BASIC SCIENCE RESEARCH
Lecture 1: Accuracy in Diagnosing Cancers
A. CANCER DEVELOPMENT
 Multi-step process
 Unique form of genetic disease
 Neoplastic transformation of cells
 Tumor cells grow through a process of clonal expansion driven
by mutation:
o 1st mutation leads to limited expansion of progeny of a single
cell
o Each subsequent mutation gives rise to a new clonal
outgrowth with greater proliferative potential
 It is the accumulation of multiple genetic alterations in affected
cells, and not necessarily the order in which these changes
accumulate, that determines tumor formation and progression
B. CANCER: DISEASE OF ABNORMAL GENE EXPRESSION
Somatic mutations in developing cancers
 Alter gene expression patterns:
o Significant change to cellular physiology
o Abnormal regulation of cell proliferation
o Acquisition of invasive behaviors
Gene expression profiling
 Differential diagnosis
o Study gene expression in normal tissue vs. tumor tissue
 Prognostication
 Prediction of responses to therapy
o Helps determine if cancer will disappear or become resistant
C. MUTATION-DRIVEN TUMORIGENESIS
 Mutation: ultimate source of variability for individual cells and
essential component of the process of natural selection
 Multiple somatic mutations contribute to the stepwise process
of neoplastic transformation and tumorigenesis  karyotypic
(chromosomal) alterations and abnormal chromosome numbers
(additional or lessening number of chromosomes)
 Tumorigenesis: process of natural selection to which cells
develop a growth advantage that allows them to proliferate and
invade under conditions where normal cells cannot 
acquisition of this ability is driven by mutation; form of somatic
evolution
D. COMPLEX DISEASE
 The ability of malignant cells to proliferate and metastasize is
the result of genetic aberrations
o Activation of proto-oncogenes
o Inactivation of tumor suppressor genes (TSRs)
o Inactivation of DNA repair mechanisms
 However, cancer‟s genetic origins do not account for its
molecular complexity
o Epigenetic modulation of mRNA expression
 i.e. silencing
E. CARCINOGENESIS
 Multiple hits and multiple factors
Knudson proposed that carcinogenesis requires 2 hits
 1st event – initiation
 Carcinogen = initiator
 2nd event – promotion
 Agent = promoter
Multiple hits occur – 5 or more
 Each hit produces a change in the genome which is transmitted
to its progeny (i.e. clone)
 Lag period: time between exposure (first hit) and development
of clinically apparent cancer
o Altered cell shows no abnormality during lag period
 6-8 mutations may be necessary for progression to an invasive
tumor
F. GENETIC CHANGES IN MALIGNANT TRANSFORMATION
PROCESS
 Deletions, rearrangements, and mutations that lead to either
inactivation or activation of specific target genes
BASIC SCIENCE RESEARCH | Block 2
BLOCK 2
o Oncogenes: activated / deactivated genes whose products
normally promote cell growth (peptide GFs, peptide GFRs,
signal transduction factors, tyr kinase, transcription factors)
Oncogenic Mutation Mechanism s
 Deletion or point mutation in coding sequence
 Gene Amplification
 Chromosomal rearrangement
o Ex. Tyr kinase  gene fusions  formation of aberrant
protein with aberrant function
 Instead of being inducible it becomes constitutive
G. MUTATIONS IN CANCER
Chromosomal abnormalities
 Aneuploidy (gain or loss of whole chromosomes)
 Chromosomal rearrangements resulting from DNA strand
breakage (translocations  hybrid/chimera, inversions and
other rearrangements)
 Gain or loss of portions of chromosomes (amplifications, large -
scale deletions  copy number variants)
Nucleotide sequence abnormalities
 Single nucleotide changes (missense, nonsense)
 Small insertions or deletions i.e. indels (frameshift muations)
H. CAUSE OR CONSEQUENCE
