to take adequate
steps like wearing mask, washing hand and medical consultant to avoid any flu.
Prevention is better than cure.

2. INTRODUCTION
Swine flu (H1N) which is caused by influenza or we may call it as swine
influenza. It is a kind of respiratory disease which is caused by influenza
viruses, that infect the respiratory tract of pigs, which resulting in febrile
respiratory disease i.e. in nasal secretions or a barking cough, decreased
appetite. Scientist have found that Swine flu (H1N1) produces most of the same
symptoms in pigs as human flu produces in people. Swine flu can last about one
to two weeks in pigs that survive. Swine influenza virus was first isolated from
pigs in 1930 in the U.S. and has been recognized by pork producers to cause
infections in pigs worldwide. In a number of instances, many people have
developed the swine flu infection when they started closely associated with pigs
(for example, farmers, pork processors), and likewise, some time consuming
pork which was non hygienic or not cooked properly, for several year pig
populations have occasionally been infected with the human flu infection. In
most instances, the cross-species i.e. human and pig infections have remained in
local areas and have not spread over the worldwide i.e. it only caused national
and worldwide infections in either pigs or humans. Unfortunately, this cross-species
situation with influenza viruses has had the potential to change. Many
investigators decided that the 2009 swine flu strain, which was first seen in
Mexico, to be termed as novel H1N1 flu because it was mainly found infecting
only humans and exhibits two main surface antigens, H1 (hemagglutinin type 1)
and N1 (neuraminidase type1). Total of eight RNA strands from novel H1N1
flu have one strand derived from human flu strains, two from avian (bird)
strains, and five from swine strains. Swine flu is transmitted form , which
causes person to person by inhalation or direct contact of droplets containing
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3. viruses from different people sneezing or coughing; it is not transmitted by
eating cooked pork products. The newest swine flu virus that has caused swine
flu is influenza A H3N2v (commonly termed H3N2v) that began as an outbreak
in 2011. The "v" in the name means the virus is a variant that normally infects
only pigs but has begun to infect humans.
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INFLUENZA
Influenza is considered as viral infection which attacks our respiratory system
like nose, throat and lungs. Influenza, commonly called the flu or swine
influenza,it is not the same as the stomach "flu" viruses that cause diarrhea and
vomiting, it’s all different from that.
SWINE INFLUENZA
Technically term "swine flu" refers to influenza in pigs. Occasionally, pigs
transmit influenza viruses to people, mainly to veterinarians. Less often,
someone infected passes the infection to others like wise it continue to spread to
many people.
The human respiratory infection caused by a specific influenza virus H1N1
strain popularly known as swine flu — was first recognized in spring 2009. A
few months after the first swine flu cases were seen, rates of confirmed H1N1-

4. related illness were increasing in much of the world. As a result, the World
Health Organization declared the infection a global pandemic.
The pandemic was declared over in August 2010. Currently, H1N1 is still
circulating in humans as a seasonal flu virus and is included in the seasonal flu
vaccine.
Swine influenza is a respiratory disease of pigs which caused by type A
influenza viruses ,that regularly cause outbreaks of influenza in pigs. Influenza
viruses that commonly circulate in swine are called “swine influenza viruses” .
Like human influenza viruses, there are different subtypes and strains of swine
influenza viruses. The main swine influenza viruses circulating in U.S. pigs in
recent years are:
 swine triple reassortant (tr) H1N1 influenza virus
 trH3N2 virus
 trH1N2 virus
(STRUCTURE OF A GENERIC INFLUENZA VIRUS)
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5. 5
SYMPTOMS
Swine flu symptoms in humans are similar to those of other flu strains:
 Fever
 Cough
 Sore throat
 Runny or stuffy nose
 Body aches
 Headache
 Chills
 Fatigue
 Diarrhea
 Vomiting
Swine flu symptoms develop about one to three days after you're exposed to the
virus and continue for about seven days.
