Bio 151 lectures for examination 2

VACCINES
REFERENCES: a) Abbas; b) Kuby; and c) Lectures of Dr Nina Gloriani, MD, PhD
Monday, September 10, 2012

EDWARD JENNER
• Father of Vaccinology &
Founder of Immunology
• Major contribution: ﬁrst
reliable method of
conferring lasting immunity
to a major contagious
disease (smallpox)
• Initiated the technique of
vaccination


LOOKING BACK...
• 18th century: Small pox

• 19th century: Rabies; typhoid; cholera; plague

• Early 20th century: BCG;Yellow fever; pertussis:
inﬂuenza; diphtheria; tetanus

• After world war II:

• OPV; MMR; Adenovirus; Salmonella Ty21a; varicella;
Injected Polio; Rabies; Japanese encephalitis; Hepatitis A;
Pneumococcus; meningococcus, Hemophilus inﬂuenza
PRP, Hepatitis B; Acellular pertussis


RECALL:
IMMUNITY


ACQUIRING PASSIVE AND
ACTIVE IMMUNITY


ACTIVE & PASSIVE
IMMUNITY


PASSIVE
IMMUNIZATION
• Administration of preformed
antibodies to treat infection
• Can be life-saving where toxins are
already circulating : tetanus,
diphtheria, snake bite
• Important where high titer speciﬁc
antibody is required

PASSIVE
IMMUNIZATION
• Preformed antibodies generally made in
horses, but occasionally obtained from
recovered patients
• Over 1000 donors used for each pool of
ISG, and sera are screened for HIV and
hepatitis B and C

COMMONLY
USED AGENTS
DISEASE AGENT
Black widow spider bite Horse antitoxin
Botulism Horse antitoxin
Diphtheria Horse antitoxin

Hepatitis A & B Pooled human IgG
Measles Pooled human IgG
Rabies Pooled human IgG
Snake bite Horse antivenin
Tetanus Pooled human IgG or horse
antitoxin

PASSIVE
IMMUNIZATION


NON-SPECIFIC IMMUNOTHERAPY
• Same compounds that act as adjuvants for vaccines also
used on their own to boost the general level of immune
activity


IMMUNOTHERAPY
OF TUMORS


ACTIVE
IMMUNITY
• The process of administering antigen to a live host to
induce an immune response for either academic or
public health reasons

• Vaccines have been developed as a prophylactic
measure to prevent disease caused by infectious agents,
provided their use caused only low levels of morbidity

• WORLD HEALTH ORGANIZATION: Expanded
Program on Immunization (EPI) and Vaccine
development programs/initiatives


GENERAL TYPE OF
VACCINES


ANTIGENIC PREPARATIONS
USED AS VACCINES


COMPARING
NOTES


LIVE ATTENUATED VACCINES


EFFECT OF VACCINES
ON VIRAL DISEASES


KILLED-WHOLE
ORGANISM VACCINE


POLIO: LIVE VERSUS KILLED
VACCINE


SUBCELLULAR
FRAGMENTS VACCINES


TOXIN-BASED
VACCINES


DNA VACCINES


THE MALARIA VACCINE
STRATEGY


VACCINES IN
GENERAL USE


VACCINES RESTRICTED TO
CERTAIN GROUPS


SAFETY CONCERNS WITH
VACCINES


WANTED VACCINES...


ADJUVANTS


EFFECTS OF ADJUVANTS
• Concentration of antigen in a site where
lymphocytes are exposed to it
• the “DEPOT” effect
• Induction of cytokines which regulate
lymphocyte function
• cytokines themselves shown to be
effective adjuvants, particularly when
coupled directly to antigen

START HERE


VACCINE
CONSIDERATIONS #1

• Type
• Indications
• Target group
• Dose
• Route of Administration
• Monitoring Side Effects

TARGET GROUPS

MONITORING
• Side effects: Fever
• LOCAL SYMPTOMS AT
INJECTION SITE: Soreness,
redness, swelling size
• GENERAL symptoms–
headache, malaise, loss of
appetite, nausea, vomiting
• Adverse Reactions
• Contraindications

VACCINE
CONSIDERATIONS #2
• SAFETY: Toxicity, teratogenicity, pyrogenicity,
adverse reactions
• IMMUNOGENICITY: Seroconversion: levels of
antibody before and after immunization;
Geometric mean titer (GMT): antibody titer
following vaccination
• EFFICACY: correlation between seroconversion
and PROTECTION (Antibodies; CMI or both)

SAFETY: ADVERSE
REACTIONS
• MILD: adverse reaction does not interfere with
normal activities; may be bothersome
• MODERATE: adverse reaction produces some
impairment of functioning but NOT hazardous
to health; uncomfortable or disturbing
• SEVERE: adverse reaction produces signiﬁcant
impairment of functioning or incapacitation; a
deﬁnite hazard to health

EFFICACY
• Streptococcus pneumoniae; Neisseria meningitidis;
Hemophilus inﬂuenzae capsular polysaccharide
(85%)
• Diphtheria and tetanus Toxoids (>90%)
• Bordetella pertussis (whooping cough) (>90%)
• Salmonella typhi (killed or live attenuated)
(80%)
• Mycobacterium tuberculosis (BCG) (0-70%)

VACCINE-
PREVENTABLE
DISEASES

VACCINES FOR
YOU
Following primary series of
immunizations, young adults should
have a further dose of TD at age
15-16 years

Health care workers need evidence of
two measles vaccinations or blood
test conforming immunity

Meningitis vaccination recommended
for students & staff in residential
colleges of the university or in any
group accommodation

