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Some aspects of immunity and disease during influenza A virus infection

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Title:
Some aspects of immunity and disease during influenza A virus infection
Creator:
Cogliano, Robert Christopher, 1946-
Publication Date:
Language:
English
Physical Description:
v, 60 leaves : ill. ; 29 cm.

Subjects

Subjects / Keywords:
Antibodies ( jstor )
Bladder ( jstor )
Death ( jstor )
Ferrets ( jstor )
Immunity ( jstor )
Infections ( jstor )
Influenza ( jstor )
Lungs ( jstor )
Organ culture techniques ( jstor )
Trachea ( jstor )
Dissertations, Academic -- immunology and medical microbiology -- UF ( mesh )
Immunology and Medical Microbiology Thesis Ph.D ( mesh )
Influenza A virus -- immunology ( mesh )
Influenza A virus -- pathogenicity ( mesh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida, 1979.
Bibliography:
Bibliography: leaves 56-59.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Robert Christopher Cogliano.

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University of Florida
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University of Florida
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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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25097453 ( OCLC )
AEJ9676 ( NOTIS )
AA00004909_00001 ( sobekcm )

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Full Text












SOME ASPECTS OF IMMUNITY AND DISEASE DURING
INFLUENZA A VIRUS INFECTION

















By

ROBERT CHRISTOPHER COGLIANO


A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY





UNIVERSITY OF FLORIDA


1979




SONE ASPECTS OF IMMUNITY AND DISEASE DURING
INFLUENZA A VIRUS INFECTION
By
ROBERT CHRISTOPHER COGLIANO
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA


ACKNOWLEDGEMENTS
I would like to thank Dr. Parker A. Small, Jr., for his support
and patience. Scientifically, his imaginative, creative and critical
thinking were always a standard for which to strive. His nature to
take time to care and try to offer advice on personal matters was much
appreciated.
My thanks also to Bob Yetter, a friend when needed, a source of
impeccable common sense when none was to be found elsewhere and one
of the best trivia experts around.
9
ii


TABLE OF CONTENTS
ACKNOWLEDGEMENTS
Page
ii
ABSTRACT iv
INTRODUCTION 1
The Virus 1
The Disease 3
Immunity to Influenza ..... 4
METHODS AND MATERIALS 11
Viruses
Intranasal Inoculation of Virus into Animals
Virus Adaptation in Mice
Assays
Tissue Cultures
Statistical Analysis
Serum Antibody Production
Antiimmunoglobulin Production .
Interferon Assay
Neutralization Titers
X-rays
Scanning Electron Microscopy
11
11
12
12
12
13
13
15
16
17
17
18
SPECIFIC LOCAL IMMUNITY IN FERRET ORGAN CULTURES
19
Introduction 19
Results 21
Mechanisms of Immunity in Ferret Organ Cultures 28
Bladder Immunity 34
Ciliary Activity in Tracheal Organ Cultures 36
Discussion 39
APPENDICES
APPENDIX A 45
APPENDIX B 55
REFERENCES 56
i i i
BIOGRAPHICAL SKETCH
60


Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the Requirements
for the Degree of Doctor of Philosophy
SOME ASPECTS OF IMMUNITY AND DISEASE DURING
INFLUENZA A VIRUS INFECTION
By
Robert Christopher Cogliano
December 1979
Chairman: Parker A. Small, Jr.
Major Department: Medical Sciences (Immunology and Medical Microbiology)
In order to study immunity to influenza virus infection in the
upper respiratory tract we have tested organ cultures from normal and
convalescent ferrets for resistance to infection. We then attempted to
delineate immune mechanisms acting within the tissue.
Ferret tracheal organ cultures prepared from animals previously
infected intranasally with influenza A virus required approximately 130
times more homologous virus (A/PR/8/34(MONI) or A/Port Chalmers/1/73
(H3N2)) to become infected in vitro than similar cultures from normal
ferrets. Also, these cultures from convalescent ferrets required about
10 times more heterologous virus (A/PR/8/34 (H0N1) or Sendai) to become
infected in vitro than similar cultures from normal animals challenged
in vitro with the heterologous virus. We conclude that these tracheal
rings are specifically immune.
Possible mechanisms of resistance of the tracheal organ cultures
were examined. Serum antibody given intraperitoneally to normal ferrets
did not bring about immunity in cultures derived from these animals.
IV


Rabbit antiferret gamma chain did not significantly ablate resistance
of cultures when put on cultures prior to virus challenge. Rabbit anti
ferret alpha chain treatment gave ambiguous results. Neither neutraliz
ing antibody nor interferon could be measured in Day 4 culture super
natants.
Once tracheal rings are infected, they continue to shed viruses for
at least 60 days, the longest period any cultures were kept. Virus shed
ding in the intact ferret lasts normally 5-7 days. Thus, recovery in the
intact ferret seems to be dependent upon factors which are not present,
or at least not functional, in the tracheal explant. This is consistent
with the hypothesis that recovery is dependent upon systemic rather than
local phenomena.
Bladder tissue from normal and previously infected ferrets was
also cultured and challenged with homologous and heterologous virus. The
bladder from previously infected ferrets exhibited specific immunity,
although the immunity was more variable.
Concurrent studies aimed at determining the therapeutic value of
serum antibody for the treatment of lethal viral pneumonia in mice showed
that serum antibody given before infection or on Days 3, 4 and 5 after
infection was life-saving but given later was ineffective in preventing
death. Since radiological change could first be detected 6-7 days after
infection we conclude that serum antibody is not likely to be useful for
treatment of animals.with lethal viral pneumonia.
v


INTRODUCTION
This dissertation deals primarily with aspects of immunity to
influenza virus and secondarily with pathogenesis and recovery from
influenza infection. The introduction begins with a discussion of the
virus, specifically its structure, replication and antigenic nature.
This is followed by a description of the disease caused by the virus and
then a summary of our knowledge of immunity. Lastly, we report the
specific questions which we addressed in this research.
The Virus
Structure
The influenza virus is taxonomically designated as belonging to
the Orthomyxoviridae family (Melnick, 1977). Structurally, influenza
virus contains a segmented RNA genome which is associated in the nucleo-
capsid with an RNA transcriptase consisting of 3 proteins and a nucleo-
protein. Exterior to the genome is the virus coded matrix protein and
an envelope which is derived from the host cell during budding. Imbedded
in the envelope of the virus are two types of virus specific glycoprotein
spikes, the hemagglutinin (HA) and the neuraminidase (NA). An eighth
protein is the nonstructural protein (NS) for which no definite role has
been established.
Replication
The RNA genome-of influenza A contains 8 segments (Ritchey et al.
1976) and the virus replicates as a class V virus according to the Baltimore
classification (Baltimore, 1971). Briefly, the negative stranded RNA
enters the cell. The segments must first be transcribed to plus stranded
1


2
RNA to be able to act as messengers. There appears to be two types of
plus strands, one which is poly-adenylated at the 3' end and one type
that is not poly-adenylated. It has been suggested that the poly-A
form acts as messenger for viral proteins while the form without poly-A
acts as template for production of progeny viral RNA (Hay and Skehel,
1979). The nucleocapsid is assembled in the cytoplasm of the host cell
while surface viral proteins are inserted into the cell membrane at var
ious sites. Concurrently, host proteins are largely excluded from the
membrane at these specific sites. From these sites at the cell surface
progeny virus bud by some unknown mechanism taking with them the nucleo
capsid and cell membrane with imbedded viral proteins. One recent report
which merits mention but has as yet not been confirmed or explained is
that DNA has also been found in the influenza virus and comprises between
5-8% of the total nucleic acid (Kantorovich-Prokudina et al, 1978).
0
Antigenic Characteristics
Influenza viruses are classified into serotypes and each serotype
is further classified into subtypes. The serotype depends upon antigenic
determinants found in the nucleoprotein. Three major serotypes have so
far been identified A, B and C. Types A and B cause the majority of
disease in man. The subtypes within each serotype are defined by anti
genic determinants on the HA and NA. It is the characteristic genetic
variability of the segments of viral RNA coding for these two viral
proteins which causes the unpredictable "shifts" and "drifts" in antigenic
character. Shift is found only in type A but drift is found in both
types A and B. These shifts and drifts in viral antigens result in the
well known pandemics and epidemics, respectively. Shift refers to a major
change in antigenic determinants of a surface protein, for example, the


3
shift of the hemagglutinin from H2N2 to H3N2. This change has been shown
to be radical in nature. By mapping tryptic digests of these viral pro
teins it can be shown that the amino acid sequences from H2 differ greatly
from those of H3 (Webster and Laver, 1975). These types of changes
have been hypothesized to happen by reassortment events after co-infection
of host cells. Animal influenza viruses have been implicated as a source
of these new and different antigenic determinants. The presence of these
new antigens on a virus then leaves the population with decreased immunity
to this virus (Rasmussen, 1964). A drift, for example a drift of H3N2
Port Chalmers (1973) to H3N2 Texas (1977), is a small change in antigenic
character (perhaps as little as a single amino acid change). This is
demonstrated, again, by tryptic digests of viral surface proteins (Webster
and Laver, 1975). Drift probably occurs by single mutational events at
a locus within the RNA segment coding for the protein.
The Disease
0
Influenza, although it has been with us for centuries, remains as
perhaps the last great pandemic of man. During the period from 1968 to
1979, more than 150,000 excess deaths are estimated to have occurred
during epidemics of influenza A in the United States (MMWR, 1979). These
excess deaths occur primarily among the young, the chronically ill and
older individuals.
The disease in humans is commonly a rhino-tracheobronchitis with
accompanying headache, malaise, fever, anorexia and myalgia. In uncom
plicated cases recovery occurs in a few days to a week. In a few cases,
however, the virus can cause a pneumonia which quickly runs its course.
Prognosis is poor in this instance. Viremia may be found only in these
severe cases (Naficy, 1963). Supportive care has proven useful in some


4
cases but more often than not death ensues in 3-7 days after onset of
illness. Prior to widespread use of antibiotics, bacterial pneumonia
was a frequent complication following influenza infections and even
today remains the major cause of influenza related deaths. Bacterial
infection after influenza was most probably the major cause of death
during the 1918 pandemic which claimed 20 million excess deaths world
wide. It has been estimated, however, that a full 20% of the 20 million
excess deaths were directly due to viral pneumonia with death occurring
in 6 or less days (Crosby, 1976).
Immunity to Influenza
That an important part of the immunity to influenza is specific
is well known. Animals with prior infection with a subtype of influenza
(e.g. H0N1) will at least for a period be solidly immune to challenge
with that same virus but are still susceptible to infection with a dif
ferent type A influenza virus (e.g. H3N2). Specific immunity can be
either cell mediated (CMI) or antibody mediated (Ab). Further, the
immune response may be either local or systemic in nature. The obvious
question then arises: Which is these, local Ab, local CHI, systemic Ab
or systemic CMI is most responsible for the specific resistance to rein
fection?
Antibody Mediated Immunity
If we turn first to Ab, most researchers can be divided into two
schools: those that conclude that systemic Ab protects the animal against
infection and those that believe that serum antibody (Sab) does not cause
protection but only correlates with it and that local antibody (Lab) is
responsible for resistance to reinfection. In the former group Stuart-
Harris (1973) reported that serum antibody was protective in man. Both


5
Allan et al. (1971) and Rott. et al. (1974) reported serum antibody to
be protective in chickens. More recently, Virelizier (1975) concluded
that in mice serum antibody was most important for protection against
reinfection by influenza. McLaren et al. (1974), using ferrets in
challenge experiments, found serum antibody against the hemagglutinin
to correlate best with protection against infection. Serum antihemag
glutinin antibody is thought to be the effector in the above examples.
However, Murphy et al. (1972), working with human volunteers, and Schulman
et al. (1968), working with mice,have concluded that serum antineuramini
dase antibody can also be protective against infection.
There are also those that have observed the reverse situations,
that is, that Sab is not protective against infection. Morris et al.
(1966) demonstrated that influenza infection in humans is not prevented
by high levels of serum antibody. Small et al. (1976) passively immunized
ferrets so that their hemagglutination-inhibition titers (HAI) were 1000
or greater, but were unable to prevent influenza infection. They con
cluded that serum antibody played no role in preventing infection of
ferrets. So it appears there is evidence both supporting and refuting
serum antibody as an important immune mechanism in preventing influenza
infection.
Many investigations support the idea that antibody produced locally
in the respiratory tract and distributed on the mucosal surface is the
primary mode of protection. Richman et al. (1974) have shown .that the
local antibody response is specific. Francis (1943) infers that nasal
wash antibody specific for infecting influenza virus is the most important
determinant of specific resistance to influenza. Frazekas de St. Groth
and Donnelley (1950a), studying the antibody response to mice receiving


6
graded doses of influenza virus (live or inactivated) by various immuni
zation routes concluded that "serum antibody does not give any informa
tion on antibody that is found at the local site." In further work
(1950b) they conclude that serum antibody does not coincide with immunity
but local antibody does. Bearc et al. (1969) concluded that a factor,
probably local antibody, exerted a considerable influence on human
resistance to infection by influenza virus. Waldman and Coggins (1972)
concluded that a vaccine's ability to stimulate nasal secretory antibody
was the deciding factor as to whether it could protect humans against
influenza infection. Potter et al. (1972) showed production of nasal
antibody after nasal infection of ferrets but not after immunization
with killed virus. They postulated this was the reason for better pro
tection of these animals upon subsequent challenge with virus. All of
the above studies show only a correlation between local antibody and
protection but do not prove that the local antibody is the cause of
immunity. However, Barber and Small (1978) in experiments using ferrets
with surgically created tracheal pouches, which allow study of infection
and protection in two separate sites, conclude that systemic factors do
not play a role in preventing upper respiratory tract infection with
influenza and that, therefore, local factors are primary in prevention
of infection.
Again there is evidence to support the opposite view, that is,,
that local antibody, is not responsible for immunity to influeirza. In a
recent report Jennings et al. (1978), using medical student volunteers,
specifically concluded that nasal wash antibody does not correlate with
protection.


7
Much of the confusion as to which is more important for protection,
serum antibody or local antibody, may stem from two problems of experi
mental design. First, those that look only at Sab and not Lab can only
show correlation with the one parameter and are unable to rule out the
other parameter, Lab. Secondly, different workers study different sites
of infection having different immune mechanisms in operation. Virelizier
(1975) and Loosli (1953) while concluding that Sab protects against influ
enza infection were studying the lung. While Sab probably does protect
the lung in their animal system, it probably does not protect the upper
respiratory tract, the tissue affected in the vast majority of cases of
human influenza. Ramphal et al. (1979) have shown in mice that serum
antibody given prior to infection will protect the lung from pneumonitis
but will not protect the trachea, thus separating the upper and lower
respiratory tract immunologically (see Appendix B).
Cell Mediated Immunity
The possible role of CMI in immunity to influenza has only recently
begun to be examined. However, it has been shown to be important in both
recovery and pathogenesis. In studies where nude mice (Sullivan et al.
1976)y or mice treated with antilymphocyte serum (Suzuki et al. 1974), were
infected with influenza virus significant prolongation of survival time
was noted. Also, virus was not eliminated from these mice. This suggests
that, in severe infections, CMI is responsible for some pathogenicity of
the virus and thatT-cells are required for.recovery from influenza
infection. This, however, has not been shown to be the case in studies
by Yap et al. (1979) where they conclude that influenza immune T-cells
inhibit rather than contribute to pulmonary pathology. However, they do
agree that CMI may play an important role in recovery from murine influenzal
pneumonia.