 Intrinsic mutation rate is insufficient to account for numerous
changes in cancer cells  early event in neoplastic
transformation is development of hypermutability or genetic
instability
o Exposure to exogenous mutagenic agents
o Spontaneous mutational mechanisms
 Diminished capacities for surveillance and repair of DNA
lesions, leading to increased rates of spontaneous mutation
and/or increased susceptibility to mutation following exposure
to some exogenous carcinogenic agent
I. “MUTATOR PHENOTYPE”
 Accumulate mutations more rapidly than normal cells
 Rate of gene amplification in malignant cells is much higher
than in normal cells
J. TNM (Tumor, Node and Metastasis) STAGING
 Standardized anatomical basis: tumour size or depth, lymph
node spread, metastasis
BASIS FOR
 Prediction of survival
 Choice of initial treatment
 Stratification of patients in clinical trials
 Uniform resulting of end result of cancer management
K. IMPACT OF USE OF BIOMARKERS
 Important in personalized/precision medicine
 Can determine which biomarker can respond to a particular
target therapy
 Before diagnosis, markers might be used for risk assessment
and screening. At diagnosis, markers can assist with staging,
grading and selection of initial therapy. Later, they can be used
to monitor therapy, select additional therapy, or monitor for
recurrent disease.
L. SOME BIOMARKERS UNDER STUDY
 Tumour specific antigens in hepatocellular carcinoma (CA)
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 MHC Class I antigen pathway defects in colorectal CA B. GERMLINE MUTATIONS
 Tumour suppressor gene mutations in breast CA  2 hit hypothesis:
 DNA methylation markers in leukemias, breast, cervical, o Germ line mutation on one allele and an acquired mutation
hepatic and GI CAs on the second allele leads to the full blown phenotype
 Serum tumour markers in pancreatic cancers o In sporadic cases, same genes can be mutated e.g. APC or
mismatch repair genes, but both allelic mutations are
M. DNA-BASED BIOMARKERS acquired
 SNPs (single-nucleotide polymorphisms)
 Chromosomal aberrations C. FAMILIAL ADENOMATOUS POLYPOSIS (FAP)
 Copy number variations  <1% of CRC (colorectal CA)
 Microsatellite instability  15 y.o. – average age 1 st polyp
 Promoter region methylation  CRC average age 39.7% CA < age 21
 Circulating DNA or tumour cells  90% CRC by age 45
 Oncogenes, tumour suppressor genes, mismatch repair genes  Conduct screening early on
 Histone deacetylation, histone methylation, promoter region
methylation  gene silencing Familial Adenomatous Polyposis Genetics
 Mitochondrial DNA  APC gene: chr 5q (patients have mutation in this gene)
encodes protein (2843 aa)
 Oncoviral markers
 Most families have separate, distinct mutations – STOP codons
N. RNA-BASED BIOMARKERS  Substitution errors not sufficient for phenotype
 Overexpressed or under-expressed transcripts (mRNA)
Attenuated FAP
 Regulatory RNAs (i.e., miRNA)
 Microarrays  APC mutations in 5‟ end, 3‟ end or in exon 9
 < 50 – 100 polyps, onset 2 decades later (mid 30‟s)
 Quantitative real-time PCR
 Polyps cluster in right colon (usual location)
 Bioinformatics and biostatistics
 Malignancy ~ 1- 20 years after FAP
O. PROTEIN BIOMARKERS
D. HEREDITARY NONPOLYPOSIS COLI (HNPCC)
 Cell surface receptors
 Tumour antigens (i.e. PSA)  Most common form of hereditary colon cancer susceptibility; 5
– 8% of all colon cancers
 Phosphorylation states
 Autosomal dominant
 Circulating tumour peptides (serum, urine, sputum, nipple
aspirates, other body fluids)  Difficult to diagnose
 Lack of clear phenotypic characteristics
 Functional proteomics
o Phosphorylation  Early onset of colorectal CA
o Glycosylation  Presence of extra colonic tumor
o Cellular location  Due to germ line mutation in mismatch repair genes
o Tissue location  Endometrial, ovarian, other cancers