VACCINES FOR
YOU
Following primary series of
immunizations, young adults should
have a further dose of TD at age
Basic Vaccinations: 15-16 years
Tetanus
Diphtheria Health care workers need evidence of
Polio
Measles
two measles vaccinations or blood
Mumps test conforming immunity
Rubella
Hepatitis B Meningitis vaccination recommended
Meningitis for students & staff in residential
colleges of the university or in any
group accommodation

VACCINES FOR
YOU
• RUBELLA

• Lyophilized preparation of highly
attenuated Wistar RA 27/3 strain
rubella virus propagated in human
diploid cells

• 0.5ml single dose , SC

• Side effects: rashes, malaise,
temperature elevation, cough, coryza,
headache, transient arthralgia &
arthritis with or w/o joint effusion

VACCINES FOR
YOU
• RUBELLA

Basic Vaccinations: • Lyophilized preparation of highly
Tetanus attenuated Wistar RA 27/3 strain
Diphtheria rubella virus propagated in human
Polio
diploid cells
Measles
Mumps • 0.5ml single dose , SC
Rubella
Hepatitis B
Meningitis • Side effects: rashes, malaise,
temperature elevation, cough, coryza,
headache, transient arthralgia &
arthritis with or w/o joint effusion

VACCINES FOR
YOU
• MMR

• Live attenuated measles (Edmonston
–Zagreb strain), mumps (Rubini
strain), rubella (Wistar RA 27/3
strain) viruses.
• Strains propagated on human diploid
cells, no antibiotics
• Vaccinate infants only after 15
months = Repeat immunization after
15 years for infants immunized
before age 12 months

VACCINES FOR
YOU
• MMR

• Live attenuated measles (Edmonston
Basic Vaccinations: –Zagreb strain), mumps (Rubini
Tetanus strain), rubella (Wistar RA 27/3
Diphtheria
strain) viruses.
Polio
Measles • Strains propagated on human diploid
Mumps cells, no antibiotics
Rubella
Hepatitis B • Vaccinate infants only after 15
Meningitis months = Repeat immunization after
15 years for infants immunized
before age 12 months

VACCINES
FOR YOU
• CHICKEN POX (VARICELLA)

• Live attenuated varicella vaccine
• For healthy infants from age 9 months
onward, healthy subjects , high risk
patients & healthy close contacts, patients
with acute leukemia, under
immunosuppressive treatment, patients
with planned organ transplantation,
patients with chronic diseases & healthy
close contacts
• Dose recommended: 2 doses 4-6 weeks
apart for adults

VACCINES
FOR YOU
• CHICKEN POX (VARICELLA)

Additional • Live attenuated varicella vaccine
Vaccinations:
• For healthy infants from age 9 months
Chicken pox
onward, healthy subjects , high risk
Hepatitis A
patients & healthy close contacts, patients
Influenza
Pertussis
with acute leukemia, under
immunosuppressive treatment, patients
Salmonella typhi with planned organ transplantation,
Cholera patients with chronic diseases & healthy
Pneumonia close contacts
• Dose recommended: 2 doses 4-6 weeks
apart for adults

VACCINES
FOR YOU
• HEPATITIS A

• Inactivated hepatitis A virus for individuals >
15 years of age in pre-ﬁlled syringe
• Virosomal hepatitis A virus antigen (RG-SB
strain) for adults & children over 12
months
• Basic immunization : Intramuscular
injection
• Booster immunization: 6, 12 or 18 months
after ﬁrst immunization
• Simultaneous active & passive immunization
at another site

VACCINES
FOR YOU
• HEPATITIS A

Additional • Inactivated hepatitis A virus for individuals >
Vaccinations: 15 years of age in pre-ﬁlled syringe
Chicken pox
• Virosomal hepatitis A virus antigen (RG-SB
Hepatitis A
strain) for adults & children over 12
Influenza
months
Pertussis
• Basic immunization : Intramuscular
Salmonella typhi injection
Cholera • Booster immunization: 6, 12 or 18 months
Pneumonia after ﬁrst immunization
• Simultaneous active & passive immunization
at another site

VACCINES
FOR YOU
• INFLUENZA

• Highly purified surface antigens
of influenza viruses types A &
B constituted strains
annually recommended by
WHO
• Polyvalent whole virus vaccine
against strains of groups A & B
• Purified split inactivated
influenza virus

VACCINES
FOR YOU
• INFLUENZA
Additional
Vaccinations: • Highly purified surface antigens
Chicken pox of influenza viruses types A &
Hepatitis A B constituted strains
Influenza
annually recommended by
Pertussis
WHO
Salmonella typhi • Polyvalent whole virus vaccine
Cholera
Pneumonia
against strains of groups A & B
• Purified split inactivated
influenza virus

VACCINES
FOR YOU • CHOLERA

• Orochol E Berna contains not < 2x109
viable organisms of the attenuated strain
Vibrio CVD 103-HgR in a lyophilized form

• For ORAL active immunization of adults &
children >2 years against cholera

• Dosage: Resuspend contents of both
chambers at the same time in cold or
lukewarm water, mix carefully for 5-10
seconds & drink immediately.

• Do not resuspend in milk, juice or
carbonated beverages.

VACCINES
FOR YOU • CHOLERA

• Orochol E Berna contains not < 2x109
viable organisms of the attenuated strain
Vibrio CVD 103-HgR in a lyophilized form
Additional
Vaccinations:
• For ORAL active immunization of adults &
Chicken pox children >2 years against cholera
Hepatitis A
Influenza
Pertussis • Dosage: Resuspend contents of both
chambers at the same time in cold or
Salmonella typhi lukewarm water, mix carefully for 5-10
Cholera seconds & drink immediately.
Pneumonia
• Do not resuspend in milk, juice or
carbonated beverages.