INFLUNZA DEFENCE
The respiratory tract mucosa is the site of infection for influenza viruses and the
site of defense against many other virus infections. Influenza A viruses are
classified by identifying two surface proteins:
 Haemagglutinin (15 known forms)
 Neuraminidase (9 forms).

6. An H1N1 virus caused the deadly 1918 pandemic. A H1N1 “swine flu”
emerged in Mexico in 2009 was a new H1N1 virus. The avian flu that generated
so much concern and speculation beginning in 2004 was a H5N1 version.
Genetic sequencing of viral DNA provides a more detailed method of
identification. The 2009 Mexican strain contained DNA from four different
virus sources: from North American swine influenza viruses, North American
avian influenza viruses, human influenza viruses and swine influenza viruses
found in Asia and Europe. The popular description “swine flu” was misleading
since the novel DNA suggested that the viral DNA came through human and/or
bird infections. Influenza viruses evolve quickly through mutation and DNA
recombination.
Viruses are initially detected and destroyed non-specifically by innate immune
mechanisms, but if the viruses escape the early defense mechanisms, they are
detected and eliminated specifically by adaptive immune mechanisms:
(i) Specific secretory-IgA (S-IgA) antibodies and CD8 cytotoxic T lymphocytes
are involved in the recovery from influenza following viral infection of mice.
(ii) Preexisting specific S-IgA and IgG Abs in the immunized animals are
involved in viral elimination by forming virus-Ig complexes shortly after re-infection.
S-IgA Abs are carried to the mucus by transepithelial transport and
provide protection against virus infection. IgGs move from the serum into the
mucosa by diffusion and are distributed on the alveolar epithelia to prevent
influenza pneumonia.
6

7. (iii) In the absence of Abs in the pre-immunized animals, the production of
specific IgA and IgG Abs by B memory cells is accelerated after re-infection.
These antibodies play a role in viral elimination from day 3 onwards after re-infection.
(iv) In the pre-immunized animals, CTL production by memory T cells is also
accelerated and these cells appear to participate in the killing of the host cells
infected with different subtype viruses from day 3 onwards after re-infection.
(v) Memory Th1 cells mediate an accelerated delayed-type hypersensitivity
response and block virus replication by secreting IFN-gamma.
These defense mechanisms suggest that a mucosal vaccine, capable of inducing
S-IgA Abs, may provide cross-protection against variant viruses within the
same subtype. In addition serum IgG Abs are needed to prevent influenza
pneumonia and CTLs, will provide broad cross-protection against different
subtype viruses.
SWINE FLU SURVIVOR DEVELOPED 'EXTRAORDINARY SUPER
IMMUNITY'
People who recover from swine flu may be left with an extraordinary natural
ability to fight off flu viruses, findings suggests.
In beating a bout of H1N1 the body makes antibodies that can kill many other
flu strains, a study in the Journal of Experimental Medicine shows.
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8. Doctors hope to harness this power to make a universal flu vaccine that would
protect against any type of influenza.
Ultimately this could replace the "best guess" flu vaccines currently used.
Such a vaccine is the "holy grail" for flu researchers. Many scientists are
already testing different prototypes to put an end to the yearly race to predict
coming flu strains and quickly mass produce a new vaccine each flu season.
A woman receives a H1N1 influenza vaccine shot from a medical
Staff at a hospital in Nonthaburi province, on the outskirts of
Bangkok January 11, 2010.
A study of antibodies from people infected with H1N1 swine flu adds proof
that scientists are closing in on a "universal" flu shot that could neutralize many
types of flu strains, including H1N1 swine flu and H5N1 bird flu, U.S.
researchers said on Monday.
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9. They said people who were infected in the H1N1 pandemic developed an
unusual immune response, making antibodies that could protect them from all
the seasonal H1N1 flu strains from the last decade, the deadly "Spanish flu"
strain from 1918 and even a strain of the H5N1 avian flu.