P. LASER CAPTURE MICRODISSECTION Amsterdam II Criteria

 Used to obtain DNA, mRNA or protein from precise locations
within a tumour which allows differentiation of markers in
malignant cells from that in other cell types
Q. IMAGING BIOMARKERS
 Functional molecular imaging modalities:
o MRI, PET, Optical imaging
 At least 3 relatives in 2 successive generations have been
diagnosed w/ cancer assoc. with HNPCC (colon, endometrium,
small bowel, ureter or renal pelvis)
 One is a first degree-relative of the other two;
 at least one of these relatives must have been diagnosed with
cancer before age 50
 FAP is excluded as the cause of colon cancer in the family

II. COLORECTAL CANCER (CRC/COLORECTAL CA) Molecular Genetics of HNPCC

A. Types of Colon Cancer
 Sporadic (80% - 90% of cases)
 Hereditary or Familial (10 – 20%)
o Familial adenomatous polyposis coli (APC)
o Hereditary non-polyposis coli (HNPCC)
 Polyps may be benign or may become malignant
Risk Early
Assessment Detection
 6 MMR genes: hMSH2, hMLH1, h MSH6, hPMS1, hPMS2,
hLH3
 Microsatellite instability (MSI) pathway
E. MICROSATELLITE INSTABILITY IN SPORADIC
COLORECTAL CANCER
 10 – 15% of sporadic CRC
 In HNPCC: Germ line+ somatic =MSI
Prognosis &  Sporadic- bi-allelic somatic mutation via methylation of MLH1
Treatment promoter