VACCINES
FOR YOU
• TYPHOID

• Vi capsular polysaccharide
typhoid vaccine: Single IM or SC
injection

• Oral Vaccine preparation > 109
viable organisms of attenuated
Salmonella typhi strain Ty21a
Berna; has cross immunity
against S. paratyphi B

VACCINES
FOR YOU
• TYPHOID

Additional • Vi capsular polysaccharide
Vaccinations: typhoid vaccine: Single IM or SC
Chicken pox injection
Hepatitis A
Influenza
Pertussis
• Oral Vaccine preparation > 109
Salmonella typhi viable organisms of attenuated
Cholera
Pneumonia
Salmonella typhi strain Ty21a
Berna; has cross immunity
against S. paratyphi B

VACCINES
FOR YOU • PNEUMOCOCCUS

• Puriﬁed polysaccharide of
Streptococcus pneumoniae

• 25ug each of 23 serotypes:
1,2,3,4,5,6B,7F,8,9V,10A, 11A, 12F, 14,
15B, 17F, 18C, 19A, 19F, 20, 22F, 23F,
33F

• Indications: Prevention of pneumococcal
infections, particularly those of
respiratory origin for ages over 2 years
who are at risk of serious
pneumococcal infection

VACCINES
FOR YOU • PNEUMOCOCCUS

• Puriﬁed polysaccharide of
Streptococcus pneumoniae
Additional
Vaccinations:
Chicken pox • 25ug each of 23 serotypes:
Hepatitis A 1,2,3,4,5,6B,7F,8,9V,10A, 11A, 12F, 14,
Influenza 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F,
Pertussis 33F

Salmonella typhi
• Indications: Prevention of pneumococcal
Cholera infections, particularly those of
Pneumonia respiratory origin for ages over 2 years
who are at risk of serious
pneumococcal infection

VACCINES
FOR YOU
• PNEUMOCOCCUS

• Dosage: Initial Injections: Single
IM or SC injection confers
protection against the 23 types of
pneumococci in the vaccine

• Revaccination should not be
carried out for at least 5 years as
severe local reactions (Arthus
phenomena type) were observed
if time between 2 injections is
too short

VACCINES
FOR YOU
• PNEUMOCOCCUS

• Dosage: Initial Injections: Single
Additional
IM or SC injection confers
Vaccinations:
Chicken pox
protection against the 23 types of
Hepatitis A pneumococci in the vaccine
Influenza
Pertussis
• Revaccination should not be
Salmonella typhi carried out for at least 5 years as
Cholera severe local reactions (Arthus
Pneumonia phenomena type) were observed
if time between 2 injections is
too short

ISSUES TO
CONSIDER
• Which vaccine to administer to whom?
• When to vaccinate: months before exposure?
• How many doses? Cost?
• Interval between doses in case of multi-dose regimen
for primary immunization
• When to expect protection? Immunologic response vs
protection?
• Possibility of adverse/side effects

EXPANDED
IMMUNIZATION
PROGRAMS


The 7 EPI Diseases
Measles Diphtheria

Poliomyelitis Hepatitis B
Pertussis

Tetanus

Tuberculosis


1976 Official launching of EPI in the Philippines

1977 BCG & DPT2 for infants in priority areas

BCG & DPT2 expanded nationwide
1979 OPV3 in selected areas reporting outbreaks
TT2 for pregnant women at 5 months gestation

1980 OPV3 & TT2 given nationwide

1982 Measles vaccine for 35% of the population

1983 Measles vaccination given nationwide

1984 DPT3 added; TT given anytime during pregnancy

1985 Comprehensive Program Review

1986 Universal Child Immunization Goal by 1990-UN

Start of Rotary International mass campaigns –Polio
1987
Plus

TT3, TT4, TT5 added; Wednesday adopted as
1989
Immunization Day

National Plan of Action for Polio Eradication
1991
launched; Polio Eradication Unit

1992 Hepatitis B immunization -40% of infants

1993 1st National Immunization Days

Neonatal Tetanus Elimination-Action Plan

Measles Control – National Plan of Action

Bacteria Disease Antigen Efficacy

Streptococcus Capsular
Pneumonia 85%
pneumoniae polysaccharide

Neisseria Capsular
meningitis 85%
meningitidis polysaccharide

Hemophilus Capsular
meningitis 85%
inﬂuenzae polysaccharide

Corynebacterium
Diphtheriae Toxoid >90%
diphtheriae

Clostridium tetani tetanus Toxoid >90%

Bordetella
Whooping cough Killed organism >90%
pertussis

Mycobacterium
Tuberculosis Live attenuated 0 – 70 %
bovis (BCG)

Killed/Live
Salmonella typhi Typhoid fever 80%
attenuated


Vaccine Reason for Immunization

Given at the earliest possible age protects
BCG against the possibility of infection from
family members

Early start with DPT reduces chance of
DPT
severe pertussis

Protection against polio increased the
OPV
earlier the OPV is given

Early start of HBV vaccination reduces
Hepatitis B chance of being infected and becoming a
carrier

At least 80% of measles can be prevented
Measles
by immunization at this age

Diseases to be immunized
Age
against

Birth Tuberculosis

6 weeks DPT; Polio; HBV



9 months Measles

Tetanus toxoid Immunization Schedule for
Women
Percent
Vaccine Minimum age/interval Duration of protection
protected

As early as possible during
TT1
pregnancy

* Infants born to the
mother protected from
TT2 At least 4 weeks later 80%
neonatal tetanus; 3 years
protection to mother

* Infants protected;
TT3 At least 6 months later 95% 5 years protection to
mother

* Infants protected;
TT4 At least one year later 99% 10 years protection to
mother

Lifetime protection for
mother; * all infants born
TT5 At least one year later 99%
to that mother will be
protected

Philippine Immunization
Flagship Programs

 Poliomyelitis Eradication
 Measles Elimination

 Maternal and Neonatal Tetanus
Elimination
 Control of Hepatitis B


TAKE-HOME
QUIZ
• Next to Smallpox, why do you think that polio
and measles are the next diseases targeted for
global elimination?
• Differentiate the 4 Immunization Flagship
Programs of the country in terms of:
• target group, vaccine to use and route, criteria
for success

IMMUNOLOGIC TOLERANCE
AND AUTOIMMUNITY

RECALL...
• Immunologic tolerance: unresponsiveness
to self-antigens
• WHAT WE WILL LEARN:
• How does the immune system maintain its
unresponsiveness to self-antigens
• What are the factors that may contribute to
the development of autoimmunity?