"It says that a universal influenza vaccine is really possible," said Patrick
Wilson of the University of Chicago, who worked on the paper published in the
Journal of Experimental Medicine.
Many teams are working on a "universal" flu shot that could protect people
from all flu strains for decades or even life.
U.S. officials say an effective universal flu vaccine would have enormous
ramifications for the control of influenza, which kills anywhere from 3,300 to
49,000 people in the United States each year.
Wilson's team started making the antibodies in 2009 from nine people who had
been infected in the first wave of the H1N1 swine flu pandemic before an H1N1
vaccine had been produced. The hope was to develop a way to protect
healthcare personnel.
Working with researchers from Emory University School of Medicine, the team
produced 86 antibodies that reacted with the H1N1 virus, and tested them on
different flu strains.
9

10. Of these, five were cross-protective, meaning they could interfere with many
strains of flu including the 1918 "Spanish flu" and a strain of H5N1 or avian
flu.
Tests of these antibodies in mice showed they were fully protected from an
otherwise lethal dose of flu.
And some of these cross-protective antibodies were similar in structure to those
discovered by other teams as having potential for a universal flu vaccine.
"It demonstrates how to make a single vaccine that could potentially provide
permanent immunity to all influenza," Wilson said in a telephone interview.
SWINE INFECTION BOOSTED IMMUNITY TO SURPRISING
10
DEGREES

11. H1N1 MOSTLY AFFECTS YOUNGER PATIENTS, CAN TURN
CRITICAL QUICKLY
A collection of studies to be published in the November 4 issue of the Journal
of the American Medical Association offer insights into the H1N1 flu strain that
has now caused the deaths of at least 4,500 people worldwide, and which World
Health Organization officials estimate will continue to be classified as a
pandemic for several years. A breakdown of the major findings:
CRITICAL ILLNESS CAUSED BY H1N1 SETS IN QUICKLY:
A study of 128 Canadian patients with confirmed or probable cases of swine flu
found that, critical illness—including organ failure, plummeting levels of
oxygen in the blood and the need for mechanical respiratory assistance—tends
to set in shortly after initial hospitalization. Most patients included in the study
experienced symptoms of the H1N1 flu virus for about four days before going
to the hospital, but upon admission generally deteriorated into critical condition
within one to two days. In this study, 14.3% of patients (24 people) died within
a month after critical illness set in; five more patients (fewer than 3%) died
within three months after the onset of critical illness.
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12. YOUNGER PATIENTS AND WOMEN MAY BE HARDEST HIT BY
H1N1:
In the Canadian study, the average age of patients with confirmed or likely
cases of the H1N1 flu virus was 32.3-years-old, and 67.3% (113 patients) were
women. Children too made up a large portion of swine flu patients—29.8% (50
patients). In contrast, few people older than 60 were admitted to the hospital for
swine flu during the study period. The concentration of illness among younger
patients is strikingly similar to the 1918 pandemic of the same flu strain, the
researchers say. “severe disease and mortality in the current outbreak is
concentrated in relatively healthy adolescents and adults between the ages of 10
and 60 years, a pattern reminiscent of the W-shaped curve previously seen only
during the 1918 H1N1 Spanish pandemic,” they write.
DEATH HIGHEST AMONG PATIENTS WHO BECOME CRITICALLY
ILL:
A study of 899 patients admitted into hospitals in Mexico with confirmed or
suspected cases of swine flu revealed that those in whom the virus caused
critical illness faced the most grim outcomes. Of the 58 patients who became
critically ill (just 6% of all admitted), 41.4% died within three months. Of those,
19 died within two weeks of admission. Again, the researchers found, the
critically ill population consisted of mostly younger patients—the median age
for those in serious condition was 44 years.