8
Cytotoxicity is seen by many as the major cell mediated effector
mechanism influencing the outcome of influenzal disease. Yap and Ada
(1977) have shown that in mice this cytotoxicity is probably due to T-
cells. Yap and Ada (1978), speaking specifically about systemically
derived cytotoxic T-cells found in the lungs of mice, conclude that
these cells can bring about recovery and are part of the consolidation
process. Ennis et al. (1978) also conclude that cytotoxic T-cells are
derived systemically and that they are part of the immunological and
pathological response to virus infection.
It has been shown in vitro that cytotoxicity against influenza
infected cells displays the same histocompatibility restriction as has
been shown for other viruses (Braciale, 1979). However, it is not
specifically known if H-2 antigens exist in vivo on the epithelial cells
which are infected by influenza virus.
The mechanism of cytotoxicity is unknown. It has been suggested
that both antibody and cells may be involved and that this might there
fore be an antibody-dependent cell cytotoxicity (Lucas and Barry, 1977,
Greenberg et al., 1977). Further, cytotoxic cells have been shown
generally to be of the Ly-23+ subclass and that this activity can be
amplified by the Ly-1+ subclass of T-cells (Cantor and Boyse, 1975).
It has become clear that CMI reactions during influenza infection
are not as specific as humoral responses. Doherty et al. (1977) have
shown in mice that.at least two populations of cytotoxic T-cells arise
during influenza infection, one specific and one cross-reactive. Also,
during secondary response, the cross-reactive population may be preferen
tially stimulated. Biddison et al. (1979) have shown that cross-reactive


9
cytotoxic T-cells exist in man and that secondary response T-cells are
preferentially cross-reactive. What is the cause of this cross-reactivity?
Webster and Hinshaw (1977) suggest that M-protein, although not expressed
on the surface of the virion or on cell surfaces, may be responsible for
the cross-reactive T-cells. M-protein is virtually the same in all type-A
influenza (the cross-reactivity does not extend across serotypes).
Cretescu et al. (1978) have shown that humans do make anti-M-protein
responses during infection, so evidently the M-protein can be recognized
by the immune system at some stage during infection. In fact Ada and
Yap (1977) and Braciale (1977) report that M-protein is expressed on the
cell surface. Other reports show that another antigen which is shared
by all subtypes within a serotype, the nucleoprotein, is expressed at
the surface of infected cells during early events (Virelizier, 1977).
This may, therefore, account for the cross-reactivity of T-cell cyto
toxicity.
Local CMI responses have been shown in a few instances. Henney and
Waldman (1970), and Waldman and Henney (1971) demonstrated that pulmonary
CMI is relatively independent of the circulating response. Cambridge et
al. (1976), using migration inhibition and cytotoxicity assays, observed
in mice that the local response by pulmonary node lymphocytes was both
greater and faster than spleen cells after influenza infection, thus
suggesting the presence of local CMI. Wyde and Cate (1978) found that
during murine inflyenza an increase in cell cytotoxicity was found in
regional lymph node cells before peripheral blood lymphocytes. They infer
from this that the cytotoxic response found in bronchioalveolar washes
at Day 6 after infection is a local CMI response. In humans, Jurgensen
et al. (1978) show that CMI in the respiratory tract was best, stimulated


10
by aerosol immunization while subcutanetous immunization stimulated
primarily systemic CMI. They presented this to support the importance
of local CMI during influenza infection.
Most definitive work proves upper respiratory tract immunity is
local (Barber and Small, 1978). Further it has been shown that CMI is
not specific enough in nature to explain the specific immunity seen in
influenza. These two statements taken together suggest local Ab as
the probable mechanism of resistance. However, reported work which
implicates Lab is not direct. We therefore assessed the role of antibody,
both local and systemic in vitro. Further, we assessed the role of Sab
in pathogenesis and recovery in vivo. We wish to relate our findings to
three areas: Are these factors (1) important in resistance?, (2) import
ant in causing pathology seen during influenza infection? and (3) import
ant in recovery from the disease?


METHODS AND MATERIALS
Animals
Mature ferrets were obtained from Marshall Research Animals, Inc.,
North Rose, New York, and housed in individual cages under conditions
which prevent cross-infection. Five-week old A/J mice were obtained
from Jackson Laboratory, Bar Harbor, Maine. Goats were obtained from
the University of Florida. New Zealand white rabbits were obtained from
Kel Farms, Gainesville, Florida.
Viruses
Influenza viruses used were A/PR/8/34(H0Nl) and A/Fort Chalmers/
1/73(H3N2). Large stocks of both viruses were obtained by injecting
viruses into allantoic cavities of ten-day old embryonated chicken eggs
which were then incubated for 3 days at 36C at which time allantoic
fluid was harvested, pooled and stored at -85 C in 1 ml aliquots. The
H0N1 virus had a chick erythrocyte hemagglutination (HA) titer of 1280
7 2
and contained 10 50% egg infectious doses/ml (EID^q). The H3N2 virus
8 2
had an HA titer of 160 and contained 10 EID^^/ml. Sendai virus was
propagated in the same manner and had an HA titer of 512 containing
109,2 EID /ml.
Intranasal Inoculation of Virus Into Animals
Ferrets were anesthetized with 0.5 cc of Ketaset (Ketamine hydro
chloride, Bristol laboratories) and infected with 0.1 ml of undiluted
virus in each naris. Mice were anesthetized with 0.2 cc nembutal
(6 mg/ml sodium pentobarbitol, Abbott Laboratories, Chicago) and infected
with 0.05 cc of undiluted virus per naris.
11


12
Virus Adaptation in Mice
To obtain virus which ivas able to cause lethal pneumonia in mice,
H3N2 influenza virus was passaged successively in mouse lung. Mice were
anesthetized, infected and three days later they were sacrificed via
cervical dislocation. The lungs aseptically removed and macerated in
5 mis of Lebovitz's L-15 medium. Homogenates of lung tissue were
centrifuged to clear debris and supernatants were used to infect the
next group of mice.
Assays
Virus was detected by inoculation of samples into the allantoic
cavity of embryonated chicken eggs that were 10 days old, as previously
described (Barber and Small, 1974). HA and HAI titers were performed
with a microtiter kit using disposable microtiter plates (Cooke Engineer
ing, Alexandria, Virginia) as described by Sever (1962). Sera used for
HAI assays were first absorbed with kaolin and chicken RBCs and heated
at 56C for 30 minutes as described previously (Barber and Small, 1974).
Tissue Cultures
Ferrets were anesthetized and exsanguinated by cardiac puncture.
Trachea and bladder were aseptically removed and placed into sterile
100 x 15 mm petri dishes containing approximately 20 mis of Hanks'
balanced salt solution with 100 units/ml penicillin and 100 yg/ml strepto
mycin. While in the petri dish the trachea was cut into individual rings
2
and the bladder was^ cut into pieces approximately 3 x 3 mm .Tissue
pieces were then put individually into 35 x 10 mm petri dishes with 3 mis
of the L-15 medium (glutamine [.3 mg/ml], 10% fetal calf serum, gentamicin
[5 yg/ml], streptomycin [100 yg/ml], penicillin [125 units/ml] and


13
mycostatin [100 units/ml]). The cultures were incubated overnight at
35C. The following day (Day 1) the 3 mis of medium was removed and
replaced with 3 mis of the medium containing the challenge virus at the
proper dilution (Figure 1). If antiimmunoglobulin (a-Ig) treatment was
done, the a-Ig serum was added after tissues were cut up but still in
100 x 15 mm dishes on Day 0. The a-Ig serum was added in equal parts with
medium and cultures were incubated at 35C. The next day the individual
pieces of tissue were put into 35 x 10 mm petri dishes with media without
antiimmunoglobulin and cultures were immediately challeneged with virus
as before.
Statistical Analysis
Viral and antibody titers were compared using the t-test (Men
denhall, 1975) and mortality compared using Fisher's exact test
(Siegel, 1956).
Serum Antibody Production
Ferrets previously infected with H3N2 influenza virus were inoc
ulated IM with 0.5 cc of A/Fort Chalmers/l/73/N3N2 killed vaccine (MRC-11)
Two weeks later they were bled via cardiac puncture and serum was col
lected. Goat serum antibody was obtained from a goat given the same
A/Port Chalmers vaccine IM weekly for three weeks. The goat was bled
via the external jugular. The sera were heat inactivated at 56C for
30 minutes and then filter sterilized before use. In some experiments
very high titer antibody was needed. This was prepared by three succes-
ive 33% ammonium sulfate precipitations and centrifugations at room temper
ature. After the last spin the precipitate was redissolved in saline and
the solution was then dialyzed against saline for two days. They were
then heat inactivated and filter sterilized.


C'
G
O
H;
2
o
r+-
9
ACUTE SERA
FOR HI
NASAL WASH
NASAL WASH
i
o
rt
O
o
o
o
X
o
H*
3
2
rt-
&
h-*
CONVALESCENT ANO
NORMAL SERA FOR HI
REMOVE VIRUS, WASH 3X
SUPERNATANT FOR
VIRUS ASSAY

o
pg
2
o
o
H*
3
C-
X
to
SUPERNATANT FOR
VIRUS ASSAY ^
SUPERNATANT FOR
VIRUS ASSAY
SUPERNATANT FOR VIRUS ASSAY
INFECT WITH VIRUS
"S.
SACRIFICE-
CHALLENGE
PUT TISSUES INTO ORGAN CULTURES
CULTURES WITH VIRUS DILUTIONS


15
Antiimmunoglobulin Production
Rabbit Antialpha Serum
Preparation of rabbit antialpha serum was performed by Richard
Kris in our laboratory. Surgery was performed on ferrets to canulate
the bile duct. Bile was collected over a period of a few days. The
bile was subjected to preparative electrophoresis on acrylamide (3% aga
rose) at pH 6.3, 5 milliamps for 18 hours. The gammaglobulin fraction,
collected in the buffer at the top of the gel, was then concentrated by
pressure dialysis. This was then gel filtered over an Ara-22 column
(Pharmacia) and the 11S fractions w'ere pooled. One-half of a mg of
this in complete Freund's adjuvant (CFA) was then injected subcutaneously
into a rabbit which had previously been injected with ferret IgA prepara
tions more than one year prior to this time. Two weeks later the rabbit
> -J\ *
was bled from the ear artery and serum was collected. This serum was
then subjected to affinity chromatography by passing it over a sepharose
4B CNBr column (Pharmacia) with ferret IgG attached. The resulting serum
was shown by ouchterlony analysis to have anti-IgA activity but no activity
toward other immunoglobulin classes.
Rabbit Antigamma Serum
Again, preparation of rabbit antigamma serum was largely performed
by R. Kris. Ferret serum was treated with KBr and centrifuged to elim-
_3
inate lipoproteins. The supernatant was dialyzed against PBS (10 M).
The pseudoglobulin fraction was passed over a DEAE column and the 7S
fractions were pooled. One-half of a mg of this 7S fraction in CFA was
injected subcutaneously into a rabbit. Three weeks later the rabbit was
boosted with 0.5 mg in Freund's incomplete adjuvant. Three weeks later


16
the rabbit was bled for serum. The anti-IgG serum was subjected to
affinity chromatography by passing it over a sepharose 4B CNBr column with
IgA attached. The resulting serum was shown by ouchterlony analysis to
have activity against ferret IgG but not ferret IgA.
Interferon Assay
Ferret kidneys were removed aseptically and put into Gey's A solu
tion. The kidney capsules were stripped and the kidneys were minced
with scissors. The minced tissue was washed twice with Gey's solution.
The tissue was put into a trypsinization flask with 100 mis Gey's A,
(Streptomycin 125 ygms/ml, penicillin 250 units/ml), 0.1% trypsin and
0.04% versene. The flask was put into the cold (4C) overnight. The
next day the flask was put on a magnetic stirring device at low speed
and 37C for 30 minutes. The loose cells were decanted and centrifuged
at 1000 rpm for 15 minutes. The supernatant was poured off and the pellet
resuspended in medium. This was centrifuged and resuspended as before.
Cell density was adjusted to approximately 10^ cells/ml and 20 mis were
dispensed into 75 cm^ culture flasks which were then incubated at 37C.
Cultures were fed after three days with MEM (10% FCS and 0.03 M HEPES)
and thereafter twice weekly. After monolayers were confluent they were
washed once with Gey's A solution and treated with trypsin/versene solu
tion until the cells came off the plastic. The culture flask was washed
twice with 10 mis of medium. Cell density was adjusted to 2.5 x 10^
cells/ml and 1 ml was dispensed into each well of a 24 well plate. The
next day samples to be assayed were incubated with ferret antiinfluenza
virus antibody (HAI = 1000) for 30 minutes at 37C. Samples were then
d:¡ luted serially and put onto cell cultures overnight. Fluid was withdrawn


17
and 0.1 ml of a dilution of VSV (giving between 30 and 300 plaques/well
on controls) was dispensed to each well for 1 hour. Methyl-cellulose
overlay was put on and two days later removed. Cell monolayers were then
stained with crystal violet for 20 minutes. Plaques were counted and 50%
plaque reduction titers were calculated.
Neutralization Titers
Samples were heat inactivated at 56C for 30 minutes. Samples were
then diluted serially and equal amounts of samples and virus (100 EID^/
mis) were incubated for 1 hour at room temperature. Ten-day old embryon-
ated chicken eggs were injected with 0.1 ml of incubated mixtures. Eggs
were incubated at 36C for two days, then harvested and hemagglutination
assays performed and results recorded. That dilution of the sample that
gave 50% neutralization of the virus was determined to be the neutrali
zation titer.
X-rays
Mice were x-rayed using Dupon Extremity A film (10 x 12 inches)
and an Extremity I cassette. The machine used was a Phillips XF 3001.
Films were takn at 40 KV, 16 MAS, and 1/20. Developing was accomplished
with a Kodak RP X-omat automatic processor, model M6A-N. Animals were
held extended and in place by extending a string across a styrofoam board
and allowing the animals to grab hold of the string and pulling them back
by the tail. The styrofoam board was approximately 1 inch thick because
thicker boards cause loss of clarity in the radiograph. This board was
rested on top of the cassette as the picture was taken. Individuals
holding the tails of the mice were always dressed in protective clothing
and wore a film badge for monitoring levels of radiation exposure.


18
Scanning Electron Microscopy
Trachea samples for scanning electron microscopy (SEM) were
placed in a buffered fixative composed of 2.5% glutaraldehyde, 0.1 M
sodium cacodylate, and 0.1% CaCl2 (pH 7.4) and allowed to fix for at
least 24 hours before further preparation. They were then removed
from the fixative and dehydrated in graded concentrations of acetone
(70 to 100%). Specimens were critical point dried in a Bomar SPC 900/Ex
critical point drying machine (Bomar Corp., Tacoma, Washington) coated
with gold-palladium in a Hummer II shadowing machine (Technics, Alexandria,
Virginia) and examined with a Novascan 30 electron microscope (Semco,
Ottawa, Canada).


SPECIFIC LOCAL IMMUNITY IN FERRET ORGAN CULTURES
Introduction
Organ Culture
We began our studies by examining local immune mechanisms by use
of ferret organ cultures. Organ cultures consist of fragments of tissue
taken from the adult animal and maintained in vitro. Cell proliferation
does not normally occur during the short time the cultures are in use
and the tissue retains many of its in vivo characteristics. Organ cultures
can provide a high degree of sensitivity and economy in determining factors
at work in tissues (Schmidt and Maassab, 1974).
Many uses have been found for organ cultures, including the study
of resistance to reinfection. Heuschele and Easterday (1970) used organ
cultures derived from chicken trachea to study resistance to reinfection
by Newcastle Disease Virus (NDV). They suggested a potential role for
organ cultures to study mechanisms of formation and/or secretion of local
antibody. Finkelstein et al. (1972) again using chicken tracheal organ
cultures demonstrated that resistance, to NDV was probably partially
mediated by interferon. Resistance after intratracheal immunization
correlated only with tracheal antibody and could be partially blocked by
exogenously applied rabbit antichicken globulin. This suggested antibody
was present on mucosal surfaces which could have been produced locally.
Schmidt and Maassatr (1974) found specific resistance to influenza virus
in tracheal organ cultures from chickens. They suggest that this immunity
was at least partially derived from a secretory immune system.
19


20
Organ cultures have been shown to be an excellent system for
studying pathogenesis of respiratory infections. Klein and Collier
(1974) studied pathogenesis of human parainfluenza type 3 virus infection
by using hamster tracheal organ cultures. Human organ cultures have been
used successfully to study the cytopathic effect of this same pathogen
(Craighead and Brennan, 1968).
The relationships between influenza strains that infect humans and
animals can be studied in organ cultures. In other words, they offer a
way of determining the host ranges of the different strains in a precise
and economical manner. This is important as it relates to the hypothesized
recombinational events occurring in simultaneously infected host cells
causing the creation of a new strain of influenza virus capable of causing
pandemics (see general Introduction). Schmidt et al. (1974) studied human,
swine, equine and avian influenza A viruses and tested them for infectivity
in chicken, ferret, equine and porcine tracheal organ cultures. Results
showed that the homologous tissue was much more easily infected than hetero
logous tissue.
Organ cultures have also been shown to be an excellent way for
screening potential live vaccines. Mostow and Tyrrell (1973) have used
ciliary activity in tracheal organ cultures as a measure of virulence and
therefore usefulness of vaccines. Vaccines which cause a small decrease
of ciliary activity have been shown to be extremely avirulent in vivo
while vaccines that are highly virulent thus causing marked decreases in
ciliary activity in organ culture are likewise virulent in vivo.