Smad4/ PIK3CA III. MICROSATELLITE INSTABILITY (MSI): “HALLMARK OF

APC/Beta- K- P53
TGF- PTEN HNPPC TUMORS”
catenin Ras/ /Bax
B- betaRII
Raf  Microsatellites: repetitive sequences consisting of variable
PRL-3 numbers of repeated units of one to four (or more) nucleotides:
numerous and randomly distributed throughout the human
genome
 MSI: alterations in simple repeated sequences, including both
expansions (insertions) and contractions (deletions), typically
Small Large resulting in frame shift mutations ; Caused by mutations in
Adenoma
Normal Adenoma Cancer Mets mismatch repair (MMR) genes

A. MISMATCH REPAIR PROTEINS

 Proteins involved w/ MMR operate in concert to recognize
mispaired or unpaired nucleotides and facilitate their removal
Genetic and repair (hMSH2, hMSH3, hMSH6,/ GTBP, hMLH1, hPMS2.
Instability hMLH3)
Cancer Progression and Biom arkers

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 B. MISMATCH REPAIR DEFECTS LEADS TO MSI CML T(9;22) BCR/ABL 1(b2a2)
 Strand slippage of the primer at a repetitive sequence BCR/ABL 1(b3a2)
generates a misaligned intermediate that is stabilized by correct
base pairing between discrete repeat units on the misaligned ALL T(9;22) BCR/ABL 1(e1a2)
strand T(1;19) E2A/PBX1 (e13/e2)
 Normally repaired through proofreading function of polymerase T(12;21) TEL/AML1 (e5/e2)
complex, or by post-replication repair mechanisms T(4:11) MLL/AF4 (e20/e4)
 If the intermediate is not repaired, subsequent rounds of MLL/AF4
replication will generate insertion or deletion mutations in th e (e10/e4)
newly synthesized DNA strands. APL T(15:17) PML/RARa(Lform)
 Mismatch repair genes correct these “slippages”  once PML/RARa(Sform)
altered  these genes cannot repair it  b ecomes AML INV 16 CBFB/MYH11 (A
microsatellite instability T(8:21) type)
CBFB/MYH 11
MMR genes (Dtype)
hMSH2 hMLH1 AML1/ETO (e5/e12
 Chrom 2p16  Chrom 3p23 Gene Fusion Markers in Leukem ia
 16 exons  19exons
 Protein (MutS) recognize base  Protein (MutL): catalyzes the E. BCR-ARL GENE FUSION FOR CML
mismatch (exon 12-14) dow nstream of MMR
 This transformation to CML is caused by a reciprocal
 Promotes binding of MutS for
repair to proceed translocation of the BCR gene on chromosome 22 (at 22q11)
Mism atch Repair (MMR) Genes and the ABL gene on chromosome 9 (at 9q34), resulting in a
fused BCR-ABL gene dubbed the “Philadelphia chromosome”.
IV. Use of Biomarkers in Companion Diagnostics The protein that results from the fused genes promotes
 Companion diagnostics are molecular diagnostics that transition to the malignant state, increasing proliferation and
accompany therapy decreasing apoptosis of the malignant cell
 It identifies if target is in patient (i.e. EGFR mutation)
F.STATE OF THE ART LEUKEMIA DIAGNOSIS
 Monitors response to treatment
Hematologic Cytogenetic Molecular
 Commonly used to treat CRC patients
Response Response Response
 Anti-EGFR therapies are commonly used in treating patients w/
Frequency Every 2 Every 6 Every 3
advanced colon cancer. These therapies heavily rely on
weeks until a months until a months
blocking the EGFR signalling pathway
complete complete
response has response has
A. EGFR-TARGETED THERAPY been been
 Monoclonal antibodies approved to treat mCRC: achieved and achieved and
 Avastin (bevacizumab) blocks the growth of blood vessels to confirmed confirmed
the tumour
 Erbitux (cetu ximab) and Vectibix (panitumumab) blocks the Every 3 Every 12
effect of hormone-like factors that promotes cancer cell growth months months
unless
B. BIOMARKERS FOUND IN EGFR PATHWAY otherwise
EGFR Is a receptor TK which is expressed in all colon required
cancer carcinomas. Methods Complete RQ-PCR
EGFR Patients with highly responsive NSCLC tumors are Blood Count
mutational found to contain somatic mutations in EGFR TK (CBC) with
analysis domain. The presence of the somatic EGFR is differential
by PCPR mutation is significantly associated with response Diagnostics (Hematologic response  complete blood count w/
to gefitinib and erfotinib, and strongly predictive of differential)
prolonged survival in NSCLC patients
KRAS Mutations are present in greater than 45% of G.CML (Chronic Myelogenous Leukemia) THERAPY
colorectal adenomas and carcinomas and the vast  Tyrosine kinase inhibitors (TKIs) imatinib (Gleevec), dasatinib
majority occur in codon 12 and 13 of the (Srycel), and nicotinib (Tasigna) inhibit activity of the BCR-ABL
oncogene. fusion protein, resulting in both hematologic response as well
BRAF This protein plays a role in in regulating MAP as cytogenetic response.
kinase/ EFKs signalling pathways which affects cell  Chronic myelogenous leukemia: biphasic disease
division, differentiation and secretion o Chronic phase (assoc. w/ t(9:22) translocation)
PI3K Acti vation can result in an increase in cell o Blast crisis (fatal for 85% of patients)
proliferation and survival. Targeting it may enhance  Allogeneic bone marrow transplant was only curative treatment
the effects of chemotherapeutics agents and available but is only applicable to a minority of patients (15-
provide novel adjuvant treatment for selected colon 20%)
cancer  A recent advance in treatment has been the introduction of the
PTEN Is a natural inhibitor of PI3K. Loss of function is BCR-ABL inhibitor ST1571 (gleevec)
seen in about 30 % of all colorectal cancer
Biom arkers in EGFR pathw ay (All found in colorectal cancer) GLIVEC (GLEEVEC)
 New drug indicated for the treatment of CML patients after
C. K-ras testing is Essential Prior to Anti-EGFR Therapy failure of interferon-a therapy
 The anti-EGFR antibodies show activity in multiple tumour
 Designed to specifically target Ph + cells
types. However, responses are seen only in the subset of
 Administer orally once daily
patients lacking a K-ras mutation (wild-type K-ras)
 Directly binds to the BCR-ABL protein target
 Recent data strongly suggest the evaluation of downstream
o Rational design: STI-571 bound to ABL (PDB: 1IEP)
markers, such as K-ras, are important in selecting which patient
will respond to therapy
 Patients w/ mutations in the K-ras oncogene are less likely to
respond to anti-EGFR therapies