ON ENCOUNTER WITH
LYMPHOCYTES...


ON ENCOUNTER WITH
LYMPHOCYTES...

The choice among lymphocyte activation, tolerance, and ignorance is determined by the nature of
the antigen-speciﬁc lymphocytes and by the nature of the antigen and how it is displayed to the
immune system

IMPORTANCE OF IMMUNOLOGIC
TOLERANCE
• the knowledge that self-antigens normally induce tolerance

• if we learn how to induce tolerance in lymphocytes speciﬁc
for a particular antigen, we may be able to use this knowledge
to prevent or control unwanted immune reactions

• APPLICATIONS:
• treat allergic and autoimmune diseases
• prevent the rejection of organ transplants
• gene therapy
• prevent immune responses against the products of newly
expressed genes or vectors

CENTRAL &
PERIPHERAL
• central tolerance:
induced when developing
lymphocytes encounter
these antigens in the
generative lymphoid organs
(bone marrow and thymus)

• peripheral tolerance:
induces when mature
lymphocytes encounter self
antigens in peripheral
tissues

AUTOIMMUNITY
• Principal factors in the
development of
autoimmunity:

• inheritance of susceptibility
genes

• may contribute to failure
of self-tolerance

• environmental triggers

• e.g. infections = may
activate self-reactive
lymphocytes

CENTRAL T-LYMPHOCYTE
TOLERANCE

• Negative
selection:
principal
mechanism of
central tolerance

• Strong recognition of self antigens immature T-cells in the thymus may lead to death of
the cells (negative selection or deletion)

• Self-antigen recognition in the thymus may also lead to the development of regulatory T-
cells that enter peripheral tissues


PERIPHERAL T-
LYMPHOCYTES & ANERGY

• Peripheral tolerance:
• induced when mature T cells recognize self
antigens in peripheral tissues, leading to
functional inactivation (anergy) or death
• induced when the self-reactive lymphocytes are
suppressed by regulatory T cells


ANERGY

• ANERGY: functional inactivation of T-lymphocytes that occurs
when these ceIls recognize antigens without adequate levels of the
costimulators (second signals) that are needed for full T-cell
activation

ACTIVATION-INDUCED CELL
DEATH : DELETION


IMMUNE SUPPRESSION


Characteristics of self and foreign (e.g. microbial)
protein antigens that determine why the self
antigens induce tolerance and microbial antigens
stimulate T-cell mediated immune responses


CENTRAL TOLERANCE IN
B-CELLS


PERIPHERAL
TOLERANCE IN B-CELLS


AUTOIMMUNE DISEASES
& MHC MOLECULES


MOLECULAR MIMICRY &
AUTOIMMUNITY


SUMMARY
• Immunologic tolerance: speciﬁc
unresponsiveness to an antigen induced by exposure of
lymphocytes to that antigen
• All individuals are tolerant of (unresponsive to) their
own (self)antigens
• Tolerance against antigens may be induced by
administering that antigen in particular ways =
strategy may be useful for treating immunologic
disease and for preventing the rejection of
transplants


SUMMARY

• Autoimmune diseases: result from a failure of self-
tolerance
• Multiple factors contribute to autoimmunity:
• immunologic abnormalities
• susceptibility genes
• infections


SUMMARY
• Central tolerance: induced by the death of immature
lymphocytes that encounter antigens in the generative
lymphoid organs while Peripheral tolerance: results
from the recognition of antigens by mature Iymphocytes in
peripheral tissues

• T-CELLS:
• Central tolerance (negative selection): result of high-
afﬁnity recognition of antigens in the thymus, which tend to
be widely disseminated self-antigens AND may eliminate
the potentially most dangerous T-cells, which express high-
afﬁnity receptors for disseminated self-antigens


SUMMARY
• T-CELLS:
• Peripheral tolerance: induced by multiple mechanisms
• Anergy (functional inactivation) results from the
recognition of antigens without costimulators (second
signals) or when T cells use inhibitory receptors to
recognize costimulators

• Deletion (death by apoptosis) occurs when T cells
repeatedly encounter self antigens

• Some self-reactive T cells suppress potentially
pathogenic T-cells

SUMMARY

•B-CELLS
• central tolerance: induced when
immature cells recognize self antigens in the
bone marrow
• peripheral tolerance: (by anergy)
induced when mature B cells recognize self
antigens without T cell help


SUMMARY
• AUTOIMMUNITY
• Many genes contribute to the development of
autoimmunity
• The strongest associations are between HLA genes
and various T cell-mediated autoimmune diseases
• Infections predispose to autoimmunity, by causing
inﬂammation and inducing the aberrant expression
of costimulators or because of cross-reactions
between microbial and self antigens

vanderbilt.edu

IMMUNE RESPONSE AGAINST
TUMORS/TRANSPLANTS
Reference; Immunology by Abbas


CALENDAR FOR BIO 151
DATE ACTIVITY
September 11 TUMORS, TRANSPLANTS & SERODIAGNOSIS

September 18 EXAMINATION 2 (VACCINES TO SEROLOGICAL
DIAGNOSTICS)
September 25- October 2 IMMUNITY TO MICROBIAL INFECTIONS: Pneumonia
a.Fungi
b.Virus
c.Bacteria