21
Ferrets and Influenza
We have elected to use ferrets for our studies of influenza, because
influenza infection in ferrets appears to resemble the human infection and
because ferret trachea are relatively long and contain a large number of
individual rings. Further, the size of the animal makes it easy to handle.
The ferret also lends itself to the organ culture system because of the
ease with which their tissues are infected by influenza virus. Bang and
Niven (1958) reported the cultivation of influenza virus in ferret nasal
mucosa. In perhaps the most extensive studies performed so far, Basarab
and Smith (1970) showed the growth patterns of influenza virus in cultures
of ferret organs. They reported virus replication in cultures of ferret
nasal mucosa, lung, trachea, oviduct and bladder.
Results
Immunity in Ferret Organ Cultures >
The basic experimental design is shown in Figure 1. In all experi
ments ferrets, subsequently identified as convalescent, were infected 21
days before being killed. Nasal washes were taken 2 and 4 days after
this infection. Serum was taken at the time of infection and of killing.
Animals were proven to be infected by both virus isolation from nasal wash
(one exception noted in Table 1) and a 4-fold or greater rise in HAI anti
body titre to the virus. At Day 0 normal and convalescent ferrets were
killed and their tissues put into organ cultures. Serum taken from normal
ferrets at the time of killing (Day 0) had no detectable HAI antibody
(<1:8) to influenza"virus. Cultures were challenged with varying dilutions
of influenza virus on Day 1. The number of organ cultures/dilution varied
in different experiments depending upon the number obtained from each


22
TABLE 1
VIRUS ISOLATION PROM TRACHEAL RINGS
3.
Virus Isolation on Day
Virus Challenge
Rings
0
4
6
8 10
Normal
12
Ferret
14
17
19
Control
1
0
0
0
0
0
0
0
0
0
2
0
0
0
0
0
0
0
0
0
3
0
0
0
0
0
0
0
0
0
4
0
0
0
0
0
0
0
0
0
5
0
0
0
0
0
0
0
0
0
-3
10
1
0
+
+
+
+
+
+
+
+
2
0
+
+
+
+
+
+
+
+
3
0
+
+
+
+
+
+
+
+
4
0
+
+
+
+
+
+
+
+
5
0
+
+
+
+
+
+
+
+
-4
10
1
0
+
+
+
+
+
+
+
2
0
+
0
+
+
+
+
+
+
3
0
+
+
+
+
+
+
+
+
4
0
+
+
4-
+
+
+
+
A I
+
5
0
+
+
+
+
+
+
+
+
-S
10
1
0
+
+
+
+
+
+
+
+
2
0
+
+
+
+
+
+
+
+
3
0
+
+
+
+
+
+
+
+
4
0
+
+
+
+
+
+
+
+
5
0
+
+
+
+
+
+
+
+
10
1
0
+
+
+
+
+
+
+
+
2
0
+
+
+
+
+
+
+
+
3
0
+
+
+
+
+
+
+
+
4
0
+
+
+
+
+
+
+
+
5
0
0
0
0
0
0
0
0
0
-7
10
1
0
0
0
0
0
0
0
0
0
2
0
0
0
0
0
0
0
+
0
3
0
+
+
+
+
+
+
+
+
4
0
+
+
+
+
+
+
+
+
5C.
0
0
+
+
+
+
+
0
0


23
TABLE 1--Continued
Infected Rings (Day 8)
21 25 31 37 40 Total Rings OCID^q
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
ND
+
0
+
+
ND
+
0
+
+
ND
+
+
0
+
ND
+
+
+
+
ND
+
+
+
+
+
+
+
+
+
+
+
0
+
+
+
+
+
+
J-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
0
+
+
+
+
0
+
f
+
+
+
0
+
+
+
+
+
+
+
+
0
+
+
+
+
0
+
+
+
0
+
+
+
+
+
0
+
+
0 0
0 0
0 0
+ + 0 + +
+ 0 + +
0/5
5/5
100,8EID5()s/ml
5/5
5/5
4/5
+
3/5


24
TABLE 1--Continued
Virus
Isolation3 on
Day
Virus Challenge
Rings
0
4
6
8 10
Convalescent
12 14
Ferret
17
19
-7
10
1
0
+
+
+
+
+
+
+
+
2
0
+
+
+
+
+
+
+
+
3
0
+
0
+
+
+
+
+
+
4
0
+
+
+
+
+
+
+
+
1
o
t-H
1
0
+
+
+
+
+
+
+
+
2
0
+
+
+
+
+
+
f
+
3
0
+
+
+
+
+
+
+
+
4
0
+
+
+
+
+
+
+
+
io~4
1
0
0
0
0
0
0
0
0
0
2
0
+
+
+
+
+
+
+
+
3
0
+
+
+
+
+
+
0
+
4
0
+
+
+
+
+
+
+
+
10
1
0
0
0
0
0
0
0
0
0
2
0
0
0
0
0
0
0
0
0
3
0
0
0
0
0
0
0
0
0
4
0
+
+
+
+
+
+
+
0
-fi
10
1
0
+
0
0
0
0
0
0
0
2
0
+
+
+
+
+
+
+
+
3C
0
0
0
0
0
0
0
0
0
4C
0
0
0
0
+
+
+
+
+
-7
10
1
0
0
0
0
0
0
0
0
0
2
0
0
0
0
0
0
0
0
0
3
0
0
0
0
0
0
0
0
0
4
0
0
0
0
0
0
0
0
0
5
0
0
0
0
0
0
0
0
0
+ = at least one of the duplicate eggs show HA 3 days after inoculation
with sample.
^Dilution of stock virus containing 10^"^ EID^/ml of A/PR/8/34 (H0N1) .
c
Ring which changed from not infected to infected.
ND = Not done.


25
TABLE 1--Continued
Infected Rings (Day 8)
21
25
31
37
60
Total Rings
+
0
+
+
+
+
+
+
+
4-
4/4
+
+
+
+
+
+
+
0
+
+
+
+
+
+
+
+
+
+
+
+
4/4
+
+
0
+
+
+
+
+
+
+
0
0
+
+
+
+
+
3/4
+
+
+
+
0
+
+
0
0
0
0
0
0
0
0
0
1/4
+
0
+
+
+
0
0
0
0
0
+
+
f
+
+
+
+
+
+
+
1/4
+
+
+
+
+
0
0
0
0
0
0
0/5
0 0
0 0
OCID
50
101,5EID50s/jnl
o


26
animal. Fluids were completely drained and replaced every other day and
supernatants were assayed for virus on Days 1, 4, 6 and 8 and in one experi
ment periodically up to 60 days.
Table 1 shows data from Experiment IV. In general, once a culture
was infected it usually remained so throughout the experiment. Three of
the 55 cultures differed significantly from this pattern. Transient con
versions from positive to negative occurred occasionally but were probably
due to the egg assay system which apparently can have false negatives but
not false positives. Mien a large number of false negatives appeared on
one day it probably can be attributed to improper handling of samples
(e.g. Day 31). Since very little variation occurred from day to day, Day 8
was selected to calculate an OCID^ (50% organ culture infectious dose).
0CID^qS were calculated by the method of Reed and Muench (1938). For the
remaining experiments cultures were usually carried through Day 10 and
0CIDj-qS calculated for Day 8.
Table 2 summarizes the results of Experiment IV (already shown in
Table 1) and Experiment V, in which tracheal organ cultures from 5 normal
and 5 convalescent ferrets were compared for relative resistance to influ
enza infection. As can be seen, convalescent ferret trachea required
between 140 and 200 times more virus to infect them than did normal cul
tures. This difference was statistically significant and hence the
experiments demonstrate the presence of immunity.
Immunity in Ferret Tracheal Organ Cultures with
Homologous and Heterologous Virus
To determine if the immunity demonstrated in tracheal organ cultures
was specific for the virus which had been used to infect the animal (homo
logous virus), half of the cultures were challenged with the homologous


TABLE 2
IMMUNITY IN TRACHEAL ORGAN CULTURES
Experi
ment
Number of
Animals^-

Number of
Rings/Animal/
Dilution of
Virus
Virus
OCID
Normal
50 Lg10 b,C ('mean SD')
, Ratio of
d 0
Convalescent Infectivity
f
P
IV
2
4 or 5
H0N1
-0.8
1.5
200 x
<0.0005
V
10
2
H0N1
0.3 (0.4)
2.45 (0.8)
140 x
<0.0005
Weighted average 150 x
^lalf the number of animals listed were normal and half were convalescent.
OCID_g obtained by using total infected and total uninfected cultures for an entire group of animals,
c
OCIDj-q = 50% organ culture infectious dose as determined by the Reed-Muench 50% endpoint method (1938) .
^11 of 12 animals were convalescent by two criteria: (a) Virus shedding; (b) 4-fold increase in specific
Ab titre after infection. (One convalescent animal in Experiment V was not tested for virus shedding.)
0
Antilog of (Log^Q 0CIDj.q convalescent--Log^ OCID^ normal) homologous virus,
f
Probability that difference occurred by chance calculated by Student's t test.


28
virus and the other half with a different virus (heterologous virus).
The results are shown in Table 3. Rings challenged with homologous
virus (H3N2) were between 20 and 200 times more resistant to infection
in culture than normal ferret tracheal organ cultures. Cultures chal
lenged with heterologous virus (H0N1 or Sendai) showed no significant
difference in one experiment and only 6-16 times more resistance in two
experiments when the 0CID^qS were compared with those from normal rings.
The immunity therefore appears to be largely specific, although a variable
amount of nonspecific immunity is sometimes observed.
Mechanisms of Immunity in Ferret Organ Cultures
Having now shown that ferret tracheal organ cultures derived from
convalescent animals are specifically immune to challenge, we wish to
delineate the mechanisms involved. Since the immunity is largely specific,
' 'J> f!*
the mechanism could be mediated by specific antibody and/or lymphocytes.
However, the character of CMI, as stated before, is not totally specific
but rather cross-reactive in nature. With this information it seemed
more likely that antibody was the mechanisms of specific immunity in
tracheal organ cultures and some other mechanism responsible for the non
specific aspect. We therefore wished to test the possibility that either
serum antibody or local antibody was responsible for the specific effect.
Effect of Passive Administration of Serum Antibody
On Organ Culture Immunity (in vivo)
The protocol diagrammed in Figure 1 was followed except instead of
m
infecting ferrets at Day -21, animals received a total of 50 ml of either
immune ferret serum (HAI 2048) or normal ferret serum (HAI <16) in two
doses at Day -7 and Day -1. A third group of ferrets received no treat
ment. Ferrets in the immune serum group had HAI titers of 64, 64, and


29
TABLE 3
SPECIFIC IMMUNITY IN FERRET TRACHEAL ORGAN CULTURE
Experi
ment
Number
of
Animals
Rings/
Animal/
Dilution
of Virus
OCID^
Log n (Mean
SD)
Virusu
Normal
Homologous
Heterologous
Homologous
VII
4
4
H0N1
4
H3N2
1.8 (0)
IX
6
2
1I0N1
2
H3N2
3.95(0.4)
XI
8
2
Sendai
2
I13N2
2.2 (0.7)
Half the number of animals listed were normal and half were convalescent.
^OCID^p obtained by using total infected and total uninfected cultures for an
entire group of animals.
0
OCID = 50% organ culture infectious dose as determined by the Reed-Muench
50% endpoint method (1938).
^Antilog of OCID^o convalescent -IjOg^0 OCID^q norma^)
0
Probability that difference occurred by chance.


30
TABLE 3--Continued
OCID50
Log1f> b,C (Mean
1 SD)
Ratio of
Infectivity
Normal
Convalescent
Homologous
Heterologous
Heterologous
Homologous
Heterdogous
Challenge
Challenge
6
p
1.3(0.7)
3.1(10.2)
0.9(10.3)
20
0.4
NS
0.05
1.4(0.2)
5.5(10.3)
2.2(10.4)
32
6
<0.005
<0.005
0.6(10.5)
4.5(10.3)
1.8(10.4)
200
16
<0.005
<0.005
Weighted average
104
9


31
128 at sacrifice. Titers of normal and no treatment groups were all 8 or
less. Table 4 illustrates the results. Both trachea and bladder cultures
were assayed for immunity. The results show that OCID^s of organ cultures
derived from animals receiving normal serum (NS) did not significantly
differ from OCID,_qS of organ cultures from animals receiving immune serum
(IS). Further, the IS group did not differ significantly from the no
treatment group. We conclude from this that serum antibody does not cause
specific immunity in ferret organ cultures.
Antialpha Treatment of Organ Cultures
To test whether tracheal organ culture immunity is caused totally
or in part by IgA in the tissue, organ cultures were exposed to rabbit
antiferret alpha chain 24 hours prior to challenge with virus. Briefly,
ferrets were infected and 21 days later sacrificed along with normal
uninfected ferrets. Trachea were cut into rings and placed in a petri
dish with antialpha or rabbit normal serum overnight. Cultures were
challenged and sampled periodically as before. Results are shown in
Table 5. Organ cultures from convalescent ferrets exposed to normal serum
showed 16-fold immunity over organ cultures from normal animals also ex
posed to normal serum (p = <.01) Organ cultures from convalescent ferrets
exposed to antialpha showed no significant immunity relative to normals.
However, it is also apparent that when cultures from normal ferrets were
treated with the antialpha they became 200 fold more resistant to infec
tion (p <.01). This result is unexplainable and therefore makes the
interpretation of the results of antialpha treatment ambiguous. We can
make no clear interpretation of these data.


32
TABLE 4
WILL PASSIVE ADMINISTRATION OF ANTIBODY3 PRODUCE FERRET
ORGAN CULTURE IMMUNITY
OCID;-q Lo^g(Mean
SD)
Virus
Number
of
Animals
No
Rx
NS
IS
b
P
c
P
Trachea
H3N2
9
3.5 .1
2.9
.4
2.9 .8
NSd
NS
Bladder
H3N2
9
3.3 .9
3.0 1.0
3.5 .5
NS
NS
aFerret anti-H3N2 antibody (64, 64, 128)
blSvs- Nokx
CIS vs. NS
dNot significant
0


33
TABLE 5
ABLATION OF RESISTANCE IN FERRET TRACHEAL ORGAN CULTURES WITH
RABBIT ANTIFERRET ALPHA AND RABBIT ANTIFERRET GAMMA
Treatment
Number of
Animals
OCID
(log1() i SD)
a
P
Antialpha
Convalescent
3
4.9
.2
NSb
Normal
3
4.0
.6
Rabbit Normal Serum
Convalescent
3
5.1
.4
<01
<.01
Normal
3
6.3
. 2_
Antigamma
Convalescent
3
4.5
.4-
<.02
Normal
3
5.7
0
*
Rabbit Normal Serum
NS
Convalescent
3
3.7
.6-
Normal
3
5.8
.3
<.01
Students t test (small sample)
bNot significant


34
Antigamma Treatment of Organ Cultures
To test whether tracheal organ culture immunity might also be
ablated by antigamma treatment we carried out the same protocol again
except that we substituted the use of rabbit antiferret gamma chain
antisera for the antialpha used above. Table 5 shows that convalescent
cultures treated with rabbit normal serum have 130-fold immunity
(p = <.01) compared to normal cultures treated with normal serum. Con
valescent cultures treated with antigamma showed 16-fold immunity
(p = <.02) when compared with normal cultures. Further, convalescent
cultures treated with antigamma were not significantly different from
convalescent cultures treated with normal serum. This suggests that IgG
may not play a significant role in immunity of tracheal organ cultures.
Neutralizing Antibody in Tracheal
Organ Cultures
Day 4 cultures which received no virus were assayed for neutral-
0
izing antibody to the homologous virus (A/Port Chalmers) and a hetero
logous virus (A/PR8). IVe were not able to show any significant neutral
izing capability in the supernatants of the cultures to either strain.
This is shown in Table 6.
Interferon in Tracheal Organ Cultures
Interferon assays were performed on culture supernatants from
Day 4 cultures. Table 6 shows that no significant interferon activity
was found (<3 units/ml) in any culture.
Bladder Immunity
To get an indication of whether specific immunity was restricted
to the respiratory tract, a second anatomically distinct site was tested.
Basarab and Smith (1970) had shown that ferret bladder tissue was


35
TABLE 6
NEUTRALIZING
IN
ANTIBODY AND
DAY 4 TRACHEAL
INTERFERON LEVELS IN
ORGAN CULTURES
Interferon
Neutralizing Antibody
Treatment
(units/ml)
H3N2
H0N1
Antialpha
Convalescent
< 3
< 8
< 8
Normal
< 3
< 8
< 8
Antigamma
Convalescent
< 3
< 8
< 8
Normal
< 3
< 8
< 8
Normal Serum
Convalescent
< 3
< 8
< 8
Normal
< 3
< 8
< 8
0


36
susceptible to in vitro infection by influenza virus. Therefore, cultures
of trachea (results shown in Tables 2 and 3) and bladder were taken from
ferrets in some experiments. Table 7 shows the OCID^s for the normal
and convalescent bladder in four experiments. In Experiment V bladder
immunity is demonstrated since convalescent bladder tissue required
approximately 200 times more virus to infect than did bladder from
normal ferrets (statistically significant at p < 0.10). From Experiment
VII it appears that nonspecific immunity exists since it took 20 and 16
times more virus, respectively, to infect convalescent tissues than to
infect normal tissues with heterologous and homologous virus. Levels of
significance varied with a p value of 0.10 for heterologous and a value
of < 0.10 for homologous; hence this is not a conclusive experiment.
The last two experiments (IX and XI) show specific immunity. In experi
ments which include tissues from a total of 14 animals it took 500 and
O
40 times more homologous virus to infect convalescent tissues than to
infect normal tissues (significance p = 0.025 and p = < 0.005). At the
same time, it took 10 and 0.25 times more heterologous virus to infect
convalescent cultures than it took to infect normal cultures (significance
level of p = 0.10 and p = < 0.005). Thus 3 of the 4 experiments suggest
specific immunity in bladder to homologous virus.
Ciliary Activity in Tracheal Organ Cultures
We attempted to correlate ciliary activity with infection of tracheal
organ cultures. Organ cultures were observed under 100X power using an
inverted microscope. Ciliary activity of rings was qualitatively evalu
ated by determining the percentage of the inner circumference of the
ring showing detectable activity and by the intensity of ciliary beating.
We observed a significant decrease of both parameters by activity of


37
TABLE 7
IMMUNITY IN FERRET BLADDER ORGAN CULTURES
Experi
ment
Number
of
Animalsd
Pieces of
Tissue/
Animal/
Dilution
of Virus
OCID
Logm bC (Mean
SD)
Vi:
rus
Normal
Homologous
Heterologous
Homologous
V
4
2 or 3
H0N1
0.2 (0.8)
VII
4
2
HON 1
H3N2
2.8(+0.4)
IX
6
1
HON 1
1I3N2
2.3(0.8)
XI
8
2
Sendai
H3N2
3.0(0.5)
Hlalf the
number of
animals listed
were normal and
half were convalescent.
^OCID-q obtained by using total infected and total uninfected cultures for an
entire group of animals.
Q
OCID = 50% organ culture infectious dose as determined by the Reed-Muench
50% endpoint method (1938).
^Antilog of (Logjy OCID^q convalescent -Log^^ OCID^ normal).
0
21 or 22 animals shown to be convalescent by both criteria: (a) Virus
shedding as determined by assay of nasal wash; (b) 4-fold increase in specific
Ab titre after infection. (One convalescent animal in Experiment V was not
tested for (a) due to lost samples.
f
Probability that difference occurred by chance.