D. DIAGNOSING LEUKEMIA
 Blood smear analysis
 Karyotyping – analysis of Chromosomes from leukemic cells
Fushion transcripts detected by signature LTx v2.0 kit
Clasification Translocation Fusion transcripts

BASIC SCIENCE RESEARCH | Block 2 Page 3 of 9

Lecture 2: Biomarkers in Heart Disease and Accuracy
in Diagnosing Cancers
I. METABOLIC IDENTIFICATION OF NOVEL BIOMARKERS
OF MYOCARDIAL ISCHEMIA
A. Overview of the Problem
- for identification and profiling of biomarkers and pathways
activated in myocardial ischemia.
- for diagnosis of Coronary Artery Disease
B. Background of the Study
- Currently, myocardial ischemia is diagnosed through:
1. History consistent with typical angina pectoris
2. Labile ECG ST- segment and T-wave changes
(spontaneously or on provocation with exercise testing)
- Techniques with wide application in diagnostic biochemical
profiling:
1. Nuclear magnetic resonance (NMR) spectroscopy
2. Mass spectrometry
3. Liquid chromatography
- Limitations:
1. High degree of inter-individual variability
2.Unpredictability of the onset of an acute coronary syndrome
C. Methods
1. Liquid Chromatography Coupled with High Sensitivity
Electrospray Mass Spectrometry
- metabolic profiling technology used for the blood samples
from the patients undergoing exercise stress testing
- approach is used and considered powerful because serial
sampling can be performed in patients before and after a
controlled ischemic insult -> allowing each patient to serve
as his or her own biological control.
2. Subjects:
- ELIGIBLE: all patients undergoing stress testing for possible
myocardial ischemia.
- EXCLUDED: patients who were undergoing
pharmacological testing.
- The cases and controls were determined using a stress-
imaging protocol which made use of single-photon computed
tomography myocardial perfusion imaging.
3. Study Protocol
- Symptoms, heart rate, BP, and a 12-lead EG were recorded
before the test, midway through each stage, and during
recovery.
- If the patient developed angina during the test, the timing,
quality, and effect on the test were noted.
- The maximal horizontal/downsloping ST segment changes
were noted.
4. HPLC and Mass Spectrometry Analysis
- Blood samples were taken immediately before, immediately
after, and 4 hours after the stress testing, and were
processed within 60 minutes since the proteins in the blood
denature quickly. Plasma was kept at -80 C, and aliquots
were drawn and thawed.
- The samples were subjected to different kinds of
chromatography in order to isolate certain metabolites that
they want to examine.
D. Conclusion
- The study has shown that the current technology is adequate
for metabolic marker identification.
- Metabolites, when taken as part of chemical pathways rather
than single entities, further enhances the understanding of
exercise performance and myocardial ischemia.
- Ad vancements on metabolomics aid the diagnosis of
myocardial ischemia.
- Lactic acid is not a reliable metabolite to diagnosis Myocardial
Ischemia since it is increased in both cases and controls.
- Emphasis were given on GABA and citric acid because they
showed decreased levels among the cases while relatively
unchanged in controls.
II. ACCURACY IN DIAGNOSING CANCER: EML4-ALK
MUTATIONS IN LUNG CANCER THAT CONFERS
RESISTANCE TO ALK INHIBITORS
A. Background
- Lung cancer is one of the leading causes of cancer-related
deaths in most countries.
- 2 Types of Lung Cancer
BASIC SCIENCE RESEARCH | Block 2
1. Non-Small Cell Lung Cancer: 85 -90%
Three sub-types:
a. Adenocarcinoma- slow-growing and usually discovered in
the outer part of the lung
b. Squamous cell carcinoma- in the center of the lung
c. Large cell carcinoma- appears anywhere in the lung;
grows more rapidly and spreads.
2. Small Cell Lung Cancer
• Small-sized cancer cells that starts near the center of the
chest in the bronchi.
• fast-growing form
• tends to spread in its early stages
• Common seen in smokers
- Anaplastic lymphoma kinase (ALK)
- one of the potent oncogenes in non-small cell lung cancer
(NSCLC)
- It fuses with several partners, including EML4 (Echinoderm
microtub ule-associated protein-like 4), resulting in a potent
transforming activity in NSCLC.
- EML4-ALK Positive Lung Cancer
- refers to a primary malignant lung tumor whose cells contain
an abnormal DNA where the EML4 gene and ALK gene fuse
- Most lung carcinomas containing EML4-ALK gene fusion are
adenocarcinomas.
B. Case Report
- 28-year-old man, no smoking history
- April 2008: Diagnosed with Lung Adenocarcinoma
- treated with Crizotinib, an orally available small-molecule
inhibitor of ALK tyrosine kinase.
C. Methods
1. Molecular Analysis
2. Deep Sequencing
3. High-throughput sequencer (Genome Analyzer II, Illumina)
D. Results and Discussion
- Based on the case report, there is tumor progression despite
sustained administration of ALK inhibitor (Crizotinib).
- Two de novo mutations within the kinase domain of EML4-ALK
form the tumor of the patient that confer resistance to multiple
ALK inhibitors.
- NO EML4-ALK cDNA had both mutations, thus, each mutation
developed independently in distinct subclones of the tumor.
- Amino acid substitutions at the gatekeeper position of several
tyrosine kinases have been detected in tumors treated with
tyrosine kinase inhibitors.
- It is therefore likely that gatekeeper alterations constitute a
universal mechanism for the acquisition of tyrosine kinase–