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13. Oxygenation treatment is largely successful for H1N1 patients with respiratory
complications: Finally, good news! A study of 68 patients with severe
respiratory symptoms of H1N1 admitted to 15 different intensive care units
across Australia and New Zealand between June 1–August 31 of this year,
found that, when treated with extracorporeal membrane oxygenation (ECMO),
the majority of patients survived. ECMO—a technique that enables doctors to
oxygenate patients’ blood outside of the body—was used to treat patients for 7-
10 days. By the end of the study period, 71% of patients (48 people) had
survived, with 32 already discharged from the hospital. “Despite the disease
severity and the intensity of treatment, the mortality rate was low,” the
researchers write. At the end of reporting, 14 patients (21%) had died, and 6
remained in intensive care.
INTO THE EYE OF THE CYTOKINE STORM
The term “cytokine storm” calls up vivid images of an immune system gone
awry and an inflammatory response flaring out of control (Fig. 1). The term has
captured the attention of the public and the scientific community alike and is
increasingly being used in both the popular media and the scientific literature.
However, while the general concept of an excessive or uncontrolled release of
proinflammatory cytokines is well known, an actual definition of what
constitutes a cytokine storm is lacking. Furthermore, there is not a good
understanding of the molecular events that precipitate a cytokine storm, of the
contribution such a “storm” makes to pathogenesis, or of what therapeutic
strategies might be used to prevent the storm or quell it once it has started.
13

14. The cytokine storm has captured the attention of the public and the scientific
community alike, and while the general notion of an excessive or uncontrolled
release of proinflammatory cytokines is well known, the concept of a cytokine
storm and the biological consequences of cytokine overproduction are not
clearly defined. Cytokine storms are associated with a wide variety of infectious
and noninfectious diseases. The term was popularized largely in the context of
avian H5N1 influenza virus infection, bringing the term into popular media. In
this review, we focus on the cytokine storm in the context of virus infection,
and we highlight how high-throughput genomic methods are revealing the
importance of the kinetics of cytokine gene expression and the remarkable
degree of redundancy and overlap in cytokine signaling. We also address
evidence for and against the role of the cytokine storm in the pathology of
clinical and infectious disease and discuss why it has been so difficult to use
knowledge of the cytokine storm and immunomodulatory therapies to improve
the clinical outcomes for patients with severe acute infections.
CYTOKINE STORM
Many of the most severe cases of influenza, including deadly cases, are caused
when the body produces too many immunity molecules called cytokines. The
sudden release of cytokines is dubbed a "cytokine storm." Normally, cytokines
help the body respond to infections, but a cytokine storm leads to widespread
inflammation.
14

15. (IMAGERY OF A CYTOKINE STORM)
"If we look at the 2009 flu pandemic, where millions of people got sick, many
of the people who were hospitalized or died are those that had significant
amounts of cytokines in their blood stream.
Scientists previously thought that cytokine storms happened when cells infected
with a virus released cytokines. But the new research showed that it's cells
lining blood vessels, called endothelial cells, that are opening the floodgates,
and that a protein called S1P1 is key to the release.
They could block S1P1 and the body would still fight the flu virus but without
the risk of dangerous inflammation. Indeed, they found that mice treated with a
drug that targeted S1P1 survived the flu without showing signs of massive
cytokine production and inflammation, severe lung infections, in which local
inflammation spills over into the systemic circulation, producing systemic
sepsis, as defined by persistent hypotension, hyper- or hypothermia,
leukocytosis or leukopenia, and often thrombocytopenia.
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16. 16
1
HUMAN ARDS. PHOTOMICROGRAPHS FROM THE LUNGS OF 2
DIFFERENT PATIENTS WITH ARDS, STAINED WITH HEMATOXYLIN
AND EOSIN (H&E) ARE SHOWN. THE ALVEOLAR SPACES ARE
FILLED WITH A MIXED MONONUCLEAR/NEUTROPHILIC
INFILTRATE, THE ALVEOLAR WALLS ARE THICKENED, AND THE
SEPTAE ARE EDEMATOUS
CYTOKINES
Cytokines are a diverse group of small proteins that are secreted by cells for the
purpose of intercellular signaling and communication. Specific cytokines have
autocrine, paracrine, and/or endocrine activity and, through receptor binding,
can elicit a variety of responses, depending upon the cytokine and the target
cell. Among the many functions of cytokines are the control of cell proliferation
and differentiation and the regulation of angiogenesis and immune and
inflammatory responses.