38
TABLE 7-
-Continued
ocid50
Lgm b,C (Mean
SD)
Ratio of
Infectivity
Normal
Convales cent''
Homologous
Heterologous
f
P
Heterologous
Homologous
Heterologous
Challenge
Challenge
2.52(0.5)
200
<0.10
0.2 (0)
4.0(0)
1.5(0.35)
16
20
0.10
<0.10
0.7(0)
5.0(1.0)
1.7 (0.7)
500
10
0.10
<0.025
2.3(0.25)
4.6(0.9)
1.7(0.2)
40
.25
<0.005
<0.005
Weighted average
190
8


39
both infected and noninfected tracheal organ cultures during the first
ten days. Upon further manipulation, this decrease in activity was not
as apparent as originally seen. It was observed that upon agitating the
tissue in it's media there was a significant increase in activity. With
this method we were unable to conclude that there was a significant dif
ference in ciliary activity between infected and noninfected cultures.
We wished to look at the surface of the rings to determine if
infection caused cell loss and if we could detect a difference between
infected and noninfected rings. To determine the amount of desquamation
occurring in tracheal organ cultures we employed SEM to observe rings
at various times, up to 28 days, after infection in culture. No more
than 50% loss of ciliated cells was seen in any culture on any day.
Further, there seemd to be no difference between infected and noninfected
cultures. We also compared the ciliated surface of trachea taken from
animals infected in vivo. Animals were sacrificed on Days 0, 3, 4 and
5. No loss of ciliated cells was noted. These results suggest that in
our organ culture system neither ciliostasis nor desquamation can be used
as an indicator of infection of cultures. The possible explanation is
that this virus is not able to cause desquamation of ciliated epithelium.
Discussion
Organ cultures were used to study immunity in tissues of ferrets
exposed to influenza A virus. Tracheal organ cultures from ferrets con
valescent from influenza infection required about 130 times mgre homologous
virus to become infected than cultures from normal ferrets. It took only
about 9 times more heterologous virus to infect convalescent cultures
than to infect normal cultures. Immunity was therefore largely specific.


40
Bang and Niven (1958) briefly reported without giving their data or
experimental details that ferret mucosal tissue from convalescent animals
was not resistant to infection. It is not clear why their results dif
fered from those presented here.
Bladder tissue cultures were used to test if the specific immunity
was localized in the respiratory tract or was more widespread. Experi
ments show that bladders from convalescent ferrets were about 190 times
more resistant to challenge with homologous virus than normal bladder,
and that convalescent tissues were only about 8 times more resistant to
heterologous challenge. Bladder specific immunity could be explained in
at least two ways: (a) the specific immunity is caused by systemic
factors, or (b) it is a local response caused by either antigenemia or
homing to bladder mucosal tissue of specific lymphocytes stimulated in
the respiratory tract. Basarab and Smith (1970) did show that influenza
virus could replicate in vivo in bladders of ferrets. And it has been
shown that during severe influenza infection virus can be recovered from
urine of patients (Naficy, 1963).
Since the immunity in both trachea and bladder appears to be largely
specific, it could be mediated by antibody and/or lymphocytes. However,
it has recently been demonstrated in cytotoxic studies using influenza-
infected target cells (Effros et al., 1977; Zweerink et al., 1977) that
CMI may be less specific than is required to account for the specific
protection measured in challenge experiments. Therefore, antibody seems
to be the more likely mechanisms for prevention of influenza in ferrets.
Using tracheal organ cultures we tested which of the following
hypotheses might be true: (1) Systemic serum antibody could have been


41
responsible and simply be trapped in the mucous secretions and/or in the
tissue itself or (2) the immunity could be locally produced in submucosal
immunocompetent cells. Organ cultures derived from animals receiving IP
injections of Sab were not significantly more resistant than organ cultures
derived from normal ferrets. This suggests that Sab plays no role in
tracheal organ culture immunity. The second alternative of locally pro
duced antibody was tested and we showed that antigamma serum did not
ablate the in vitro resistance of tracheal rings. Antialpha serum seemed
to ablate the response but results from antialpha treatment of normal
rings made a clear interpretation impossible. Although we were not able
to clearly implicate IgA in resistance we were able to show that IgG does
not contribute significantly to resistance. This agrees well with
studies which quantitate immunoglobulin levels in the respiratory tract.
.i
Waldman et al. (1973) showed that IgA levels increased dramatically as
you ascend from the lower respiratory tract to the upper. Further,
histological studies show a preponderance of IgA and IgM positive plasma
cells in the lamina propria of secretory tissues (Bienenstock et al.,
1978). However, little IgM is found in secretions (Waldman et al., 1970)
so it seems likely that IgA is the primary immunoglobulin able to act in
the upper respiratory tract.
An attempt was made to measure interferon and neutralizing antibody
in supernatants of organ cultures. Both assays, done with samples taken
from cultures on Day'4, were negative. Interferon levels werq less than
3 units/inl and neutralizing antibody titers were less than 8 in all cases.
This does not necessarily mean that these substances were not present in


42
measurable quantities in the tissue or in close proximity to the rings,
but we were not able to find significant amounts once dilution occurs
in the total volume of the media on each culture.
We observed that ciliary activity could not be an accurate indicator
of infection of tracheal organ cultures since agitation of cultures
seemed to significantly restore activity which had been seemingly lost
over the first ten days. This suggests that mucous could entirely cover
surface areas which then would appear to have lost ciliary activity, or
mucous could slow the beating of cilia thus causing a further decrease
in activity. We are unable to say why these observations differ from
those of Mostow and Tyrrell (1973).
Further study using SEM showed that desquamation was not complete
in tissue from tracheal organ cultures and, in fact, no culture showed
.J
greater than 50% loss of ciliated cells. Also, tracheas taken from
infected ferrets showed no loss of ciliated cells. We are unable to say
why these observations do not agree with those seen in mice by Ramphal
et al. (1979). Possibly our egg grown virus is not virulent enough to
cause total desquamation. Or perhaps ferret trachea is more resistant to
loss of ciliated cell than mouse trachea.
It is difficult to assess the value of in vitro studies in the
in vivo situation, especially when animal models are used to study human
disease. In the case of organ cultures used for this study two important
factors are offered for consideration. First, Rosztodzy et al. (1975)
have shown that human fetal tissues have similar susceptability to
challenge in culture with influenza virus as ferret tissues. Secondly,
Mostow and Tyrrell (1973) have shown that attenuated human influenza
viruses have similar activity in ferret organ cultures to that found in


43
vivo in humans. These studies suggest that ferret organ cultures are
a tool for studying influenza in humans.
Turning from prevention of infection to recovery from infection,
recent studies showed that mice with deficient CMI (nude mice; Sullivan
et al., 1976) or mice treated with ALS (Suzuki et al., 1974) shed virus
over longer periods than did normal mice, suggesting CMI may play a
critical role in recovery. In Experiment IV we showed that once a
ferret tracheal organ culture was infected it remained so; that is, it
did not recover. If CMI is responsible for recovery, it would follow
that CMI was not functional in the tracheal organ culture.
Irrespective of the mechanism of prevention or recovery from
influenza, it seems that the ferret tracheal organ culture enables one
to separate the two mechanisms. Immunity to reinfection can be demon-
strated in the same piece of tissue that lacks the ability to recover.
Hence it seems that prevention and recovery are mediated by different
mechanisms.
The role of antibody in recovery will be addressed in
Appendix A.


APPENDICES


APPENDIX A
SERUM ANTIBODY IN THERAPY OF INFLUENZAL PNEUMONIA
Introduction
As was stated before, the major cause of death related to influenza
is subsequent bacterial pneumonia. This is treatable by antibiotics.
However, in a small number of cases viral pneumonia is the direct cause
of death. Why in these few cases does the lung become involved to such
a great extent? Immune factors are evidently one of the major influences
in this determination. There is good support for the idea that Sab, if
present, is a factor which prevents lung involvement. This is a likely
explanation taking into account the intimate association of the lung
parenchyma and the pulmonary circulation. Loosli (1953) showed the
importance of circulating antibody in the prevention of death of mice.
Virelizier (1975) reported that HAI antibodys have an important role in
protection. More recently, Ramphal et al. (1979) reported that Sab pro
tects the lung from pneumonitis but does not protect the ciliated epi
thelium of the trachea. So it appears that if Sab against the infecting
influenza virus is present prior to infection, the lung will be protected
against viral pneumonia but the animal can still have an upper respiratory
infection. When drift from one subtype of influenza virus to another
subtype occurs very few cases of viral pneumonia are found. The reason
for this may be that Sab formed during infection by the closel'y related
strain is cross-reactive with the second strain of virus thus providing
lung protection. During a shift, when viral pneumonia is found to a
greater extent, cross-reacting Sab is not available because of the major
45


46
shift of viral antigens thus the lung is left unprotected and lung involve
ment is more likely.
Since it has been proven that Sab does protect against death, by
decreasing viral pneumonitis, we wished to determine if Sab could be used
therapeutically to treat diagnosed or suspected viral pneumonia. If a
cure could be affected, we wished to determine how soon before death Sab
could be effective for treatment.
Results
The virus used in these experiments was passaged in mouse lung
11 times. A lethal dose 50 (LD,_q) was performed in mice and we found
3 9
that 0.05 cc of the fluid contained 10 LD^qS. In all subsequent experi
ments this virus was diluted so that mice received approximately 10 LD^^S.
In our first experiments we wished to ensure that the Sab we were
using could be life-saving upon subsequent infection with the eleventh
passaged influenza virus. Briefly, one group of A/J mice were injected
intraperitoneally (IP) with a total of 0.5 cc of goat antiinfluenza
antibody (HAI 16000) in equal doses on two days. A second group received
no Sab. Both groups were infected on the day the first group received
its second dose of Sab. Animals were observed for deaths. Results
are shown in Table A-l. No deaths were recorded in the group receiving
Sab (0/5 dead) while those that did not receive Sab all died by two
weeks time (5/5 dead). We conclude that the dose of virus used is
lethal for our mice and that the dose of Sab was sufficient to protect
mice from lethal pneumonitis when given before infection.
We asked could Sab given after infection be life-saving? The
experiment consisted of infecting mice and then subsequently giving Sab
on specific days. In a preliminary experiment mice were infected and


TABLE A-1
Sab PROTECTS AGAINST VIRAL PNEUMONIA
Number of
Animals Dead
Treatment
Number of Animals Infected
No serum
5/5 -
p = .004
Saba
0/5 -
£
Sab given in two doses prior to infection,
0.25 cc/dose.


48
on Days 3, 4 and 5 after infection one group of these mice were given
Sab (0.75 cc total) and a second group was given nothing. Animals were
observed for death. Results for Experiment I are shown in Table A-2.
Mice not receiving Sab all died between Days 7 and 10 (4/4 dead) but
those receiving Sab on Days 3, 4 and 5 after infection all survived
for at least 21 days (0/5 dead). We conclude that Sab given on Days 3,
4 and 5 after a lethal influenza infection can be life-saving.
The next experiment was to determine how close to death mice could
be when administered Sab and still survive lethal infection. As before,
mice were infected and groups of mice were given (a) nothing, (b) Sab
on Days 3, 4 and 5, (c) Sab on Days 5, 6 and 7, or (d) Sab on Days 7, 8
and 9. Animals were observed for death. Results are shown in Table A-2
under column Experiment II. Again those mice that did not receive Sab
all died on Day 8 (7/7 dead). Mice that received Sab on Days 5, 6 and
7 all died between Days 5 and 8 (6/6 dead) and mice given Sab on Days 7,
8 and 9 all died between Days 7 and 9 (6/6 dead). The only group to be
affected by the Sab was the group receiving Sab on Days 3, 4 and 5 (1/7
dead). This one death occurred immediately after Sab injection on Day 4.
This was a significant decrease in mortality when compared with controls
(p = 0.002). This experiment was then repeated exactly as before but with
larger numbers of mice. Results are shown in Table A-2 under Experiment
III. Again, mice that did not receive Sab all died between Days 7 and 10.
Those receiving Sal on Days 7, 8 and 9 all died between Days 6 and 10
after infection. In the group that received Sab on Days 5, 6 and
7 all but one animal died with deaths occurring between Days 6 and 8.
The one group that differed significantly from the control group was the


49
TABLE A-2
EFFECT OF Sab GIVEN AFTER LETHAL INFLUENZA VIRUS INFECTION
Treatment
Experi
ment I
Experiment II
Experiment III
No Serum
4/4a
7/7
10/10 -
Sabb
p = 0.000006
Days 3, 4, 5
0/5
1/7 -
1/13 -
p = .002
Days 5, 6, 7
NDC
6/6 -
11/12
Days 7, 8, 9
ND
6/6
14/14
aMortality (number of animals dead/number of animals infected)
bDose of Sab 0.25 cc
c
Not done
I


50
tiro un that received Sab on Days 3, 4, and 5 where only a single death
was recorded (1/13 p = 0.000006).
The results so far obtained seemed to suggest if the lethal
infection by influenza could be diagnosed and treated by Days 3 or 4,
a cure could be affected. The determining factor in clinical diagnosis
of viral pneumonia, however, is often radiological evidence of consolida
tion. We therefore attempted to discern if radiological changes were
evident by Day 3. If so, then it seemed possible that in a clinical
situation there might be a chance that the diagnosis might be made and
that subsequent treatment with Sab might benefit the patient. A control
group was infected with the virus and x-rayed on Days 3, 5, 7, and 9.
Photographs of representative radiographs are presented in Figure A-l.
It can be seen that no radiological evidence of lung involvement is
.. ** **
present until Day 7 or after. This indicates that, since by Day 7, Sab
cannot modify the lethal effect of infection with the virus that it
seems unlikely that Sab can be a useful treatment for lethal penumonitis.
Discussion
We have shown that high titered Sab (HAI 16000) given to mice
before a lethal infection of virus (10 LD^qS, which kills in 7-12 days)
can be life-saving. Further, when Sab is given after a similar infection
but before radiological changes appear in mouse lung the treatment is
also life-saving. However, if Sab is given after radiological changes
are noted, death occurs as if no treatment were given.
Because of our original success with Sab treatment of mice prior to
infection (Ramphai et al., 1979, see Appendix B) and because totally
effective treatment of lethal viral pneumonia is not available we had


Figure A-l. Radiological appearance of mouse lungs after
infection with lethal influenza virus (A/Port Chalmers, 10 LD,.^)
A.
Day
B.
Day
C.
Day
D.
Day


52
B




54
hoped to show that Sab given after infection could be useful in a life-
threatening situation. However, clinically, x-ray evidence of consolida
tion is the primary diagnostic tool in determining lung involvement dur
ing viral disease. Because of this and because we were not able to
modify the lethal effects of the advanced disease state, therapeutic use
of Sab seems less promising.
It is probable at this stage of disease that immune processes
have begun to overwhelm the majority of lung tissue. Epithelial cells
of alveoli have been disrupted and cellular infiltrates and edema are
present and increasing. Alveoli become filled and at this point Sab
acting to neutralize virus or coat infected cells has negligible effect.
In fact, Sab may increase vascular pressure which in turn increases
edematous flow into alveolar spaces thus worsening the problem.