IMMUNITY TO MICROBIAL INFECTIONS: Schistosomiasis

October 9 EXAMINATION 3: Take-Home (24 hours)

Questions available October 9, 2012 at 12 noon...Submission
should be October 10, 2012 at 12 noon (at DB, received and
stamped by DB staff)

*** Printed and well-cited


WHAT ARE TUMORS? webmd.com

• Simply means a mass of
cells

• Can be either benign or
malignant

• Benign tumors: not a
threat to life or long-
term health; made up of
related but different cells

• Malignant tumors:
threat to life or long-
term health; clonal or all
identical to the cell of
origin

IMMUNE RESPONSE TO
TUMORS

• T cells generally mount
effective surveillance against
tumors associated with
oncogenic viruses or UV
induction (these are strongly
immunogenic)

• More weakly immunogenic
tumors are not controlled by joseph-birch.livejournal.com

T cell surveillance, although
sometimes low grade
responses are evoked


IMMUNE RESPONSE TO
TUMORS
• NK cells play a role in
containing tumor growth
and metastases;

• The importance of the
immune system in
preventing tumor growth
can be seen in
immunocompromised
patients - AIDS patients
have a much higher chance
of developing many types
of cancer

WHAT IS CANCER?
• diseases in which there is loss
of regulation of the
proliferative process

• characterized by excessive,
uncontrolled growth of
abnormal cells, which invade
and destroy other tissues nursingcrib.com

• hyperproliferation of cells
that have violated the basic
rules of social cell behavior
(i.e, lost the ability to be
controlled by normal cell
signals)

CANCER
• Control is important to
ensure that cells only
divide when needed
(organs and tissues
should maintain their
correct shapes and sizes)

• Should this system fail,
several backup safety
mechanisms prevent
topnews.in

the cell from dividing
uncontrollably

DEFENSE AGAINST
UNCONTROLLED PROLIFERATION

• A cell can initiate cell
death (suicide) when a
defect is detected through
a process called apoptosis
• A cell can repair the error
and become a normal cell genengnews.com

• Immune system (e.g.,
natural killer cells can
detect abnormal cells and
kill them)

WHAT ARE ORGANS
TRANSPLANTS?
• surgical operation in
which a failing or damaged
organ in the human body is
removed and replaced
with a functioning one
• donated organ may be
from a deceased donor, a
living donor, or an
animal

• NOTE: In some cases an
artiﬁcial organ is used

WHAT ARE ORGANS
TRANSPLANTS?
• Cadaveric organ donation involves
removing organs from a recently
deceased donor

• Living organ donation involves the
donation of one of a paired organ (such
as kidneys) or a portion of an organ
(such as a lobe of the liver or lung)

• The donor's organ system is still
able to function after the donation

• Living donors are often related to
the patient, but that is not always
the case

SO WHAT’S IT FOR
IMMUNOLOGY?

• CANCER: enhancing immunity against
the tumors holds much promise for
treatment
•TRANSPLANTS: immune
responses against transplants are a
barrier for a successful transplantation


WHAT WE WILL LEARN...

• What are the antigens in tumors and tissue
transplants that are recognized as foreign
by the immune system?
• How does the immune system recognize
and react to tumors and transplants?
• How can the immune responses to tumors
and grafts be manipulated to enhance
tumor rejection and inhibit graft rejection?


IMMUNE SURVEILLANCE
• 1950s: a physiologic function of the
adaptive immune system is to prevent the
outgrowth of transformed cells or to
destroy these cells before they become
harmful tumors

london-research-institute.org.uk
aihealthsolutions.ca


EVIDENCES SUPPORTING
THE CONCEPT OF
IMMUNO SURVEILLANCE


TUMOR ANTIGENS
• Malignant
tumors
express
various types
of molecules
that may be
recognized by
the immune
system as
foreign
antigens


IMMUNE MECHANISM FOR
TUMOR REJECTION
• The principal immune mechanism of tumor
eradication is killing of tumor cells by cytolytic
T-lymphocytes (CTLs) speciﬁc for tumor
antigens
• endogenously synthesized cytosolic proteins
displayed as Class I MHC-associated
peptides
• SO: recognized by class I MHC-restricted
CD8+ CTLs (kills the cell producing the
antigens)

TUMOR REJECTION
• CTLs responses against tumor cells are often induced
by recognition of tumor antigens on host-antigen
presenting cells (APCs) which ingest tumor cells or
their antigens and present the antigens to T-cells


TUMOR REJECTION

• anti-tumor CD4+ T-
cell responses and
antibodies have been
detected in patients (but
may not be protective)
• activated
macrophages and NK
cells are capable of
killing tumor cells in vitro
(unclear protection)

EVASION OF
IMMUNE RESPONSE
BY TUMORS

• Immune response often fail to
check tumor growth because
these responses are
ineffective or because tumors
evolve to evade immune attack

• Immune responses against
tumors may be weak because
many tumor antigens are
weakly immunogenic,
perhaps because they differ
only slightly from self
antigens

EVASION OF IMMUNE
RESPONSE BY TUMORS
• Growing tumors also develop mechanisms for
evading immune responses
• stop expressing the antigens that are targets of
immune attack (antigen loss variants) =
continue to grow and spread
• stop expressing class I MHC molecules =
cannot display antigens to CD8+ T-cells (cross-
priming)
• may produce molecules, e.g. transforming
growth factor-beta, that suppress immune
responses