inhibitor resistance in oncogenic tyrosine kinases.
- In contrast to gatekeeper substitutions, activating mutations at
the position adjacent, on the N-terminal side, to the αC helix
(e.g., C1156 in ALK) have not been confirmed for other tyrosine
kinases in cancer specimens.
E. Conclusion
- EML4 (echinoderm microtubule-associated protein-like 4) – ALK
(anaplastic lymphoma kinase) fusion-type tyrosine kinase is an
oncoprotein found in 4 to 5% of non–small-cell lung cancers.
- It was determined that the resistance to inhibitors are due to
the nucleotide changes both 4374 G→A and 4493 C→A which
was Confirmed by using Sanger sequencing.
- The substitutions result in cysteine→tyrosine (C→Y) and
leucine→methionine (L→M) changes at the positions
corresponding to amino acids 1156 and 1196, respectively, of
wild-type human ALK.
- Crizotinib inhibited the growth of BA/F3 cells expressing primary
EML4-ALK, in a concentration-dependent manner
- Exposure to Crizotinib:
 Inhibited tyrosine phosphorylation of EML4-ALK
 Not substantially affect C1156Y and L119M mutants
- Exposure to PDD (pyrimidinediamine derivative):
 Inhibited the tyrosine phosphorylation of EML4-ALK, in a
concentration-dependent manner lesser effect on the mutants.
- EML4-ALK C1156Y mutant cells were slightly more resistant to
PDD than were those expressing the L1196M mutant form.
- A/F3 cells with secondary mutations were markedly less
sensitive to PDD than were those expressing the primary EML4 -
ALK.
Page 4 of 9
- These mutations develop during clinical treatment with
Crizotinib but their generation probably renders EML4- ALK
resistant not only to Crizotinib but also to other ALK inhibitors.
Lecture 3: Vaccines
I. PASSIVE IMMUNIZATION
 Immunoglobulins are concentrated antibody preparations
(given IM or IV) which provide immediate short-term
protection against disease
 Given to individuals who are at high risk of experiencing
severe disease or of developijng serious complications from
the disease
 They provide immediate protection but this is short lasting
(few weeks or months)
 They do not stimulate the immune system to produce any
antibodies
II. ACTIVE IMMUNIZATION
A. Traditional Vaccines
 Key principle: Pattern Recognition (antigens found on
the surface of viruses are patterns recognized by
antibodies)
 Drawbacks: inability to grow enough agent, safety
concerns, reversion of attenuated strains, incomplete
inactivation (higher chances of it causing the disease), shelf
life may require refrigeration
1. Live Vaccines- Attenuated (weakend) strains which
replicate in the host
 Advantages: single dose often sufficient, strong
immune response, local and systemic immunity
produced
 Disadvantages: potential to revert to virulence,
contraindicated in immunosuppressed patients, poor
stability
2. Inactivated Vaccines - either suspensions of whole intact
killed organisms or acellular and subunit vaccines
 Advantages: stable, constituents clearly defined,
unable to cause infection
 Disadvantages: need several doeses, local reactions
common, adjuvant needed, shorter lasting immunity
B. Recombinant DNA Vaccines
 Genes or portions of genes encoding major antigenic
determinants can be cloned in expression vectors and large
amounts of the product purified and used as a subunit or
peptide vaccine, respectively.
 Advantages: unable to cause infection, safe to produce,
lasting immunity
 Disadvantages: requires booster shots, high cost of R&D,
requires cold storage
 Example: A subunit vaccine against HSV
 Vectors- carriers used to carry the gene of interest of the
virus
genes that encode the protein envelope of the dengue
virus.
D. Peptide Vaccines
 Peptide vaccines incorporate one or more short long
amino acid sequences as viral antigens
 It does not require the transfection of vectors encoding
genes
E. DNA vaccines
 A DNA sequence used as a vaccine. This DNA sequence
code for antigenic protein of pathogen. As this DNA
inserted into cells it is translated to form antigenic protein.
As this protein is foreign to cells, so immune response
raised against this protein. In this way, DN A vaccine
provide immunity against that pathogen.
 This type of vaccine lets the patient‟s cells produce the
proteins.
 Methods of delivery: syringe delivery, gene gun delivery
 Ad vantages: avoid the risk of using actual infectious
organism, refrigiration is not required, use only the DNA
from infectious organisms
 Disadvantages: antigen may not be properly processed,
integration into host genome may not be efficient, over
expression, extended expression may lead to chronic
inflammation
III. Immunotherapy
 Treatment for individual who has been already infected with a
chronic viral infection
 The virus has evaded immune surveillance
 Priming the immune system to target the pathogen
 Involves isolating antigen presenting cells eg. Dendritic cells
 Loading the cells with recombinant viral antigen
 Return the activated dendritic cells to the patient ie.
Vaccination – educate the dendritic cells
Lecture 4: Viruses
I. VIRUS
A. Definition
 Organized associations of macromolecules. Consist of:
o Nucleic acid
 Blueprint for progeny virions: Infectious virus particles
 Viruses are non-living; do not have the machinery for
reproduction, replication, energy production
o Protein - end product of translation
 More than 60% of diseases in the tropics are caused by viruses
B. Classes of Viruses (Genome)
1. DNA Viruses