APPENDIX B
SERUM ANTIBODY PREVENTS LETHAL MURINE INFLUENZA
PNEUMONITIS BUT NOT TRACHEITIS
Reuben Ramphal, Robert C. Cogliano, Joseph IV. Shands, Jr., and
Parker A. Small, Jr.
This paper reports studies showing the effects of serum antibody
upon influenza infection at two different sites: the trachea and lung.
Tracheal desquamation, pulmonary consolidation, death, and virus
shedding were examined after infection of mice with a lethal A/Port
Chalmers/1/73(H3N2) influenza virus. Immune serum administered intra-
peri toneally before infection prevented death and pulmonary consolidation
and also significantly lowered lung virus shedding as compared with
controls receiving normal serum. However, this protection did not Z .
extend to the ciliated epithelium of the trachea because serum antibody
did not prevent desquamation of the trachea or significantly decrease
viral yield from the trachea. These results indicate that serum antibody
is protective against severe pulmonary parenchymal disease but not for
disease of the ciliated epithelium.
55


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BIOGRAPHICAL SKETCH
Robert Christopher Cogliano, son of Vincent and Mary, was born
in Boston, Massachusetts,on the third day of August, 1946. He was
raised with his 5 brothers and sisters in Pembroke, Massachusetts,and
attended college at Bridgewater State College, Bridgewater, Massa
chusetts. Robert graduated in June of 1968 and entered military
service February, 1969. He served as a pilot in the United States
Air Force for five years. Robert started his graduate studies in
September, 1974, and will now go on to a position at New York Uni
versity Medical Center.
60


I certify that I
conforms to acceptable
adequate, in scope and
Doctor of Philosophy.
have read this study and that in my opinion it
standards of scholarly presentation and is fully
quality, as a dissertation for the de
//
//Parker A. Small,
Professor of Immunology
Microbiology
Medical
I certify that I
conforms to acceptable
adequate, in scope and
Doctor of Philosophy.
have read this study and that in my opinion it
standards of scholarly presentation and is fully
quality, as a dissertation for the degree of
Professor of Immunology and Medical
Microbiology
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy. ^
r>-:
George E. Gifford
Professor of Immunology and Medical
Microbiology
I certify that I
conforms to acceptable
adequate, in scope and
Doctor of Philosophy.
have read this study and that in my opinion it
standards of scholarly presentation and is fully
quality, as a dissertation for the degree of
Paul A. Klein
Associate Professor of Pathology


This dissertation was submitted to the Graduate Faculty of the College of
Medicine and to the Graduate Council, and was accepted as partial fulfill
ment of the requirements for the degree of Doctor of Philosophy.
December 1979
Dean, College of Medicine~
Dean,
in I f
hool


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SONE ASPECTS OF IMMUNITY AND DISEASE DURING
INFLUENZA A VIRUS INFECTION
By
ROBERT CHRISTOPHER COGLIANO
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1979

ACKNOWLEDGEMENTS
I would like to thank Dr. Parker A. Small, Jr., for his support
and patience. Scientifically, his imaginative, creative and critical
thinking were always a standard for which to strive. His nature to
take time to care and try to offer advice on personal matters was much
appreciated.
My thanks also to Bob Yetter, a friend when needed, a source of
impeccable common sense when none was to be found elsewhere and one
of the best trivia experts around.
•9
ii

TABLE OF CONTENTS
ACKNOWLEDGEMENTS
Page
ii
ABSTRACT iv
INTRODUCTION 1
The Virus 1
The Disease . 3
Immunity to Influenza ..... 4
METHODS AND MATERIALS 11
Viruses
Intranasal Inoculation of Virus into Animals
Virus Adaptation in Mice
Assays
Tissue Cultures
Statistical Analysis
Serum Antibody Production
Antiimmunoglobulin Production . .
Interferon Assay
Neutralization Titers
X-rays
Scanning Electron Microscopy
11
11
12
12
12
13
13
15
16
17
17
18
SPECIFIC LOCAL IMMUNITY IN FERRET ORGAN CULTURES
19
Introduction 19
Results 21
Mechanisms of Immunity in Ferret Organ Cultures 28
Bladder Immunity 34
Ciliary Activity in Tracheal Organ Cultures 36
Discussion 39
APPENDICES
APPENDIX A 45
APPENDIX B 55
REFERENCES 56
i i i
BIOGRAPHICAL SKETCH
60

Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the Requirements
for the Degree of Doctor of Philosophy
SOME ASPECTS OF IMMUNITY AND DISEASE DURING
INFLUENZA A VIRUS INFECTION
By
Robert Christopher Cogliano
December 1979
Chairman: Parker A. Small, Jr.
Major Department: Medical Sciences (Immunology and Medical Microbiology)
In order to study immunity to influenza virus infection in the
upper respiratory tract we have tested organ cultures from normal and
convalescent ferrets for resistance to infection. We then attempted to
delineate immune mechanisms acting within the tissue.
Ferret tracheal organ cultures prepared from animals previously
infected intranasally with influenza A virus required approximately 130
times more homologous virus (A/PR/8/34(MONI) or A/Port Chalmers/1/73
(H3N2)) to become infected in vitro than similar cultures from normal
ferrets. Also, these cultures from convalescent ferrets required about
10 times more heterologous virus (A/PR/8/34 (H0N1) or Sendai) to become
infected in vitro than similar cultures from normal animals challenged
in vitro with the heterologous virus. We conclude that these tracheal
rings are specifically immune.
Possible mechanisms of resistance of the tracheal organ cultures
were examined. Serum antibody given intraperitoneally to normal ferrets
did not bring about immunity in cultures derived from these animals.
IV

Rabbit antiferret gamma chain did not significantly ablate resistance
of cultures when put on cultures prior to virus challenge. Rabbit anti¬
ferret alpha chain treatment gave ambiguous results. Neither neutraliz¬
ing antibody nor interferon could be measured in Day 4 culture super¬
natants.
Once tracheal rings are infected, they continue to shed viruses for
at least 60 days, the longest period any cultures were kept. Virus shed¬
ding in the intact ferret lasts normally 5-7 days. Thus, recovery in the
intact ferret seems to be dependent upon factors which are not present,
or at least not functional, in the tracheal explant. This is consistent
with the hypothesis that recovery is dependent upon systemic rather than
local phenomena.
Bladder tissue from normal and previously infected ferrets was
also cultured and challenged with homologous and heterologous virus. The
bladder from previously infected ferrets exhibited specific immunity,
although the immunity was more variable.
Concurrent studies aimed at determining the therapeutic value of
serum antibody for the treatment of lethal viral pneumonia in mice showed
that serum antibody given before infection or on Days 3, 4 and 5 after
infection was life-saving but given later was ineffective in preventing
death. Since radiological change could first be detected 6-7 days after
infection we conclude that serum antibody is not likely to be useful for
treatment of animals.with lethal viral pneumonia.
v

INTRODUCTION
This dissertation deals primarily with aspects of immunity to
influenza virus and secondarily with pathogenesis and recovery from
influenza infection. The introduction begins with a discussion of the
virus, specifically its structure, replication and antigenic nature.
This is followed by a description of the disease caused by the virus and
then a summary of our knowledge of immunity. Lastly, we report the
specific questions which we addressed in this research.
The Virus
Structure
The influenza virus is taxonomically designated as belonging to
the Orthomyxoviridae family (Melnick, 1977). Structurally, influenza
virus contains a segmented RNA genome which is associated in the nucleo-
capsid with an RNA transcriptase consisting of 3 proteins and a nucleo-
protein. Exterior to the genome is the virus coded matrix protein and
an envelope which is derived from the host cell during budding. Imbedded
in the envelope of the virus are two types of virus specific glycoprotein
spikes, the hemagglutinin (HA) and the neuraminidase (NA). An eighth
protein is the nonstructural protein (NS) for which no definite role has
been established.
Replication
The RNA genome«of influenza A contains 8 segments (Ritchey et al.
1976) and the virus replicates as a class V virus according to the Baltimore
classification (Baltimore, 1971). Briefly, the negative stranded RNA
enters the cell. The segments must first be transcribed to plus stranded
1

2
RNA to be able to act as messengers. There appears to be two types of
plus strands, one which is poly-adenylated at the 3' end and one type
that is not poly-adenylated. It has been suggested that the poly-A
form acts as messenger for viral proteins while the form without poly-A
acts as template for production of progeny viral RNA (Hay and Skehel,
1979). The nucleocapsid is assembled in the cytoplasm of the host cell
while surface viral proteins are inserted into the cell membrane at var¬
ious sites. Concurrently, host proteins are largely excluded from the
membrane at these specific sites. From these sites at the cell surface
progeny virus bud by some unknown mechanism taking with them the nucleo¬
capsid and cell membrane with imbedded viral proteins. One recent report
which merits mention but has as yet not been confirmed or explained is
that DNA has also been found in the influenza virus and comprises between
5-8% of the total nucleic acid (Kantorovich-Prokudina et al», 1978).
0
Antigenic Characteristics
Influenza viruses are classified into serotypes and each serotype
is further classified into subtypes. The serotype depends upon antigenic
determinants found in the nucleoprotein. Three major serotypes have so
far been identified A, B and C. Types A and B cause the majority of
disease in man. The subtypes within each serotype are defined by anti¬
genic determinants on the HA and NA. It is the characteristic genetic
variability of the segments of viral RNA coding for these two viral
proteins which causes the unpredictable "shifts" and "drifts" in antigenic
character. Shift is found only in type A but drift is found in both
types A and B. These shifts and drifts in viral antigens result in the
well known pandemics and epidemics, respectively. Shift refers to a major
change in antigenic determinants of a surface protein, for example, the

3
shift of the hemagglutinin from H2N2 to H3N2. This change has been shown
to be radical in nature. By mapping tryptic digests of these viral pro¬
teins it can be shown that the amino acid sequences from H2 differ greatly
from those of H3 (Webster and Laver, 1975). These types of changes
have been hypothesized to happen by reassortment events after co-infection
of host cells. Animal influenza viruses have been implicated as a source
of these new and different antigenic determinants. The presence of these
new antigens on a virus then leaves the population with decreased immunity
to this virus (Rasmussen, 1964). A drift, for example a drift of H3N2
Port Chalmers (1973) to H3N2 Texas (1977), is a small change in antigenic
character (perhaps as little as a single amino acid change). This is
demonstrated, again, by tryptic digests of viral surface proteins (Webster
and Laver, 1975). Drift probably occurs by single mutational events at
a locus within the RNA segment coding for the protein.
The Disease
0
Influenza, although it has been with us for centuries, remains as
perhaps the last great pandemic of man. During the period from 1968 to
1979, more than 150,000 excess deaths are estimated to have occurred
during epidemics of influenza A in the United States (MMWR, 1979). These
excess deaths occur primarily among the young, the chronically ill and
older individuals.
The disease in humans is commonly a rhino-tracheobronchitis with
accompanying headache, malaise, fever, anorexia and myalgia. In uncom¬
plicated cases recovery occurs in a few days to a week. In a few cases,
however, the virus can cause a pneumonia which quickly runs its course.
Prognosis is poor in this instance. Viremia may be found only in these
severe cases (Naficy, 1963). Supportive care has proven useful in some

4
cases but more often than not death ensues in 3-7 days after onset of
illness. Prior to widespread use of antibiotics, bacterial pneumonia
was a frequent complication following influenza infections and even
today remains the major cause of influenza related deaths. Bacterial
infection after influenza was most probably the major cause of death
during the 1918 pandemic which claimed 20 million excess deaths world¬
wide. It has been estimated, however, that a full 20% of the 20 million
excess deaths were directly due to viral pneumonia with death occurring
in 6 or less days (Crosby, 1976).
Immunity to Influenza
That an important part of the immunity to influenza is specific
is well known. Animals with prior infection with a subtype of influenza
(e.g. H0N1) will at least for a period be solidly immune to challenge
with that same virus but are still susceptible to infection with a dif¬
ferent type A influenza virus (e.g. H3N2). Specific immunity can be
either cell mediated (CMI) or antibody mediated (Ab). Further, the
immune response may be either local or systemic in nature. The obvious
question then arises: Which is these, local Ab, local CHI, systemic Ab
or systemic CMI is most responsible for the specific resistance to rein¬
fection?
Antibody Mediated Immunity
If we turn first to Ab, most researchers can be divided into two
schools: those that conclude that systemic Ab protects the animal against
infection and those that believe that serum antibody (Sab) does not cause
protection but only correlates with it and that local antibody (Lab) is
responsible for resistance to reinfection. In the former group Stuart-
Harris (1973) reported that serum antibody was protective in man. Both

5
Allan et al. (1971) and Rott. et al. (1974) reported serum antibody to
be protective in chickens. More recently, Virelizier (1975) concluded
that in mice serum antibody was most important for protection against
reinfection by influenza. McLaren et al. (1974), using ferrets in
challenge experiments, found serum antibody against the hemagglutinin
to correlate best with protection against infection. Serum antihemag¬
glutinin antibody is thought to be the effector in the above examples.
However, Murphy et al. (1972), working with human volunteers, and Schulman
et al. (1968), working with mice,have concluded that serum antineuramini¬
dase antibody can also be protective against infection.
There are also those that have observed the reverse situations,
that is, that Sab is not protective against infection. Morris et al.
(1966) demonstrated that influenza infection in humans is not prevented
•j -it «.*
by high levels of serum antibody. Small et al. (1976) passively immunized
ferrets so that their hemagglutination-inhibition titers (HAI) were 1000
or greater, but were unable to prevent influenza infection. They con¬
cluded that serum antibody played no role in preventing infection of
ferrets. So it appears there is evidence both supporting and refuting
serum antibody as an important immune mechanism in preventing influenza
infection.
Many investigations support the idea that antibody produced locally
in the respiratory tract and distributed on the mucosal surface is the
primary mode of protection. Richman et al. (1974) have shown .that the
local antibody response is specific. Francis (1943) infers that nasal
wash antibody specific for infecting influenza virus is the most important
determinant of specific resistance to influenza. Frazekas de St. Groth
and Donnelley (1950a), studying the antibody response to mice receiving

6
graded doses of influenza virus (live or inactivated) by various immuni¬
zation routes concluded that "serum antibody does not give any informa¬
tion on antibody that is found at the local site." In further work
(1950b) they conclude that serum antibody does not coincide with immunity
but local antibody does. Bearc et al. (1969) concluded that a factor,
probably local antibody, exerted a considerable influence on human
resistance to infection by influenza virus. Waldman and Coggins (1972)
concluded that a vaccine's ability to stimulate nasal secretory antibody
was the deciding factor as to whether it could protect humans against
influenza infection. Potter et al. (1972) showed production of nasal
antibody after nasal infection of ferrets but not after immunization
with killed virus. They postulated this was the reason for better pro¬
tection of these animals upon subsequent challenge with virus. All of
the above studies show only a correlation between local antibody and
protection but do not prove that the local antibody is the cause of
immunity. However, Barber and Small (1978) in experiments using ferrets
with surgically created tracheal pouches, which allow study of infection
and protection in two separate sites, conclude that systemic factors do
not play a role in preventing upper respiratory tract infection with
influenza and that, therefore, local factors are primary in prevention
of infection.
Again there is evidence to support the opposite view, that is,,
that local antibody, is not responsible for immunity to influeirza. In a
recent report Jennings et al. (1978), using medical student volunteers,
specifically concluded that nasal wash antibody does not correlate with
protection.

7
Much of the confusion as to which is more important for protection,
serum antibody or local antibody, may stem from two problems of experi¬
mental design. First, those that look only at Sab and not Lab can only
show correlation with the one parameter and are unable to rule out the
other parameter, Lab. Secondly, different workers study different sites
of infection having different immune mechanisms in operation. Virelizier
(1975) and Loosli (1953) while concluding that Sab protects against influ¬
enza infection were studying the lung. While Sab probably does protect
the lung in their animal system, it probably does not protect the upper
respiratory tract, the tissue affected in the vast majority of cases of
human influenza. Ramphal et al. (1979) have shown in mice that serum
antibody given prior to infection will protect the lung from pneumonitis
but will not protect the trachea, thus separating the upper and lower
respiratory tract immunologically (see Appendix B).
Cell Mediated Immunity
The possible role of CMI in immunity to influenza has only recently
begun to be examined. However, it has been shown to be important in both
recovery and pathogenesis. In studies where nude mice (Sullivan et al.
1976)y or mice treated with antilymphocyte serum (Suzuki et al. 1974), were
infected with influenza virus significant prolongation of survival time
was noted. Also, virus was not eliminated from these mice. This suggests
that, in severe infections, CMI is responsible for some pathogenicity of
the virus and that«T-cells are required for.recovery from influenza
infection. This, however, has not been shown to be the case in studies
by Yap et al. (1979) where they conclude that influenza immune T-cells
inhibit rather than contribute to pulmonary pathology. However, they do
agree that CMI may play an important role in recovery from murine influenzal
pneumonia.

8
Cytotoxicity is seen by many as the major cell mediated effector
mechanism influencing the outcome of influenzal disease. Yap and Ada
(1977) have shown that in mice this cytotoxicity is probably due to T-
cells. Yap and Ada (1978), speaking specifically about systemically
derived cytotoxic T-cells found in the lungs of mice, conclude that
these cells can bring about recovery and are part of the consolidation
process. Ennis et al. (1978) also conclude that cytotoxic T-cells are
derived systemically and that they are part of the immunological and
pathological response to virus infection.
It has been shown in vitro that cytotoxicity against influenza
infected cells displays the same histocompatibility restriction as has
been shown for other viruses (Braciale, 1979). However, it is not
specifically known if H-2 antigens exist in vivo on the epithelial cells
which are infected by influenza virus.
The mechanism of cytotoxicity is unknown. It has been suggested
that both antibody and cells may be involved and that this might there¬
fore be an antibody-dependent cell cytotoxicity (Lucas and Barry, 1977,
Greenberg et al., 1977). Further, cytotoxic cells have been shown
generally to be of the Ly-23+ subclass and that this activity can be
amplified by the Ly-1+ subclass of T-cells (Cantor and Boyse, 1975).
It has become clear that CMI reactions during influenza infection
are not as specific as humoral responses. Doherty et al. (1977) have
shown in mice that.at least two populations of cytotoxic T-cells arise
during influenza infection, one specific and one cross-reactive. Also,
during secondary response, the cross-reactive population may be preferen¬
tially stimulated. Biddison et al. (1979) have shown that cross-reactive

9
cytotoxic T-cells exist in man and that secondary response T-cells are
preferentially cross-reactive. What is the cause of this cross-reactivity?
Webster and Hinshaw (1977) suggest that M-protein, although not expressed
on the surface of the virion or on cell surfaces, may be responsible for
the cross-reactive T-cells. M-protein is virtually the same in all type-A
influenza (the cross-reactivity does not extend across serotypes).
Cretescu et al. (1978) have shown that humans do make anti-M-protein
responses during infection, so evidently the M-protein can be recognized
by the immune system at some stage during infection. In fact Ada and
Yap (1977) and Braciale (1977) report that M-protein is expressed on the
cell surface. Other reports show that another antigen which is shared
by all subtypes within a serotype, the nucleoprotein, is expressed at
the surface of infected cells during early events (Virelizier, 1977).
This may, therefore, account for the cross-reactivity of T-cell cyto¬
toxicity.
Local CMI responses have been shown in a few instances. Henney and
Waldman (1970), and Waldman and Henney (1971) demonstrated that pulmonary
CMI is relatively independent of the circulating response. Cambridge et
al. (1976), using migration inhibition and cytotoxicity assays, observed
in mice that the local response by pulmonary node lymphocytes was both
greater and faster than spleen cells after influenza infection, thus
suggesting the presence of local CMI. Wyde and Cate (1978) found that
during murine inflyenza an increase in cell cytotoxicity was found in
regional lymph node cells before peripheral blood lymphocytes. They infer
from this that the cytotoxic response found in bronchioalveolar washes
at Day 6 after infection is a local CMI response. In humans, Jurgensen
et al. (1978) show that CMI in the respiratory tract was best, stimulated

10
by aerosol immunization while subcutanetous immunization stimulated
primarily systemic CMI. They presented this to support the importance
of local CMI during influenza infection.
Most definitive work proves upper respiratory tract immunity is
local (Barber and Small, 1978). Further it has been shown that CMI is
not specific enough in nature to explain the specific immunity seen in
influenza. These two statements taken together suggest local Ab as
the probable mechanism of resistance. However, reported work which
implicates Lab is not direct. We therefore assessed the role of antibody,
both local and systemic in vitro. Further, we assessed the role of Sab
in pathogenesis and recovery in vivo. We wish to relate our findings to
three areas: Are these factors (1) important in resistance?, (2) import¬
ant in causing pathology seen during influenza infection? and (3) import¬
ant in recovery from the disease?