IMMUNOTHERAPY
FOR TUMORS

• MAIN GOAL:
• provide antitumor effectors (antibodies
and T-cells) to patients
• actively immunize patients against their
tumors
• stimulate the patients’ own antitumor
immune response


IMMUNOTHERAPY
VS CHEMOTHERAPY

• Chemotherapy/
Irradiation: may have
devastating effects on
normal non-tumor tissues
• Immunotherapy: highly-
speciﬁc for the tumor health.umn.edu


STRATEGY 1:
PASSIVE IMMUNITY
• immune effectors are injected into cancer
patients
• e.g. monoclonal antibodies to tumor antigens +
potent toxins
• HOW: antibodies bind to tumor antigens and
activate host effector mechanisms (e.g.
phagocytes or complement system)
• HOW: toxins delivered to tumor cells

EXAMPLE: BREAST CANCER
IMMUNOTHERAPY
• ALL information from: Critical Reviews in Oncology and
Hematology, October 2010 (Emde et al. Therapeutic strategies and
mechanisms of tumorigenesis of HER2-overexpressing breast cancer)

• HER2 (tyrosine kinase): overexpressed in approximately 25% of breast
cancers; acts as a signal amplifier for its siblings = transmembrane
receptors that collectively bind with 11 distinct growth factors of the
EGF family

• THUS: overexpression of HER2 confers aggressive invasive
growth in preclinical models and in patients

• “Specific therapies targeting HER2 include monoclonal
antibodies, antibody–drug conjugates, small molecule tyrosine
kinase inhibitors, as well as heat shock protein and sheddase
inhibitors. Two of these drugs have shown impressive – yet
mostly transient – efficacy in patients with HER2
overexpressing breast cancer.”

Signal transduction pathways instigated
by HER2, co-receptors and EGF-like
growth factors (epidermal) and NRGs
(neuregulins)


Clinically approved and experimental
therapeutic strategies targeting ErbB-2/
HER2 in carcinomas:

a. Trastuzumab: humanized monoclonal
antibody directed against the extracellular
domain of HER2; recruits immune effector
mechanisms and can induce apoptosis, block
angiogenesis and inhibit tumor cell proliferation.
b. Pertuzumab: prevent heterodimerization of
HER2 with other family members

RECENTS:
a. Lapatinib: tyrosine kinase inhibitors;
reversible inhibitor

b. Neratinib: irreversible inhibitor; variably
inhibit a broad range of tyrosine kinases

c. 17-AAG: block the ATP/ADP binding pocket
of HSP90 and target HER2 for proteasomal
degradation (Note; HSP90: molecular chaperone
required for proper folding of protein kinases like
HER2

STRATEGIES FOR
ENHANCING ANTITUMOR
IMMUNE RESPONSE

• 1. Tumor-
antigen pulsed
DC: mimic normal
pathway of cross-
presentation to
induce speciﬁc T-cell
response


STRATEGIES FOR
ENHANCING ANTITUMOR
IMMUNE RESPONSE

• 2. DNA or
transfected DC:
the host produce the
tumor antigen thus
inducing speciﬁc T-
cell response


STRATEGIES FOR
ENHANCING ANTITUMOR
IMMUNE RESPONSE
• 3. tumor cell
expressing
costimulators
(e.g. B7) or
cytokines (e.g.
IL-2): cytokines or
costimulators to
stimulate the
generation of own
tumor-speciﬁc
immune response

IMMUNE RESPONSE
AGAINST TRANSPLANTS


IMMUNE RESPONSE
AGAINST TRANSPLANTS
• transplants exchanged wenliang.myweb.uga.edu

between animals of the
same and other inbred
are accepted
• grafts among different
strains are rejected

• GENES for graft rejection: products are
expressed in ALL tissues


IMMUNE RESPONSE
AGAINST TRANSPLANTS
• Allogeneic (allografts) and
Xenogeneic (xenografts) grafts
are always rejected
• Alloantigens (alloreactive
antibodies and T-cells)
• Xenoantigens (xenoreactive
antobodies and T-cells)
• CLINICAL Scenario: mostly
allogeneic

Transplantation
Antigens

• The antigens of
allografts that
serve as the
principal targets of
rejection are
proteins encoded
in the MHC
• Human MHC =
human
leukocyte
antigen or HLA

INDUCTION OF IMMUNE
RESPONSES AGAINST
TRANSPLANTS
• T-cells may
recognize
allogeneic
MHC molecules
in the graft by
professional
APCs, or graft
alloantigens
may be
processed and
presented by
hosts’
professional
APCs

INDUCTION OF IMMUNE
RESPONSES AGAINST
TRANSPLANTS
• T-cells may
recognize
allogeneic
MHC molecules
if the graft does not contain professional APCs, how does it stimulate T cells?
in the graft by
professional
APCs, or graft
alloantigens
may be
processed and
presented by
hosts’
professional
APCs

INDUCTION OF IMMUNE
RESPONSES AGAINST
TRANSPLANTS
• T-cells may
recognize
allogeneic
MHC molecules
if the graft does not contain professional APCs, how does it stimulate T cells?
in the graft by
professional
APCs, or graft
alloantigens
may be
processed and
presented by
hosts’
graft cells are ingested by professional APCs in the recipient and
professional
the donor alloantigens are processed and presented by the self
MHC molecules on recipient APCs
APCs

MIXED LYMPHOCYTE
REACTION (MLR)
wenliang.myweb.uga.edu

• an in vitro model
of T-cell
recognition in
alloantigens


MIXED LYMPHOCYTE
REACTION (MLR)