17. Many cytokines have multiple and sometimes unrelated functions that depend
on the target cell or on the presence or absence of other cytokines. Some have
limited sequence similarity and engage distinct receptors yet transduce signals
through common intracellular pathways (e.g., type I and type III interferons
[IFNs]). In part because of this diversity of structure and function, the
classification and naming of cytokines have been a challenge. The complex
network of the cytokine response is best considered a series of overlapping
networks, each with a degree of redundancy and with alternate pathways. This
combination of overlap and redundancy has important implications with respect
to identifying the key steps in the cytokine response to infection and in targeting
specific cytokines for therapeutic intervention. While many infections are
characterized by broadly similar cytokine profiles, their clinical presentations
can be quite different. Prior to delving into a discussion of cytokine storms, it is
worth taking a brief look at the cytokines at the heart of the cytokine storm.
CYTOKINE STORM PATHOLOGY
Inflammation associated with a cytokine storm begins at a local site and spreads
throughout the body via the systemic circulation. Rubor (redness), tumor
(swelling or edema), calor (heat), dolor (pain), and “functio laesa” (loss of
function) are the hallmarks of acute inflammation. When localized in skin or
other tissue, these responses increase blood flow, enable vascular leukocytes
and plasma proteins to reach extravascular sites of injury, increase local
temperatures (which is advantageous for host defense against bacterial
infections), and generate pain, thereby warning the host of the local responses.
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18. These responses often occur at the expense of local organ function, particularly
when tissue edema causes a rise in extravascular pressures and a reduction in
tissue perfusion. Compensatory repair processes are initiated soon after
inflammation begins, and in many cases the repair process completely restores
tissue and organ function. When severe inflammation or the primary etiological
agent triggering inflammation damages local tissue structures, healing occurs
with fibrosis, which can result in persistent organ dysfunction.
The cytokine storm is best exemplified by severe lung infections, in which local
inflammation spills over into the systemic circulation, producing systemic
sepsis, as defined by persistent hypotension, hyper- or hypothermia,
leukocytosis or leukopenia, and often thrombocytopenia (84). Viral, bacterial,
and fungal pulmonary infections all cause the sepsis syndrome, and these
etiological agents are difficult to differentiate on clinical grounds. In some
cases, persistent tissue damage without severe microbial infection in the lungs
also is associated with a cytokine storm and clinical manifestations that mimic
sepsis syndrome. In addition to lung infections, the cytokine storm is a
consequence of severe infections in the gastrointestinal tract, urinary tract,
central nervous system, skin, joint spaces, and other sites.
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19. CALMING THE STORM
A cytokine storm is an overproduction of immune cells and their activating
compounds (cytokines), which, in a flu infection, is often associated with a
surge of activated immune cells into the lungs. The resulting lung inflammation
and fluid buildup can lead to respiratory distress and can be contaminated by a
secondary bacterial pneumonia—often enhancing the mortality in patients.
This little-understood phenomenon is thought to occur in at least several types
of infections and autoimmune conditions, but it appears to be particularly
relevant in outbreaks of new flu variants. Cytokine storm is now seen as a likely
major cause of mortality in the 1918-20 “Spanish flu”—which killed more than
50 million people worldwide—and the H1N1 “swine flu” and H5N1 “bird flu”
of recent years. In these epidemics, the patients most likely to die were
relatively young adults with apparently strong immune reactions to the
infection—whereas ordinary seasonal flu epidemics disproportionately affect
the very young and the elderly.