METHODS AND MATERIALS
Animals
Mature ferrets were obtained from Marshall Research Animals, Inc.,
North Rose, New York, and housed in individual cages under conditions
which prevent cross-infection. Five-week old A/J mice were obtained
from Jackson Laboratory, Bar Harbor, Maine. Goats were obtained from
the University of Florida. New Zealand white rabbits were obtained from
Kel Farms, Gainesville, Florida.
Viruses
Influenza viruses used were A/PR/8/34(H0Nl) and A/Fort Chalmers/
1/73(H3N2). Large stocks of both viruses were obtained by injecting
viruses into allantoic cavities of ten-day old embryonated chicken eggs
which were then incubated for 3 days at 36°C at which time allantoic
fluid was harvested, pooled and stored at -85°C in 1 ml aliquots. The
H0N1 virus had a chick erythrocyte hemagglutination (HA) titer of 1280
7 2
and contained 10 ' 50% egg infectious doses/ml (EID.-^). The H3N2 virus
8 2
had an HA titer of 160 and contained 10 ‘ EID^^/ml. Sendai virus was
propagated in the same manner and had an HA titer of 512 containing
109,2 EID /ml.
Intranasal Inoculation of Virus Into Animals
Ferrets were anesthetized with 0.5 cc of Ketaset (Ketamine hydro¬
chloride, Bristol laboratories) and infected with 0.1 ml of undiluted
virus in each naris. Mice were anesthetized with 0.2 cc nembutal
(6 mg/ml sodium pentobarbitol, Abbott Laboratories, Chicago) and infected
with 0.05 cc of undiluted virus per naris.
11

12
Virus Adaptation in Mice
To obtain virus which ivas able to cause lethal pneumonia in mice,
H3N2 influenza virus was passaged successively in mouse lung. Mice were
anesthetized, infected and three days later they were sacrificed via
cervical dislocation. The lungs aseptically removed and macerated in
5 mis of Lebovitz's L-15 medium. Homogenates of lung tissue were
centrifuged to clear debris and supernatants were used to infect the
next group of mice.
Assays
Virus was detected by inoculation of samples into the allantoic
cavity of embryonated chicken eggs that were 10 days old, as previously
described (Barber and Small, 1974). HA and HAI titers were performed
with a microtiter kit using disposable microtiter plates (Cooke Engineer¬
ing, Alexandria, Virginia) as described by Sever (1962). Sera used for
HAI assays were first absorbed with kaolin and chicken RBCs and heated
at 56°C for 30 minutes as described previously (Barber and Small, 1974).
Tissue Cultures
Ferrets were anesthetized and exsanguinated by cardiac puncture.
Trachea and bladder were aseptically removed and placed into sterile
100 x 15 mm petri dishes containing approximately 20 mis of Hanks'
balanced salt solution with 100 units/ml penicillin and 100 yg/ml strepto¬
mycin. While in the petri dish the trachea was cut into individual rings
2
and the bladder was^ cut into pieces approximately 3 x 3 mm . .Tissue
pieces were then put individually into 35 x 10 mm petri dishes with 3 mis
of the L-15 medium (glutamine [.3 mg/ml], 10% fetal calf serum, gentamicin
[5 yg/ml], streptomycin [100 yg/ml], penicillin [125 units/ml] and

13
mycostatin [100 units/ml]). The cultures were incubated overnight at
35°C. The following day (Day 1) the 3 mis of medium was removed and
replaced with 3 mis of the medium containing the challenge virus at the
proper dilution (Figure 1). If antiimmunoglobulin (a-Ig) treatment was
done, the a-Ig serum was added after tissues were cut up but still in
100 x 15 mm dishes on Day 0. The a-Ig serum was added in equal parts with
medium and cultures were incubated at 35°C. The next day the individual
pieces of tissue were put into 35 x 10 mm petri dishes with media without
antiimmunoglobulin and cultures were immediately challeneged with virus
as before.
Statistical Analysis
Viral and antibody titers were compared using the t-test (Men¬
denhall, 1975) and mortality compared using Fisher's exact test
(Siegel, 1956).
Serum Antibody Production
Ferrets previously infected with H3N2 influenza virus were inoc¬
ulated IM with 0.5 cc of A/Fort Chalmers/l/73/N3N2 killed vaccine (MRC-11)
Two weeks later they were bled via cardiac puncture and serum was col¬
lected. Goat serum antibody was obtained from a goat given the same
A/Port Chalmers vaccine IM weekly for three weeks. The goat was bled
via the external jugular. The sera were heat inactivated at 56°C for
30 minutes and then filter sterilized before use. In some experiments
very high titer antibody was needed. This was prepared by three succes-
ive 33% ammonium sulfate precipitations and centrifugations at room temper
ature. After the last spin the precipitate was redissolved in saline and
the solution was then dialyzed against saline for two days. They were
then heat inactivated and filter sterilized.

Figure 1. Experimental protocol; experimental sequence in clays.
ACUTE SERA
FOR HI
¡2* INFECT WITH VIRUS
NASAL WASH ^ 5
NASAL WASH ^—
PUT TISSUES INTO ORGAN CULTURES
CULTURES WITH VIRUS DILUTIONS
33
O

15
Antiimmunoglobulin Production
Rabbit Antialpha Serum
Preparation of rabbit antialpha serum was performed by Richard
Kris in our laboratory. Surgery was performed on ferrets to canulate
the bile duct. Bile was collected over a period of a few days. The
bile was subjected to preparative electrophoresis on acrylamide (3% aga¬
rose) at pH 6.3, 5 milliamps for 18 hours. The gammaglobulin fraction,
collected in the buffer at the top of the gel, was then concentrated by
pressure dialysis. This was then gel filtered over an Ara-22 column
(Pharmacia) and the 11S fractions vs'ere pooled. One-half of a mg of
this in complete Freund's adjuvant (CFA) was then injected subcutaneously
into a rabbit which had previously been injected with ferret IgA prepara¬
tions more than one year prior to this time. Two weeks later the rabbit
• > -J\ «*
was bled from the ear artery and serum was collected. This serum was
then subjected to affinity chromatography by passing it over a sepharose
4B CNBr column (Pharmacia) with ferret IgG attached. The resulting serum
was shown by ouchterlony analysis to have anti-IgA activity but no activity
toward other immunoglobulin classes.
Rabbit Antigamma Serum
Again, preparation of rabbit antigamma serum was largely performed
by R. Kris. Ferret serum was treated with KBr and centrifuged to elim-
_3
inate lipoproteins. The supernatant was dialyzed against PBS (10 M).
The pseudoglobulin fraction was passed over a DTiAE column and the 7S
fractions were pooled. One-half of a mg of this 7S fraction in CFA was
injected subcutaneously into a rabbit. Three weeks later the rabbit was
boosted with 0.5 mg in Freund's incomplete adjuvant. Three weeks later

16
the rabbit was bled for serum. The anti-IgG serum was subjected to
affinity chromatography by passing it over a sepharose 4B CNBr column with
IgA attached. The resulting serum was shown by ouchterlony analysis to
have activity against ferret IgG but not ferret IgA.
Interferon Assay
Ferret kidneys were removed aseptically and put into Gey's A solu¬
tion. The kidney capsules were stripped and the kidneys were minced
with scissors. The minced tissue was washed twice with Gey's solution.
The tissue was put into a trypsinization flask with 100 mis Gey's A,
(Streptomycin 125 ygms/ml, penicillin 250 units/ml), 0.1% trypsin and
0.04% versene. The flask was put into the cold (4°C) overnight. The
next day the flask was put on a magnetic stirring device at low speed
and 37°C for 30 minutes. The loose cells were decanted and centrifuged
at 1000 rpm for 15 minutes. The supernatant was poured off and the pellet
resuspended in medium. This was centrifuged and resuspended as before.
Cell density was adjusted to approximately 10^ cells/ml and 20 mis were
dispensed into 75 cm^ culture flasks which were then incubated at 37°C.
Cultures were fed after three days with MEM (10% FCS and 0.03 M HEPES)
and thereafter twice weekly. After monolayers were confluent they were
washed once with Gey's A solution and treated with trypsin/versene solu¬
tion until the cells came off the plastic. The culture flask was washed
twice with 10 mis of medium. Cell density was adjusted to 2.5 x 10^
cells/ml and 1 ml was dispensed into each well of a 24 well plate. The
next day samples to be assayed were incubated with ferret antiinfluenza
virus antibody (HAI = 1000) for 30 minutes at 37°C. Samples were then
d:¡ luted serially and put onto cell cultures overnight. Fluid was withdrawn

17
and 0.1 ml of a dilution of VSV (giving between 30 and 300 plaques/well
on controls) was dispensed to each well for 1 hour. Methyl-cellulose
overlay was put on and two days later removed. Cell monolayers were then
stained with crystal violet for 20 minutes. Plaques were counted and 50%
plaque reduction titers were calculated.
Neutralization Titers
Samples were heat inactivated at 56°C for 30 minutes. Samples were
then diluted serially and equal amounts of samples and virus (100 EID^/
mis) were incubated for 1 hour at room temperature. Ten-day old embryon-
ated chicken eggs were injected with 0.1 ml of incubated mixtures. Eggs
were incubated at 36°C for two days, then harvested and hemagglutination
assays performed and results recorded. That dilution of the sample that
gave 50% neutralization of the virus was determined to be the neutrali¬
zation titer.
X-rays
Mice were x-rayed using Dupon Extremity A film (10 x 12 inches)
and an Extremity I cassette. The machine used was a Phillips XF 3001.
Films were takn at 40 KV, 16 MAS, and 1/20. Developing was accomplished
with a Kodak RP X-omat automatic processor, model M6A-N. Animals were
held extended and in place by extending a string across a styrofoam board
and allowing the animals to grab hold of the string and pulling them back
by the tail. The styrofoam board was approximately 1 inch thick because
thicker boards cause loss of clarity in the radiograph. This board was
rested on top of the cassette as the picture was taken. Individuals
holding the tails of the mice were always dressed in protective clothing
and wore a film badge for monitoring levels of radiation exposure.

18
Scanning Electron Microscopy
Trachea samples for scanning electron microscopy (SEM) were
placed in a buffered fixative composed of 2.5% glutaraldehyde, 0.1 H
sodium cacodylate, and 0.1% CaCl2 (pH 7.4) and allowed to fix for at
least 24 hours before further preparation. They were then removed
from the fixative and dehydrated in graded concentrations of acetone
(70 to 100%). Specimens were critical point dried in a Bomar SPC 900/Ex
critical point drying machine (Bomar Corp., Tacoma, Washington) coated
with gold-palladium in a Hummer II shadowing machine (Technics, Alexandria,
Virginia) and examined with a Novascan 30 electron microscope (Semco,
Ottawa, Canada).

SPECIFIC LOCAL IMMUNITY IN FERRET ORGAN CULTURES
Introduction
Organ Culture
We began our studies by examining local immune mechanisms by use
of ferret organ cultures. Organ cultures consist of fragments of tissue
taken from the adult animal and maintained in vitro. Cell proliferation
does not normally occur during the short time the cultures are in use
and the tissue retains many of its in vivo characteristics. Organ cultures
can provide a high degree of sensitivity and economy in determining factors
at work in tissues (Schmidt and Maassab, 1974).
Many uses have been found for organ cultures, including the study
of resistance to reinfection. Heuschele and Easterday (1970) used organ
cultures derived from chicken trachea to study resistance to reinfection
by Newcastle Disease Virus (NDV). They suggested a potential role for
organ cultures to study mechanisms of formation and/or secretion of local
antibody. Finkelstein et al. (1972) again using chicken tracheal organ
cultures demonstrated that resistance, to NDV was probably partially
mediated by interferon. Resistance after intratracheal immunization
correlated only with tracheal antibody and could be partially blocked by
exogenously applied rabbit antichicken globulin. This suggested antibody
was present on mucosal surfaces which could have been produced locally.
Schmidt and Maassatr (1974) found specific resistance to influenza virus
in tracheal organ cultures from chickens. They suggest that this immunity
was at least partially derived from a secretory immune system.
19

20
Organ cultures have been shown to be an excellent system for
studying pathogenesis of respiratory infections. Klein and Collier
(1974) studied pathogenesis of human parainfluenza type 3 virus infection
by using hamster tracheal organ cultures. Human organ cultures have been
used successfully to study the cytopathic effect of this same pathogen
(Craighead and Brennan, 1968).
The relationships between influenza strains that infect humans and
animals can be studied in organ cultures. In other words, they offer a
way of determining the host ranges of the different strains in a precise
and economical manner. This is important as it relates to the hypothesized
recombinational events occurring in simultaneously infected host cells
causing the creation of a new strain of influenza virus capable of causing
pandemics (see general Introduction). Schmidt et al. (1974) studied human,
swine, equine and avian influenza A viruses and tested them for infectivity
in chicken, ferret, equine and porcine tracheal organ cultures. Results
showed that the homologous tissue was much more easily infected than hetero¬
logous tissue.
Organ cultures have also been shown to be an excellent way for
screening potential live vaccines. Mostow and Tyrrell (1973) have used
ciliary activity in tracheal organ cultures as a measure of virulence and
therefore usefulness of vaccines. Vaccines which cause a small decrease
of ciliary activity have been shown to be extremely avirulent in vivo
while vaccines that are highly virulent thus causing marked decreases in
ciliary activity in organ culture are likewise virulent in vivo.

21
Ferrets and Influenza
We have elected to use ferrets for our studies of influenza, because
influenza infection in ferrets appears to resemble the human infection and
because ferret trachea are relatively long and contain a large number of
individual rings. Further, the size of the animal makes it easy to handle.
The ferret also lends itself to the organ culture system because of the
ease with which their tissues are infected by influenza virus. Bang and
Niven (1958) reported the cultivation of influenza virus in ferret nasal
mucosa. In perhaps the most extensive studies performed so far, Basarab
and Smith (1970) showed the growth patterns of influenza virus in cultures
of ferret organs. They reported virus replication in cultures of ferret
nasal mucosa, lung, trachea, oviduct and bladder.
Results
Immunity in Ferret Organ Cultures • 3-•
The basic experimental design is shown in Figure 1. In all experi¬
ments ferrets, subsequently identified as convalescent, were infected 21
days before being killed. Nasal washes were taken 2 and 4 days after
this infection. Serum was taken at the time of infection and of killing.
Animals were proven to be infected by both virus isolation from nasal wash
(one exception noted in Table 1) and a 4-fold or greater rise in HAI anti¬
body titre to the virus. At Day 0 normal and convalescent ferrets were
killed and their tissues put into organ cultures. Serum taken from normal
ferrets at the time of killing (Day 0) had no detectable HAI antibody
(<1:8) to influenza"virus. Cultures were challenged with varying dilutions
of influenza virus on Day 1. The number of organ cultures/dilution varied
in different experiments depending upon the number obtained from each

22
TABLE 1
VIRUS ISOLATION PROM TRACHEAL RINGS
3.
Virus Isolation on Day
Virus Challenge
Rings
0
4
6
8 10
Normal
12
Ferret
14
17
19
Control
1
0
0
0
0
0
0
0
0
0
2
0
0
0
0
0
0
0
0
0
3
0
0
0
0
0
0
0
0
0
4
0
0
0
0
0
0
0
0
0
5
0
0
0
0
0
0
0
0
0
-3
10
1
0
+
+
+
+
+
+
+
+
2
0
+
+
+
+
+
+
+
+
3
0
+
+
+
+
+
+
+
+
4
0
+
+
+
+
+
+
+
+
5
0
+
+
+
+
+
+
+
+
-4
10
1
0
+
+
+
+
+
+
+
2
0
+
0
+
+
+
+
+
+
3
0
+
+
+
+
+
+
+
+
4
0
+
+
4-
+
+
+
+
â– A I
+
5
0
+
+
+
+
+
+
+
+
-S
10
1
0
+
+
+
+
+
+
+
+
2
0
+
+
+
+
+
+
+
+
3
0
+
+
+
+
+
+
+
+
4
0
+
+
+
+
+
+
+
+
5
0
+
+
+
+
+
+
+
+
10
1
0
+
+
+
+
+
+
+
+
2
0
+
+
+
+
+
+
+
+
3
0
+
+
+
+
+
+
+
+
4
0
+
+
+
+
+
+
+
+
5
0
0
0
0
0
0
0
0
0
-7
10
1
0
0
0
0
0
0
0
0
0
2
0
0
0
0
0
0
0
+
0
3
0
+
+
+
+
+
+
+
+
4
0
+
+
+
+
+
+
+
+
5C.
0
0
+
+
+
+
+
0
0