Parts of a vector Description

Replication origin (ori) Generate multiple copies of
plasmid
Cloning site Region of insertion of
foreign DNA
Promoter (SV40) Recognized by mammalian
cells and will be responsible
for producing the protein Central Dogma
coded by the gene
Antibiotic resistance Verification of transfection  DNA is the genetic material; can either be :
marker (kanr/neor) of plasmid into the cell o ssDNA – single stranded
o dsDNA - double stranded
C. ChimeriVax Technology  Some viruses follow the central dogma in this way
 The technology has a yellow-fever backbone. They  Some viruses have their own (viral) DNA polymerases to use
eliminated the genes that encode for the protein envelope  Some viruses use the host (cellular) DNA polymerases to
replicate their material
of the yellow fever virus and exchanged that with the
 Most eukaryotic DN A viruses replicate in the nucleus EXCEPT
for the Poxvirus  which replicates in the cytoplasm

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2. RNA Viruses  Nature of the receptors utilized by a virus determines in part
 RNA is the genetic material; can either be: its host range and tissue tropism
o ssRNA – single stranded (can be + or – strand)
 Yellow fever virus and Polio virus: (+) ssRNA Virus Influenza Virus Attachment to Cells
o Can immediately synthesize (translated) viral proteins Structure
 Measles virus and Rabies virus: (-) ssRNA o Structural Proteins on its surface
o When it enters the cell  must first create a (+) RNA o i.e. Hemagglutinin, neuraminidase, M2 ion channel
strand template (complementary RNA strand) before it can o Hemagglutinin protein (H types)  Most prominent
make proteins  is used to attach to influenza receptors on cell surface
o dsRNA- double stranded; i.e. Rotavirus o When this influenza virus infects swine  these proteins can
 Arena virus: some of the genera belonging to the family such recombine and form different viral strains  pandemic strain
as Bunyaviridae are (+/-) ssRNA  Main receptor for influenza: SIALIC ACID
o α(2,6) preferentially used by human strains; α(2,3) by avian
FAMILY VIRUS HOST
Picornaviridae Poliovirus, human Cattle monkeys and B. FUSION
rhinovirus (common eyes  Viral „fusion proteins‟ facilitate entry of virus to the host
cold), Hep A virus
 Ligand-triggered, conformational change in the fusion protein
Caliciviridae Norw alk virus Vertebrae hosts is coupled to apposition and merger of the two bilayers
(Rabbits, sw ine, cats)
Flaviviridae DENGUE, yellow fever, Sw ine, cattle, 1. Class I Fusion Proteins
JE, MVE, TBE, Hepatitis prim ates and birds,  Perpendicular to membrane – spikes  form trimers; mostly
C som e cases alpha-helical
hum ans  Hair Pinning: Fusion protein attracts other fusion proteins that
Viruses and their Hosts
will attach  fusion proteins then pull the membrane of the host
cell closer to the virus  fusion can occur  pore formation 
3. RNA (RETROVIRUSES) viral genome can then enter the host cell (All this happens in an
 Retroviruses (HIV) endosome)
o RNA  reverse transcriptase  DNA Intermediate  RNA 
viral proteins
2. Class II Fusion Proteins
 Hepadnaviruses (Hepatitis B)
 Mostly beta-sheet  Form dimers; Parallel to membrane
o DNA  RNA Intermediate  reverse transcriptase 
DNA 3. Class III Fusion Proteins
 Perpendicular to the membrane  Form trimers; Mi x of alpha-
II. INFECTION helices and Beta-sheets
A. Initiation of Infection: Collision of Virions and Cells
 Virions are inanimate: no structure for locomotion
 Driven by Brownian motion, laws of diffusion, electrostatic Lecture 5: Zika Virus Reports
interaction I. Rapid Development of a DNA Vaccine for Zika Virus
 Require a SUSCEPTIBLE HOST CELL A. Introduction: Gene-based Vaccine Delivery
 Similar to DNA-based WNV (West Nile Virus) vaccine
B. Finding the "right" cell
 Advantages of DNA Vaccines:
 Step 1: adhere to cell surface (electrostatic interaction)
➢ Rapidly tests multiple candidate antigen designs
 Step 2: Attach to specific receptor molecules on the cell surface ➢ Rapidly produces GMP material
o More than one receptor may be involved
➢ Established safety profile
o Infection will only occur if there is a specific receptor
➢ Straight forward regulatory pathway into clinical
 Step 3: Transfer genome inside the cell evaluation
C. Cellular receptors for Viruses B. Methodology
 Essential for all viruses EXCEPT those of yeasts (no 1. Antigen Design
extracellular phases) and plants (enter cells by mechanical  Goal: To identify constructs that produced particles that
damage) capture the antigenic complexity of infectious virions
 Receptors and co-receptors  Guided by prior knowledge of humoral imm unity to
 1985: one receptor identified: sialic acid for influenza virus flaviviruses
 Different Viruses can bind to the same receptor and Viruses of  Vaccine-elicited neutralizing antibodies (NAb) are
the Same Family may bind to different receptors associated with protection from flavivirus -mediated
disease
III. REPLICATION CYCLE OF VIRUSES  Expression of prM and E protein are sufficient for production
and release of subviral particles (SVP)
A. VIRUS ATTACHMENT
 Has antigenic and functional properties similar to those in
infectious virions

2. Identifying Vaccine Candidates

 prM-E constructs were synthesized and screened for
expression and efficiency of particle release from transfected
cells
 Construct was selected from a French Polynesian isolate
identical to strains in the Americas
 prM-E sequences were inserted into a CMV-immediate
Viral attachm ent early promoter-containing vector (VRC8400)
 prM: plays a critical role in folding of E protein and release
o Accessory Receptor of particles from cells
 Bind w/ low affinity  ZIKV exists as a single serotype
 Not required for virus entry but may accelerate rate of  Suggests a single vaccine antigen will provide protection
binding and uptake of virus against all ZIKV strains
o High Affinity Receptor binding  Improving expression
 Required for virus entry  VRC5283 construct: ZIKV prM signal sequence was
 Cells failing to express the appropriate receptor cannot be exchanged with the analogous region of Japanese
infected by the virus encephalitis virus (JEV) (See Figure 1A)