For the past eight years, Rosen’s and Oldstone’s laboratories have collaborated
in analyzing the cytokine storm and finding treatments for it. In 2011, led by
Teijaro, who was then a research associate in the Oldstone Laboratory, the
TSRI team identified endothelial cells lining blood vessels in the lungs as the
central orchestrators of the cytokine storm and immune cell infiltration during
H1N1 flu infection.
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20. In a separate study, the TSRI researchers found that they could quiet this
harmful reaction in flu-infected mice and ferrets by using a candidate drug
compound to activate immune-damping receptors (S1P1 receptors) on the same
endothelial cells. This prevented most of the usual mortality from H1N1
infection—and did so much more effectively than the existing antiviral drug
oseltamivir, although the combination of both therapies worked even better.
“That was really the first demonstration that inhibiting the cytokine storm is
protective,” said Teijaro.
MAPPING A PATH FORWARD
For the new study, Teijaro and his colleagues set out to map the major elements
of the cytokine storm in H1N1 infection. To do so, they used gene knock-out
techniques to breed mice that lack one or more molecular sensors of influenza
virus infection and then observed the response to infection by H1N1 influenza
virus.
The experiments showed that knocking out any one infection-sensing pathway
has relatively modest effects on damping the cytokine and immune cell lung-infiltration
response. In each case, an experimental drug compound (CYM5442)
that activates S1P1 receptors knocked it down much more.
“What this shows is that our drug is working not through one selective pathway
but much more broadly,” said Teijaro. “Many different cytokines are induced in
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21. this reaction, so just blocking one is surely not enough to reduce the lung
disease.”
While CYM5442’s effect is broad, its action is selective on cells that bear the
sphingosine-1-phosphate 1 receptor (S1P1R). Teijaro pointed out that it is also
milder than those of steroids, which act indiscriminately on all lymphoid cells,
and other strong immunosuppressant drugs, which may block the immune
response so completely that an infecting virus ends up replicating out of control.
An optimized version of CYM5442, initially developed by Rosen and fellow
TSRI chemist Ed Roberts, has been licensed to the pharmaceutical company
Receptos. It is now in Phase 3 clinical trials for treating relapsing-remitting
multiple sclerosis and Phase 2 trials for ulcerative colitis. Other S1P1 receptor
agonists are in development for inflammatory conditions. A less-specific S1P
receptor agonist—which hits S1P1, but also hits S1P3, S1P4 and S1P5, with
potential off-target effects—is already approved for treating multiple sclerosis.
“We’d like to understand all the pathways through which S1P1 agonists work
and, by pinpointing specific stop/start points, figure out how best to target those
pathways with future drugs,” said Teijaro, who plans further studies with his
colleagues to determine what other cell types are involved in orchestrating and
possibly quieting the cytokine storm. “I’m hoping our work can further
contribute to TSRI’s long track record of success in employing small molecule
probes coupled to genetic and biochemical tools to provide biological insight
into pathological disease processes.”
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22. UNIVERSAL H1N1 INFLUENZA VACCINE
Vaccination is the only public-health means for reducing the impact of
influenza morbidity and mortality by offsetting the uncertainties of timing and
virulence arising from uncontrollable complexities of population, behavioral,
viral, and environmental factors. Vaccination is also considered a cornerstone
approach for pandemic preparedness. The principle approach to influenza
vaccine design focuses on raising antibodies that prevent hemagglutination.
Generally, vaccination does induce hemagglutinating antibodies but these
antibodies are neither cross-reactive with other strains, nor persistent.
Furthermore, vaccination against influenza is only moderately effective. A
meta-analysis using data from randomized, controlled trials conducted over 12
seasons and published between 1967 and 2011 demonstrated that trivalent
influenza vaccination (TIV) in adults aged 18–65 years provided only moderate
protection (59%) over eight seasons and significantly lower levels in other
seasons.2 Live attenuated influenza vaccine (LAIV), which stimulates both
cellular and humoral immunity, showed higher efficacy (83%) in children aged
6 to 17 years, but not in adults. To improve on the shortcomings of existing
influenza vaccination approaches, novel vaccine approaches that aim to provide
universal protection are needed.