23
TABLE 1--Continued
Infected Rings (Day 8)
21 25 31 37 40 Total Rings OCID^q
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
ND
+
0
+
+
ND
+
0
+
+
ND
+
+
0
+
ND
+
+
+
+
ND
+
+
+
+
+
+
+
+
+
+
+
0
+
+
+
+
+
+
J-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
0
+
+
+
+
0
+
f
+
+
+
0
+
+
+
+
+
+
+
+
0
+
+
+
+
0
+
+
+
0
+
+
+
+
+
0
+
+
0 0
0 0
0 0
+ + 0 + +
+ 0 + +
0/5
5/5
10“0,8EID5()s/ml
5/5
5/5
4/5
+
3/5

24
TABLE 1--Continued
Virus
Isolation3 on
Day
Virus Challenge
Rings
0
4
6
8 10
Convalescent
12 14
Ferret
17
19
-7
10
1
0
+
+
+
+
+
+
+
+
2
0
+
+
+
+
+
+
+
+
3
0
+
0
+
+
+
+
+
+
4
0
+
+
+
+
+
+
+
+
tO
1
o
t-H
1
0
+
+
+
+
+
+
+
+
2
0
+
+
+
+
+
+
+
3
0
+
+
+
+
+
+
+
+
4
0
+
+
+
+
+
+
+
+
io~4
1
0
0
0
0
0
0
0
0
0
2
0
+
+
+
+
+
+
+
+
3
0
+
+
+
+
+
+
0
+
4
0
+
+
+
+
+
+
+
+
10
1
0
0
0
0
0
0
0
0
0
2
0
0
0
0
0
0
0
0
0
3
0
0
0
0
0
0
0
0
0
4
0
+
+
+
+
+
+
+
0
-fi
10
1
0
+
0
0
0
0
0
0
0
2
0
+
+
+
+
+
+
+
+
3C
0
0
0
0
0
0
0
0
0
4C
0
0
0
0
+
+
+
+
+
-7
10
1
0
0
0
0
0
0
0
0
0
2
0
0
0
0
0
0
0
0
0
3
0
0
0
0
0
0
0
0
0
4
0
0
0
0
0
0
0
0
0
5
0
0
0
0
0
0
0
0
0
+ = at least one of the duplicate eggs show HA 3 days after inoculation
with sample.
^Dilution of stock virus “containing 10^"^ EID^/ml of A/PR/8/34 (H0N1) .
c
Ring which changed from not infected to infected.
ND = Not done.

25
TABLE 1--Continued
Infected Rings (Day 8)
21
25
31
37
60
Total Rings
+
0
+
+
+
+
+
+
+
4-
4/4
+
+
•f
+
+
+
+
0
+
+
+
+
+
+
+
+
+
+
+
+
4/4
+
+
0
+
+
+
+
+
+
+
0
0
+
+
+
+
+
3/4
+
+
+
+
0
+
+
0
0
0
0
0
0
0
0
0
1/4
+
0
+
+
+
0
0
0
0
0
+
+
•f
+
+
+
+
+
+
+
1/4
+
+
+
+
+
0
0
0
0
0
0
0/5
0 0
0 0
OCID
50
101,5EID5()s/ml
o

26
animal. Fluids were completely drained and replaced every other day and
supernatants were assayed for virus on Days 1,4,6 and 8 and in one experi¬
ment periodically up to 60 days.
Table 1 shows data from Experiment IV. In general, once a culture
was infected it usually remained so throughout the experiment. Three of
the 55 cultures differed significantly from this pattern. Transient con¬
versions from positive to negative occurred occasionally but were probably
due to the egg assay system which apparently can have false negatives but
not false positives. Mien a large number of false negatives appeared on
one day it probably can be attributed to improper handling of samples
(e.g. Day 31). Since very little variation occurred from day to day, Day 8
was selected to calculate an OCID^ (50% organ culture infectious dose).
0CID^qS were calculated by the method of Reed and Muench (1938). For the
remaining experiments cultures were usually carried through Day 10 and
0CIDj-qS calculated for Day 8.
Table 2 summarizes the results of Experiment IV (already shown in
Table 1) and Experiment V, in which tracheal organ cultures from 5 normal
and 5 convalescent ferrets were compared for relative resistance to influ¬
enza infection. As can be seen, convalescent ferret trachea required
between 140 and 200 times more virus to infect them than did normal cul¬
tures. This difference was statistically significant and hence the
experiments demonstrate the presence of immunity.
Immunity in Ferret Tracheal Organ Cultures with
Homologous and Heterologous Virus
To determine if the immunity demonstrated in tracheal organ cultures
was specific for the virus which had been used to infect the animal (homo¬
logous virus), half of the cultures were challenged with the homologous

TABLE 2
IMMUNITY IN TRACHEAL ORGAN CULTURES
Experi¬
ment
Number of
Animals^
•
Number of
Rings/Animal/
Dilution of
Virus
Virus
OCID
Normal
50 L°g10 b,C ('mean ± SD')
, Ratio of
d 0
Convalescent Infectivity
f
P
IV
2
4 or 5
H0N1
-0.8
1.5
200 x
<0.0005
V
10
2
H0N1
0.3 (±0.4)
2.45 (±0.8)
140 x
<0.0005
Weighted average 150 x
^lalf the number of animals listed were normal and half were convalescent.
OCID_g obtained by using total infected and total uninfected cultures for an entire group of animals,
c
OCIDj-q = 50% organ culture infectious dose as determined by the Reed-Muench 50% endpoint method (1938) .
^11 of 12 animals were convalescent by two criteria: (a) Virus shedding; (b) 4-fold increase in specific
Ab titre after infection. (One convalescent animal in Experiment V was not tested for virus shedding.)
0
Antilog of (Log^Q 0CIDj.q convalescent--Log^ OCID^ normal) homologous virus,
f
Probability that difference occurred by chance calculated by Student's t test.

28
virus and the other half with a different virus (heterologous virus).
The results are shown in Table 3. Rings challenged with homologous
virus (H3N2) were between 20 and 200 times more resistant to infection
in culture than normal ferret tracheal organ cultures. Cultures chal¬
lenged with heterologous virus (H0N1 or Sendai) showed no significant
difference in one experiment and only 6-16 times more resistance in two
experiments when the 0CID^qS were compared with those from normal rings.
The immunity therefore appears to be largely specific, although a variable
amount of nonspecific immunity is sometimes observed.
Mechanisms of Immunity in Ferret Organ Cultures
Having now shown that ferret tracheal organ cultures derived from
convalescent animals are specifically immune to challenge, we wish to
delineate the mechanisms involved. Since the immunity is largely specific,
the mechanism could be mediated by specific antibody and/or lymphocytes.
However, the character of CMI, as stated before, is not totally specific
but rather cross-reactive in nature. With this information it seemed
more likely that antibody was the mechanisms of specific immunity in
tracheal organ cultures and some other mechanism responsible for the non¬
specific aspect. We therefore wished to test the possibility that either
serum antibody or local antibody was responsible for the specific effect.
Effect of Passive Administration of Serum Antibody
On Organ Culture Immunity {in vivo)
The protocol diagrammed in Figure 1 was followed except instead of
m
infecting ferrets at Day -21, animals received a total of 50 ml of either
immune ferret serum (HAI 2048) or normal ferret serum (HAI <16) in two
doses at Day -7 and Day -1. A third group of ferrets received no treat¬
ment. Ferrets in the immune serum group had HAI titers of 64, 64, and

29
TABLE 3
SPECIFIC IMMUNITY IN FERRET TRACHEAL ORGAN CULTURE
Experi¬
ment
Number
of
Animals
Rings/
Animal/
Dilution
of Virus
OCID^
Log n 5 (Mean
± SD)
Virusu
Normal
Homologous
Heterologous
Homologous
VII
4
4
H0N1
4
H3N2
1.8 (±0)
IX
6
2
FIONl
2
H3N2
3.95(±0.4)
XI
8
2
Sendai
2
I13N2
2.2 (±0.7)
Half the number of animals listed were normal and half were convalescent.
^OCID^p obtained by using total infected and total uninfected cultures for an
entire group of animals.
0
OCID = 50% organ culture infectious dose as determined by the Reed-Muench
50% endpoint method (1938).
^Antilog of OCID^o convalescent OCID^q norma^) •
0
Probability that difference occurred by chance.

30
TABLE 3--Continued
OCID50
Log1f> b,C (Mean
1 SD)
Ratio of Infectivity^
Normal
Convalescent
Homologous
Heterologous
Heterologous
Homologous
Heterdogous
Challenge
Challenge
6
p
1.3(±0.7)
3.1(10.2)
0.9(10.3)
20
0.4
NS
0.05
1.4(±0.2)
5.5(10.3)
2.2(10.4)
32
6
<0.005
<0.005
0.6(10.5)
4.5(10.3)
1.8(10.4)
200
16
<0.005
<0.005
Weighted average
104
9

31
128 at sacrifice. Titers of normal and no treatment groups were all 8 or
less. Table 4 illustrates the results. Both trachea and bladder cultures
were assayed for immunity. The results show that OCID^s of organ cultures
derived from animals receiving normal serum (NS) did not significantly
differ from OCID,_qS of organ cultures from animals receiving immune serum
(IS). Further, the IS group did not differ significantly from the no
treatment group. We conclude from this that serum antibody does not cause
specific immunity in ferret organ cultures.
Antialpha Treatment of Organ Cultures
To test whether tracheal organ culture immunity is caused totally
or in part by IgA in the tissue, organ cultures were exposed to rabbit
antiferret alpha chain 24 hours prior to challenge with virus. Briefly,
ferrets were infected and 21 days later sacrificed along with normal
uninfected ferrets. Trachea were cut into rings and placed in a petri
dish with antialpha or rabbit normal serum overnight. Cultures were
challenged and sampled periodically as before. Results are shown in
Table 5. Organ cultures from convalescent ferrets exposed to normal serum
showed 16-fold immunity over organ cultures from normal animals also ex¬
posed to normal serum (p = <.0$. Organ cultures from convalescent ferrets
exposed to antialpha showed no significant immunity relative to normals.
However, it is also apparent that when cultures from normal ferrets were
treated with the antialpha they became 200 fold more resistant to infec¬
tion (p <.01). This result is unexplainable and therefore makes the
interpretation of the results of antialpha treatment ambiguous. We can
make no clear interpretation of these data.

32
TABLE 4
WILL PASSIVE ADMINISTRATION OF ANTIBODY3 PRODUCE FERRET
ORGAN CULTURE IMMUNITY
OCID[-q Log¡^(Mean ±
SD)
Virus
Number
of
Animals
No„
Rx
NS
IS
b
P
c
P
Trachea
H3N2
9
3.5 ± .1
2.9 ±
.4
2.9 ± .8
NSd
NS
Bladder
H3N2
9
3.3 ± .9
3.0 ± 1.0
3.5 ± .5
NS
NS
aFerret anti-H3N2 antibody (64, 64, 128)
blSvs- %x
CIS vs. NS
dNot significant
0

33
TABLE 5
ABLATION OF RESISTANCE IN FERRET TRACHEAL ORGAN CULTURES WITH
RABBIT ANTIFERRET ALPHA AND RABBIT ANTIFERRET GAMMA
Treatment
Number of
Animals
OCID
(log1() í SD)
a
P
Antialpha
Convalescent
3
4.9 ±
.2
NSb
Normal
3
4.0 ±
.6-
Rabbit Normal Serum
Convalescent
3
5.1 ±
.4
<•01
<.01
Normal
3
6.3 ±
. 2_
Antigamma
Convalescent
3
4.5 ±
.4-
<.02
Normal
3
5.7 ±
0
*»•
Rabbit Normal Serum
NS
Convalescent
3
3.7 ±
.6-
Normal
3
5.8 ±
.3
<.01
Students t test (small sample)
bNot significant

34
Antigamma Treatment of Organ Cultures
To test whether tracheal organ culture immunity might also be
ablated by antigamma treatment we carried out the same protocol again
except that we substituted the use of rabbit antiferret gamma chain
antisera for the antialpha used above. Table 5 shows that convalescent
cultures treated with rabbit normal serum have 130-fold immunity
(p = <.01) compared to normal cultures treated with normal serum. Con¬
valescent cultures treated with antigamma showed 16-fold immunity
(p = <.02) when compared with normal cultures. Further, convalescent
cultures treated with antigamma were not significantly different from
convalescent cultures treated with normal serum. This suggests that IgG
may not play a significant role in immunity of tracheal organ cultures.
Neutralizing Antibody in Tracheal
Organ Cultures
Day 4 cultures which received no virus were assayed for neutral-
0
izing antibody to the homologous virus (A/Port Chalmers) and a hetero¬
logous virus (A/PR8). IVe were not able to show any significant neutral¬
izing capability in the supernatants of the cultures to either strain.
This is shown in Table 6.
Interferon in Tracheal Organ Cultures
Interferon assays were performed on culture supernatants from
Day 4 cultures. Table 6 shows that no significant interferon activity
was found (<3 units/ml) in any culture.
Bladder Immunity
To get an indication of whether specific immunity was restricted
to the respiratory tract, a second anatomically distinct site was tested.
Basarab and Smith (1970) had shown that ferret bladder tissue was

35
TABLE 6
NEUTRALIZING
IN
ANTIBODY AND
DAY 4 TRACHEAL
INTERFERON LEVELS IN
ORGAN CULTURES
Interferon
Neutralizing Antibody
Treatment
(units/ml)
H3N2
H0N1
Antialpha
Convalescent
< 3
< 8
< 8
Normal
< 3
< 8
< 8
Antigamma
Convalescent
< 3
< 8
< 8
Normal
< 3
< 8
< 8
Normal Serum
Convalescent
< 3
< 8
< 8
Normal
< 3
< 8
< 8
0

36
susceptible to in vitro infection by influenza virus. Therefore, cultures
of trachea (results shown in Tables 2 and 3) and bladder were taken from
ferrets in some experiments. Table 7 shows the OCID^s for the normal
and convalescent bladder in four experiments. In Experiment V bladder
immunity is demonstrated since convalescent bladder tissue required
approximately 200 times more virus to infect than did bladder from
normal ferrets (statistically significant at p < 0.10). From Experiment
VII it appears that nonspecific immunity exists since it took 20 and 16
times more virus, respectively, to infect convalescent tissues than to
infect normal tissues with heterologous and homologous virus. Levels of
significance varied with a p value of 0.10 for heterologous and a value
of < 0.10 for homologous; hence this is not a conclusive experiment.
The last two experiments (IX and XI) show specific immunity. In experi¬
ments which include tissues from a total of 14 animals it took 500 and
O
40 times more homologous virus to infect convalescent tissues than to
infect normal tissues (significance p = 0.025 and p = < 0.005). At the
same time, it took 10 and 0.25 times more heterologous virus to infect
convalescent cultures than it took to infect normal cultures (significance
level of p = 0.10 and p = < 0.005). Thus 3 of the 4 experiments suggest
specific immunity in bladder to homologous virus.
Ciliary Activity in Tracheal Organ Cultures
We attempted to correlate ciliary activity with infection of tracheal
organ cultures. Organ cultures were observed under 100X power using an
inverted microscope. Ciliary activity of rings was qualitatively evalu¬
ated by determining the percentage of the inner circumference of the
ring showing detectable activity and by the intensity of ciliary beating.
We observed a significant decrease of both parameters by activity of

37
TABLE 7
IMMUNITY IN FERRET BLADDER ORGAN CULTURES
Experi¬
ment
Number
of
Animalsd
Pieces of
Tissue/
Animal/
Dilution
of Virus
OCID
Logm b’C (Mean
± SD)
Vi:
rus
Normal
Homologous
Heterologous
Homologous
V
4
2 or 3
HON 1
0.2 (±0.8)
VII
4
2
HON 1
H3N2
2.8(+0.4)
IX
6
1
HON 1
H3N2
2.3(±0.8)
XI
8
2
Sendai
II3N2
3.0(±0.5)
^alf the number of animals listed were normal and half were convalescent.
^OCID-q obtained by using total infected and total uninfected cultures for an
entire group of animals.
Q
OCID _ = 50% organ culture infectious dose as determined by the Reed-Muench
50% endpoint method (1938).
^Antilog of (Logjy OCIDj.0 convalescent -Log^ OCID^ normal).
0
21 or 22 animals shown to be convalescent by both criteria: (a) Virus
shedding as determined by assay of nasal wash; (b) 4-fold increase in specific
Ab titre after infection. (One convalescent animal in Experiment V was not
tested for (a) due to lost samples.
f
Probability that difference occurred by chance.