• T-cells from one individual are cultured with
leukocytes of another individual and the responses
of the T-cells are assayed
• magnitude of response is proportional to the
extent of MHC differences between these individuals
• rough predictor of the outcomes of grafts
exchanged between these individuals


IMMUNE MECHANISM OF
GRAFT REJECTION

• occurs within minutes of transplantation; characterized by
thrombosis of graft vessels and ischemic necrosis of the graft

• mediated by circulating antibodies speciﬁc for antigens on graft
endothelial cells

IMMUNE MECHANISM OF
GRAFT REJECTION

not common BUT major
barrier to xenotransplantation

• occurs within minutes of transplantation; characterized by
thrombosis of graft vessels and ischemic necrosis of the graft

• mediated by circulating antibodies speciﬁc for antigens on graft
endothelial cells

IMMUNE MECHANISM OF
GRAFT REJECTION

• occurs within days or weeks after transplantation

• principal cause of early graft failure

• mediated mainly y T-cells which react against alloantigens in the graft
or may react agains cells in graft vessels leading to vascular damage

IMMUNE MECHANISM OF
GRAFT REJECTION

antibodies may also
play a role

• occurs within days or weeks after transplantation

• principal cause of early graft failure

• mediated mainly y T-cells which react against alloantigens in the graft
or may react agains cells in graft vessels leading to vascular damage

IMMUNE MECHANISM OF
GRAFT REJECTION

• occurs over months or years that leads to progressive loss of graft function

• manifested as ﬁbrosis or narrowing of vessels (arteriosclerosis)

• T-cells secrete cytokines which stimulate proliferation of ﬁbroblasts and
vascular smooth muscle cells in the graft

PREVENTION & TREATMENT
OF GRAFT REJECTION

e-steroid.com


BONE MARROW
TRANSPLANTS
• elicit strong rejection
reactions: must perform cross-
matches

• carry the risk of graft-versus-
host disease: If mature allogeneic
T cells are transplanted with the
marrow cells, these mature T cells
can attack the recipient's tissues

• lead to temporary
immunodeﬁciency in
recipients

QUESTIONS?

elitechgroup.com

SERODIAGNOSIS
Reference; Jawetz Medical Microbiology
(http://accessmedicine.com/resourceTOC.aspx?resourceID=6)

ehrs.upenn.edu

BIOSAFETY

2008.igem.org
busytrade.com

OVERVIEW OF
DIAGNOSTICS
• 3 CATEGORIES:
• Direct: clinical specimen is examined directly for
the presence of virus particles, virus antigen or viral
nucleic acids

• Indirect: the specimen into cell culture, eggs or
animals in an attempt to grow the virus

• Serology: detection of rising titres of antibody
between acute and convalescent stages of infection,
or the detection of Ig

ANTIGEN DETECTION:
DIRECT
• Applications:

• immunofluorescence testing of
nasopharyngeal aspirates for
respiratory viruses e.g.. RSV, flu
A, flu B, and adenoviruses

• detection of rotavirus antigen in
feces

rotavirus, adenovirus, astroviruses, Norwalk-like viruses • the pp65 CMV antigenaemia test

• the detection of HSV and VZV in
skin scraping

ANTIGEN DETECTION:
DIRECT
geneaid.com
wclassproducts.com

norgenbiotek.com


ANTIGEN DETECTION:
INDIRECT


ANTIGEN DETECTION:
INDIRECT (enhanced)


ANTIBODY DETECTION:
SEROLOGY
http://virology-online.com

usada.org


COMMON TESTS
• Classical Techniques: • Newer Techniques:
• Complement ﬁxation tests • Radioimmunoassay (RIA)
(CFT)
• Enzyme linked
• Hemagglutination inhibition immunosorbent assay (EIA)
tests (HAI)
• Particle agglutination
• Immunoﬂuorescence
techniques (IF) • Western Blot (WB)
• Neutralization tests • Recombinant immunoblot
assay (RIBA)
• Single Radial Hemolysis

DIAGNOSING A PRIMARY
(ACUTE) INFECTION

• A signiﬁcant rise in titre of IgG/total
antibody between acute and convalescent
sera
• CFT and HAI, it is normally taken as a four-fold or
greater increase in titre
• diagnosis is usually retrospective because by the
time the convalescent serum is taken, the patient
had probably recovered


(ACUTE) INFECTION

• Presence of IgM
• EIA, RIA, and IF may be used (rapid)
• BUT:
• interference by rheumatoid factor,
• re-infection by the virus
• unexplained persistence of IgM years after the
primary infection

(ACUTE) INFECTION

• Seroconversion
• changing from a previously antibody negative state
to a positive state
• e.g. seroconversion against HIV following a needle-
stick injury, or against rubella following contact with
a known case


(ACUTE) INFECTION

• A single high titre of IgG (or total
antibody)
• this is a very unreliable means of serological
diagnosis since the cut-off is very difﬁcult to deﬁne


DIAGNOSING A RE-
INFECTION / RE-ACTIVATION

• It is often very difﬁcult to differentiate re-infection/re-
activation from a primary infection

• When important: rubella infection in the ﬁrst
trimester of pregnancy: primary infection is associated
with a high risk of fetal damage whereas re-infection is
not

• RE-INFECTION: sharp large rise in antibody titres

• RE-INFECTION/ RE-ACTIVATION: IgM is usually
low or absent

USE DEPENDS ON
PATHOGEN
• onset of clinical symptoms coincide with the
development of antibodies: e.g. rubella and hepatitis A
(IgM or rising titres of IgG in the serum of the patient would
indicate active disease)

• pathogen produce clinical disease before the
appearance of antibodies: e.g. respiratory and diarrheal
viruses (serological diagnosis would be retrospective and
therefore will not be that useful)