Scientist became interested in the concept of cross-reactive T cell epitopes for
influenza during the 2009 H1N1 pandemic. At that time, the Centers for
Disease Control and Prevention reported that seasonal flu vaccines did not elicit
cross-reactive neutralizing antibodies against the emerging pandemic (H1N1)
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23. 2009.3 When early clinical reports released during the 2009 A(H1N1)
pandemic suggested the novel influenza was more virulent among children and
adults under 65 years than the elderly, researcher hypothesized that cellular
responses to cross-reactive T cell epitopes might explain the unexpected disease
distribution. The apparent lack of B cell epitope conservation in novel H1N1
and absence of cross-reactive antibodies raised by the seasonal vaccine H1N1
strain at the time supported this idea.
Thus, researcher set out to identify cross-conserved T cell epitopes in the
pandemic and the 2008–2009 seasonal vaccine hemagglutinin (HA) and
neuraminidase (NA) antigens, as soon as the first pandemic influenza sequences
became available, using immunoinformatic methods. The HLA class II epitope
predictions were later confirmed experimentally using peripheral blood
mononuclear cells from human donors not exposed to the pandemic virus,
illustrating that pre-existing CD4+ T cells elicit cross-reactive effector
responses against the pandemic H1N1 virus. In addition, they demonstrated that
the computational tools were 90% accurate in predicting CD4+ T cell epitopes
and their HLA-DR-dependent response profiles in donors that were chosen at
random for HLA haplotype.
As HA and NA antigens are the principle components of seasonal trivalent
inactivated and subunit influenza vaccines and CD4+ T cells support both
humoral and cellular influenza immunity, we have now performed a
significantly expanded immunoinformatic analysis of the H1-HA and N1-NA
sequence space to identify HLA class II-restricted immunogenic consensus
sequences covering isolates dating back to 1980 from the end of the 2009
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24. pandemic. The novel antigens were validated in HLA binding and T cell assays
in preparation for future vaccine efficacy studies in HLA transgenic mice. They
provide a detailed report on the methods used to define these highly cross-conserved
influenza vaccine epitopes. The method may be of interest for the
design of future H7N9, H5N1, and H3N2 vaccines.
DISCUSSION
Cross-reactive T cell epitopes such as the ones identified here may have played
a significant role in containing the human impact of the 2009 influenza H1N1
pandemic. Despite studies showing pandemic H1N1 was highly pathogenic in
laboratory animals and shared few B cell epitopes with most seasonal H1N1
viruses, the virus triggered only mild symptoms in middle-aged and elderly
adults and, fortunately, failed to cause widespread morbidity and mortality. One
explanation for this unexpected observation is that pre-existing influenza-specific
CD4+ T cells generated cross-reactive cellular responses against the
virus that were capable of limiting disease severity and virus spread in
individuals lacking cross-protective humoral immunity.
An important safety feature of the vaccine design approach described here is
that T cell epitopes that have a high degree of cross-conservation with human
genome are taken into consideration and eliminated from the list of epitopes to
be tested and included in vaccine constructs, as there is at least initial evidence
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25. that such epitopes may be immunopathogenic or tolerated by the immune
system, or they may stimulate regulatory T cell responses.
Accumulating evidence suggests that the sequences identified here may
stimulate influenza-specific T helper cells that can limit disease through
activation of cellular and humoral immune mechanisms reported to be critical
for immunity. Not only do CD4+ T cells play a role in the rate of viral
clearance, but memory helper T cells specific to a previous influenza strain
contribute to distinct cross-strain antibody responses. Thus, influenza vaccine
strategies that focus the T cell response on cross-reactive sequences may
harness cellular and humoral mechanisms with the potential to provide group-common
25
protection against diseases.

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