38
TABLE 7-
-Continued
ocid50
L°gm b,C (Mean
± SD)
Ratio of
Infectivity
Normal
Convales cent''
Homologous
Heterologous
f
P
Heterologous
Homologous
Heterologous
Challenge
Challenge
2.52(±0.5)
200
<0.10
0.2 (±0)
4.0(±0)
1.5(±0.35)
16
20
0.10
<0.10
0.7(±0)
5.0(±1.0)
1.7 (±0.7)
500
10
0.10
<0.025
2.3(±0.25)
4.6(±0.9)
1.7(±0.2)
40
.25
<0.005
<0.005
Weighted average
190
8

39
both infected and noninfected tracheal organ cultures during the first
ten days. Upon further manipulation, this decrease in activity was not
as apparent as originally seen. It was observed that upon agitating the
tissue in it's media there was a significant increase in activity. With
this method we were unable to conclude that there was a significant dif¬
ference in ciliary activity between infected and noninfected cultures.
We wished to look at the surface of the rings to determine if
infection caused cell loss and if we could detect a difference between
infected and noninfected rings. To determine the amount of desquamation
occurring in tracheal organ cultures we employed SEM to observe rings
at various times, up to 28 days, after infection in culture. No more
than 50% loss of ciliated cells was seen in any culture on any day.
Further, there seemd to be no difference between infected and noninfected
cultures. We also compared the ciliated surface of trachea taken from
animals infected in vivo. Animals were sacrificed on Days 0, 3, 4 and
5. No loss of ciliated cells was noted. These results suggest that in
our organ culture system neither ciliostasis nor desquamation can be used
as an indicator of infection of cultures. The possible explanation is
that this virus is not able to cause desquamation of ciliated epithelium.
Discussion
Organ cultures were used to study immunity in tissues of ferrets
exposed to influenza A virus. Tracheal organ cultures from ferrets con¬
valescent from influenza infection required about 130 times mgre homologous
virus to become infected than cultures from normal ferrets. It took only
about 9 times more heterologous virus to infect convalescent cultures
than to infect normal cultures. Immunity was therefore largely specific.

40
Bang and Niven (1958) briefly reported without giving their data or
experimental details that ferret mucosal tissue from convalescent animals
was not resistant to infection. It is not clear why their results dif¬
fered from those presented here.
Bladder tissue cultures were used to test if the specific immunity
was localized in the respiratory tract or was more widespread. Experi¬
ments show that bladders from convalescent ferrets were about 190 times
more resistant to challenge with homologous virus than normal bladder,
and that convalescent tissues were only about 8 times more resistant to
heterologous challenge. Bladder specific immunity could be explained in
at least two ways: (a) the specific immunity is caused by systemic
factors, or (b) it is a local response caused by either antigenemia or
homing to bladder mucosal tissue of specific lymphocytes stimulated in
the respiratory tract. Basarab and Smith (1970) did show that influenza
virus could replicate in vivo in bladders of ferrets. And it has been
shown that during severe influenza infection virus can be recovered from
urine of patients (Naficy, 1963).
Since the immunity in both trachea and bladder appears to be largely
specific, it could be mediated by antibody and/or lymphocytes. However,
it has recently been demonstrated in cytotoxic studies using influenza-
infected target cells (Effros et al., 1977; Zweerink et al., 1977) that
CMI may be less specific than is required to account for the specific
protection measured in challenge experiments. Therefore, antibody seems
to be the more likely mechanisms for prevention of influenza in ferrets.
Using tracheal organ cultures we tested which of the following
hypotheses might be true: (1) Systemic serum antibody could have been

41
responsible and simply be trapped in the mucous secretions and/or in the
tissue itself or (2) the immunity could be locally produced in submucosal
immunocompetent cells. Organ cultures derived from animals receiving IP
injections of Sab were not significantly more resistant than organ cultures
derived from normal ferrets. This suggests that Sab plays no role in
tracheal organ culture immunity. The second alternative of locally pro¬
duced antibody was tested and we showed that antigamma serum did not
ablate the in vitro resistance of tracheal rings. Antialpha serum seemed
to ablate the response but results from antialpha treatment of normal
rings made a clear interpretation impossible. Although we were not able
to clearly implicate IgA in resistance we were able to show that IgG does
not contribute significantly to resistance. This agrees well with
studies which quantitate immunoglobulin levels in the respiratory tract.
.f ^
Waldman et al. (1973) showed that IgA levels increased dramatically as
you ascend from the lower respiratory tract to the upper. Further,
histological studies show a preponderance of IgA and IgM positive plasma
cells in the lamina propria of secretory tissues (Bienenstock et al.,
1978). However, little IgM is found in secretions (Waldman et al., 1970)
so it seems likely that IgA is the primary immunoglobulin able to act in
the upper respiratory tract.
An attempt was made to measure interferon and neutralizing antibody
in supernatants of organ cultures. Both assays, done with samples taken
from cultures on Day'4, were negative. Interferon levels werq less than
3 units/inl and neutralizing antibody titers were less than 8 in all cases.
This does not necessarily mean that these substances were not present in

42
measurable quantities in the tissue or in close proximity to the rings,
but we were not able to find significant amounts once dilution occurs
in the total volume of the media on each culture.
We observed that ciliary activity could not be an accurate indicator
of infection of tracheal organ cultures since agitation of cultures
seemed to significantly restore activity which had been seemingly lost
over the first ten days. This suggests that mucous could entirely cover
surface areas which then would appear to have lost ciliary activity, or
mucous could slow the beating of cilia thus causing a further decrease
in activity. We are unable to say why these observations differ from
those of Mostow and Tyrrell (1973).
Further study using SEM showed that desquamation was not complete
in tissue from tracheal organ cultures and, in fact, no culture showed
greater than 50% loss of ciliated cells. Also, tracheas taken from
infected ferrets showed no loss of ciliated cells. We are unable to say
why these observations do not agree with those seen in mice by Ramphal
et al. (1979). Possibly our egg grown virus is not virulent enough to
cause total desquamation. Or perhaps ferret trachea is more resistant to
loss of ciliated cell than mouse trachea.
It is difficult to assess the value of in vitro studies in the
in vivo situation, especially when animal models are used to study human
disease. In the case of organ cultures used for this study two important
factors are offered for consideration. First, Rosztodzy et al. (1975)
have shown that human fetal tissues have similar susceptability to
challenge in culture with influenza virus as ferret tissues. Secondly,
Mostow and Tyrrell (1973) have shown that attenuated human influenza
viruses have similar activity in ferret organ cultures to that found in

43
vivo in humans. These studies suggest that ferret organ cultures are
a tool for studying influenza in humans.
Turning from prevention of infection to recovery from infection,
recent studies showed that mice with deficient CMI (nude mice; Sullivan
et al., 1976) or mice treated with ALS (Suzuki et al., 1974) shed virus
over longer periods than did normal mice, suggesting CMI may play a
critical role in recovery. In Experiment IV we showed that once a
ferret tracheal organ culture was infected it remained so; that is, it
did not recover. If CMI is responsible for recovery, it would follow
that CMI was not functional in the tracheal organ culture.
Irrespective of the mechanism of prevention or recovery from
influenza, it seems that the ferret tracheal organ culture enables one
to separate the two mechanisms. Immunity to reinfection can be demon-
strated in the same piece of tissue that lacks the ability to recover.
Hence it seems that prevention and recovery are mediated by different
mechanisms.
The role of antibody in recovery will be addressed in
Appendix A.

APPENDICES

APPENDIX A
SERUM ANTIBODY IN THERAPY OF INFLUENZAL PNEUMONIA
Introduction
As was stated before, the major cause of death related to influenza
is subsequent bacterial pneumonia. This is treatable by antibiotics.
However, in a small number of cases viral pneumonia is the direct cause
of death. Why in these few cases does the lung become involved to such
a great extent? Immune factors are evidently one of the major influences
in this determination. There is good support for the idea that Sab, if
present, is a factor which prevents lung involvement. This is a likely
explanation taking into account the intimate association of the lung
parenchyma and the pulmonary circulation. Loosli (1953) showed the
importance of circulating antibody in the prevention of death of mice.
Virelizier (1975) reported that HAI antibodys have an important role in
protection. More recently, Ramphal et al. (1979) reported that Sab pro¬
tects the lung from pneumonitis but does not protect the ciliated epi¬
thelium of the trachea. So it appears that if Sab against the infecting
influenza virus is present prior to infection, the lung will be protected
against viral pneumonia but the animal can still have an upper respiratory
infection. When drift from one subtype of influenza virus to another
subtype occurs very few cases of viral pneumonia are found. The reason
for this may be that Sab formed during infection by the closel'y related
strain is cross-reactive with the second strain of virus thus providing
lung protection. During a shift, when viral pneumonia is found to a
greater extent, cross-reacting Sab is not available because of the major
45

46
shift of viral antigens thus the lung is left unprotected and lung involve
ment is more likely.
Since it has been proven that Sab does protect against death, by
decreasing viral pneumonitis, we wished to determine if Sab could be used
therapeutically to treat diagnosed or suspected viral pneumonia. If a
cure could be affected, we wished to determine how soon before death Sab
could be effective for treatment.
Results
The virus used in these experiments was passaged in mouse lung
11 times. A lethal dose 50 (LD,_q) was performed in mice and we found
3 9
that 0.05 cc of the fluid contained 10 ' LD^qS. In all subsequent experi
ments this virus was diluted so that mice received approximately 10 LD^^S.
In our first experiments we wished to ensure that the Sab we were
using could be life-saving upon subsequent infection with the eleventh
passaged influenza virus. Briefly, one group of A/J mice were injected
intraperitoneally (IP) with a total of 0.5 cc of goat antiinfluenza
antibody (HAI 16000) in equal doses on two days. A second group received
no Sab. Both groups were infected on the day the first group received
its second dose of Sab. Animals were observed for deaths. Results
are shown in Table A-l. No deaths were recorded in the group receiving
Sab (0/5 dead) while those that did not receive Sab all died by two
weeks time (5/5 dead). We conclude that the dose of virus used is
lethal for our mice and that the dose of Sab was sufficient to protect
mice from lethal pneumonitis when given before infection.
We asked could Sab given after infection be life-saving? The
experiment consisted of infecting mice and then subsequently giving Sab
on specific days. In a preliminary experiment mice were infected and

47
TABLE A-1
Sab PROTECTS AGAINST VIRAL PNEUMONIA
Number of
Animals Dead
Treatment
Number of Animals Infected
No serum
5/5 -
p = .004
Saba
0/5 -
£
Sab given in two doses prior to infection,
0.25 cc/dose.

48
on Days 3, 4 and 5 after infection one group of these mice were given
Sab (0.75 cc total) and a second group was given nothing. Animals were
observed for death. Results for Experiment I are shown in Table A-2.
Mice not receiving Sab all died between Days 7 and 10 (4/4 dead) but
those receiving Sab on Days 3, 4 and 5 after infection all survived
for at least 21 days (0/5 dead). We conclude that Sab given on Days 3,
4 and 5 after a lethal influenza infection can be life-saving.
The next experiment was to determine how close to death mice could
be when administered Sab and still survive lethal infection. As before,
mice were infected and groups of mice were given (a) nothing, (b) Sab
on Days 3, 4 and 5, (c) Sab on Days 5, 6 and 7, or (d) Sab on Days 7, 8
and 9. Animals were observed for death. Results are shown in Table A-2
under column Experiment II. Again those mice that did not receive Sab
all died on Day 8 (7/7 dead). Mice that received Sab on Days 5, 6 and
7 all died between Days 5 and 8 (6/6 dead) and mice given Sab on Days 7,
8 and 9 all died between Days 7 and 9 (6/6 dead). The only group to be
affected by the Sab was the group receiving Sab on Days 3, 4 and 5 (1/7
dead). This one death occurred immediately after Sab injection on Day 4.
This was a significant decrease in mortality when compared with controls
(p = 0.002). This experiment was then repeated exactly as before but with
larger numbers of mice. Results are shown in Table A-2 under Experiment
III. Again, mice that did not receive Sab all died between Days 7 and 10.
Those receiving Sai¿ on Days 7, 8 and 9 all died between Days 6 and 10
after infection. In the group that received Sab on Days 5, 6 and
7 all but one animal died with deaths occurring between Days 6 and 8.
The one group that differed significantly from the control group was the

49
TABLE A-2
EFFECT OF Sab GIVEN AFTER LETHAL INFLUENZA VIRUS INFECTION
Treatment
Experi¬
ment I
Experiment II
Experiment III
No Serum
4/4a
7/7
10/10 -
Sabb
p = 0.000006
Days 3, 4, 5
0/5
1/7 -
1/13 -
p = .002
Days 5, 6, 7
NDC
6/6 -
11/12
Days 7, 8, 9
ND
6/6
14/14
aMortality (number of animals dead/number of animals infected)
bDose of Sab 0.25 cc
c
Not done
•«I

50
tiro un that received Sab on Days 3, 4, and 5 where only a single death
was recorded (1/13 p = 0.000006).
The results so far obtained seemed to suggest if the lethal
infection by influenza could be diagnosed and treated by Days 3 or 4,
a cure could be affected. The determining factor in clinical diagnosis
of viral pneumonia, however, is often radiological evidence of consolida¬
tion. We therefore attempted to discern if radiological changes were
evident by Day 3. If so, then it seemed possible that in a clinical
situation there might be a chance that the diagnosis might be made and
that subsequent treatment with Sab might benefit the patient. A control
group was infected with the virus and x-rayed on Days 3, 5, 7, and 9.
Photographs of representative radiographs are presented in Figure A-l.
It can be seen that no radiological evidence of lung involvement is
present until Day 7 or after. This indicates that, since by Day 7, Sab
cannot modify the lethal effect of infection with the virus that it
seems unlikely that Sab can be a useful treatment for lethal penumonitis.
Discussion
We have shown that high titered Sab (HAI 16000) given to mice
before a lethal infection of virus (10 LD^qS, which kills in 7-12 days)
can be life-saving. Further, when Sab is given after a similar infection
but before radiological changes appear in mouse lung the treatment is
also life-saving. However, if Sab is given after radiological changes
are noted, death occurs as if no treatment were given.
Because of our original success with Sab treatment of mice prior to
infection (Ramphai et al., 1979, see Appendix B) and because totally
effective treatment of lethal viral pneumonia is not available we had

Figure A-l. Radiological appearance of mouse lungs after
infection with lethal influenza virus (A/Port Chalmers, 10 LD,.^)
A.
Day
B.
Day
C.
Day
D.
Day

52
A
B


54
hoped to show that Sab given after infection could be useful in a life-
threatening situation. However, clinically, x-ray evidence of consolida¬
tion is the primary diagnostic tool in determining lung involvement dur¬
ing viral disease. Because of this and because we were not able to
modify the lethal effects of the advanced disease state, therapeutic use
of Sab seems less promising.
It is probable at this stage of disease that immune processes
have begun to overwhelm the majority of lung tissue. Epithelial cells
of alveoli have been disrupted and cellular infiltrates and edema are
present and increasing. Alveoli become filled and at this point Sab
acting to neutralize virus or coat infected cells has negligible effect.
In fact, Sab may increase vascular pressure which in turn increases
edematous flow into alveolar spaces thus worsening the problem.

APPENDIX B
SERUM ANTIBODY PREVENTS LETHAL MURINE INFLUENZA
PNEUMONITIS BUT NOT TRACHEITIS
Reuben Ramphal, Robert C. Cogliano, Joseph IV. Shands, Jr., and
Parker A. Small, Jr.
This paper reports studies showing the effects of serum antibody
upon influenza infection at two different sites: the trachea and lung.
Tracheal desquamation, pulmonary consolidation, death, and virus
shedding were examined after infection of mice with a lethal A/Port
Chalmers/1/73(H3N2) influenza virus. Immune serum administered intra-
peri toneally before infection prevented death and pulmonary consolidation
and also significantly lowered lung virus shedding as compared with
controls receiving normal serum. However, this protection did not Z -
extend to the ciliated epithelium of the trachea because serum antibody
did not prevent desquamation of the trachea or significantly decrease
viral yield from the trachea. These results indicate that serum antibody
is protective against severe pulmonary parenchymal disease but not for
disease of the ciliated epithelium.
55

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BIOGRAPHICAL SKETCH
Robert Christopher Cogliano, son of Vincent and Mary, was born
in Boston, Massachusetts,on the third day of August, 1946. He was
raised with his 5 brothers and sisters in Pembroke, Massachusetts,and
attended college at Bridgewater State College, Bridgewater, Massa¬
chusetts. Robert graduated in June of 1968 and entered military
service February, 1969. He served as a pilot in the United States
Air Force for five years. Robert started his graduate studies in
September, 1974, and will now go on to a position at New York Uni¬
versity Medical Center.
60

I certify that I
conforms to acceptable
adequate, in scope and
Doctor of Philosophy.
have read this study and that in my opinion it
standards of scholarly presentation and is fully
quality, as a dissertation for the de
//
//Parker A. Small,
Professor of Immunology
Microbiology
Medical
I certify that I
conforms to acceptable
adequate, in scope and
Doctor of Philosophy.
have read this study and that in my opinion it
standards of scholarly presentation and is fully
quality, as a dissertation for the degree of
Professor of Immunology and Medical
Microbiology
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy. ^
J/-S
George E. Gifford
Professor of Immunology and Medical
Microbiology
I certify that I
conforms to acceptable
adequate, in scope and
Doctor of Philosophy.
have read this study and that in my opinion it
standards of scholarly presentation and is fully
quality, as a dissertation for the degree of
Paul A. Klein
Associate Professor of Pathology

This dissertation was submitted to the Graduate Faculty of the College of
Medicine and to the Graduate Council, and was accepted as partial fulfill¬
ment of the requirements for the degree of Doctor of Philosophy.
December 1979
Dean, College of Medicine~
Dean,
in I '
hool

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