• pathogen produce clinical disease months or years
after seroconversion: e.g. HIV and rabies (the mere
presence of antibody is sufﬁcient to make a deﬁnitive
diagnosis)

READING ASSIGNMENT:

Q: WHAT DO YOU THINK
ARE SOME PROBLEMS
WITH SEROLOGY


medicineworld.org

THE SEROLOGICAL
immunology.utoronto.ca
DIAGNOSTICS


COMPLEMENT
FIXATION TEST
http://virology-online.com

path.cam.ac.uk

dshs.state.tx.us


HEMAGGLUTINATION/
HEMAGGLUTINATION INHIBITION
TESTS (HAI)
motifolio.com

virology.ws


HEMAGGLUTINATION/
HEMAGGLUTINATION INHIBITION
TESTS (HAI)

qmark2.leeds.ac.uk path.cam.ac.uk


IMMUNOFLUORESCENCE
TECHNIQUE (IF)

• DIRECT: known labeled antibody interacts
with unknown antigen
• INDIRECT: known antigen is attached to a
slide, unknown serum is added and then
washed = An/Ab ﬁxed onto slide and can be
detected by antibody-speciﬁc reagent


IMMUNOFLUORESCENCE
TECHNIQUE (IF)
Respiratory Syncytial Virus (RSV)

(a) Indirect technique: polyclonal rabbit
serum and a conjugated sheep anti-rabbit
serum; both were extensively absorbed to
remove unwanted antibodies

(b) Direct technique: mixture of three
monoclonal conjugated antibodies and Evans
blue counterstain (which, paradoxically, appears
red) = staining only those antigenic
concentrations for which there are antibodies
in the mixture

CREDITS: (Both micrographs were taken by
Joyce McQuillin personally, SCIENCE DIRECT)

NEUTRALIZATION
TESTS
• potential of the serum of
neutralizing the biological http://www.sanidadanimal.info

activity of an antigen can also
be known

• assessment of the capability
of a serum against bacterial
toxins, bacteria or viruses biobest.co.uk

• laborious, need cell cultures,
sterile conditions, and usually
require more time


NEUTRALIZATION
TESTS

Positive MDCC-MSB1 infected cells culture
showing ﬂuorescence at the point where CAV
antigen was detected

biobest.co.uk

culture negative to CA


SINGLE RADIAL
HEMOLYSIS

• E.g. Rubella antibody.

• LEFT PLATE: Control Plate

• RIGHT PLATE: well in the middle of the plate contains the 15 miu/ml control serum = clear
zone of lysis surrounding the well on the test plate which is absent on the control plate

• NOTE: Specimens which gives a zone of lysis equal or greater than the 15 miu/ml control
well are regarded as positive for rubella antibody. = Should a zone of similar size is present on
the control plate, then the result is not valid

RADIOIMMUNOASSAY
(RIA)
• used to quantitate antigens or haptens taht can
be radioactively-labeled
• based on competition for speciﬁc antibody
between the labeled (known) and the unlabeled
(unknown) concentration of the material
• complex that form between An/Ab can then be
separated and the amount of radioactivity
measured

RADIOIMMUNOASSAY
(RIA)
• concentration of the unlabeled (unknown)
antigen or hapten is determined by comparing
the results with those obtained using several
concentrations of a predetermined standard
antigen
• highly sensitive for:
• assay of hormones or drugs from serum
• measure amount of IgE with known allergen

users.rcn.com


RADIOALLERGOSORBENT
ASSAY (RAST)

lookfordiagnosis.com

pennstatehershey.adam.com


ENZYME
IMMUNOASSAY (EIA)
• depends on the conjugation of an enzyme to either
an antigen or an antibody
• enzyme is detected by assaying for enzyme activity
with its substrate
• no need for radioactive labels
piercenet.com


ELISA
technologyinscience.blogspot.com

• can detect the
ANTIGEN-
ANTIBODY reaction
by the use of enzyme-
linked antibodies

• based on the use of
labeled antibodies
(usually done with the
enzyme peroxidase) so
that the resulting
conjugates have both
immunological and
enzymatic activity

ELISA
• one of the components of the conjugate
(antibody or antigen) is attached to the plate, so
the antigen-antibody reaction can be easily
measured when adding the reaction substrate
• This substrate produces a colored reaction
product when it comes into contact with the
enzyme
• The color change can be seen or quantiﬁed with
a colorimeter

ELISA: ANTIGEN
DETECTION
• Sandwich ELISA

• plates are usually coated with an
antibody (monoclonal or polyclonal
antibody) against the unknown antigen

• sample that needs to be tested is added
to the wells, and if the antigen is
present, the antigen-antibody reaction
will take place

• add another antibody linked to an
enzyme.

• when the reaction substrate is added it
turns a color

ELISA: ANTIBODY
DETECTION
• INDIRECT ELISA: coating of
the ELISA plate with the antigen
against the speciﬁc antibodies
that may be present in the serum

• NEXT: addition of serum,
incubation and washing; addition
of the conjugate, incubation and
washing and ﬁnally, the addition of
the substrate, stopping the
reaction and reading the results


ELISA: ANTIBODY
DETECTION
• COMPETITIVE ELISA: have an
antibody (monoclonal of polyclonal) of
a known antigen which has previously
been bound to the plate

• competitive because the serum is
incubated with the antigen previous to
its incubation with the antiserum
bound to the plate = both compete for
the antigen

• NEXT: addition of the conjugate,
incubation, washing, and ﬁnally,
substrate addition and reading the
results

ELISA & HIV

virology-online.com

hivinfosource.org

DENGUE
SEROLOGY


END OF EXAM 2
COVERAGE

Bio 151 lectures for examination 2

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