BASIC SCIENCE RESEARCH | Block 2 Page 6 of 9

  VRC5288 construct: Second chimeric ZIKV/JEV prm -E
construct was designed
 Also encodes JEV signal sequence
 Comprised the stem and transmembrane regions
 Both vectors exhibited expression by mammalian cells
with more efficient SVP release into supernatants (See
Figure 1B and 1C)
 Electron microscopic analysis of negative‑stained
purified VRC5288 SVP preparations revealed roughly
spherical particles consistent with the appearance of
other flavivirus SVPs (See Fig. 1D)
3. Assessing Immunogenicity (in Rhesus macaques)
 Mice were immunized intramuscularly once with 50 μg of
DNA
 Used electroporation
 Serum was evaluated for binding to ZIKV SVPs and
neutralizing activity
 Vaccination elicited ZIKV-specific NAbs after single
immunization
 Evaluated after delivering vaccine intramuscularly
 Groups received 2 doses of vaccine at 0 and 4 weeks while
another group received 1 dose at 0 weeks
 After single dose of DNA, binding and neutralizing activity
already present
 Week 2: detectable
 Week 3: peak
 All ZIKV vaccine groups had higher NAb responses than
macaques receiving control vector
 Macaques with single dose had lower NAb titers than
those that received two doses
 Data indicates that both constructs elicit substantial ZIKV-
specific NAb in macaques
 8 weeks after 1 st immunization
 Animals challenged subcutaneously
 Blood collected daily
C. Results
 Control animals peak virus load on day 3 or 4
 Animals receiving 2 doses - largely protected from viremia
 1 animal, which received two 4mg doses of VRC5288, a
low-level positive PCR in one of the two assays
performed on day 3 and another positive blip on day 7
 All 6 animals that received single dose (1 mg of VRC5288)
were viremic with peak VL on day 3
 VL was significantly reduced compared to animals w/ two
doses of VRC8400 (control)
*cut off values is at <100 genome copies/mL (low level
viremia may have occurred in other animals)
 17/18 w/ two doses of vaccine - no detectable viremia
 Probability Analysis
 We can anticipate a 70% protection from viremia if a
reciprocal EC serum NAb titer of 1000 is achieved in the
RVP assay
 Corresponds roughly to a reciprocal EC MN titer of ~100
 Animals receiving a single dose of 1 mg VRC5288 had
prechallenge reciprocal EC NAb titers measured by the R VP
assay between 203 and 417
 There were two animals which had the highest NAb activity
 MN titers at the 6-week timepoint were <10 in the 1 mg
single dose group animals that uniformly had breakthrough
infection
 The larger dynamic range of the RVP assay will allow a
more precise definition of the protective threshold needed to
prevent viremia in a particular model or against a particular
challenge inoculum
 There was a pressing concern raised about vaccination
against flavi viruses that there is the possibility of enhanced
disease if there is incomplete or waning immunity
 1 mg single-dose group that received VRC5288
 Low, sub-protective levels of NAb that resulted in
breakthrough infections
 Reduced levels of viremia compared to unvaccinated
controls
 No visible signs of illness or enhancement of replication
 1 animal in the mock-immunized control group and one in
the single-dose 1 mg VRC5288 group with detectable levels
of ZIKV antibody binding, but no neutralizing activity
 Had preexisting WNV-specific Nabs
BASIC SCIENCE RESEARCH | Block 2
 Levels of virus replication were near the group average
 There was no evidence of diseas e enhancement for those
who had flavivirus exposure prior.
D. Conclusion
 Vaccine development for ZIKV must be specific and guided
by an e xpanded understanding of ZIKV virology,
pathogenesis, immunity, and transmission
 Strategic, matching technical and manufacturing feasibility
with the target populations that will benefit most from
vaccination
 It should be staged (“Why? and what do we mean by this?”)
 A rapid response to the global health emergency may
require a different vaccine approach than the longer term
goal of achieving durable immunity in the general
population
E. Associated Studies
1.) Davis et al. (2001)
...suggested the development of WN Virus Vaccine using
recombinant DNA technology for the construction of
recombinant plasmid (pCBWN) that expresses the WN virus
premembrane(prM) and envelope(E) proteins. These proteins
could also serve as noninfectious recombinant antigens
(NRAs) for diagnosis of WN virus infection.
a. Methodology
1. Recombinant plasmid pCBWN DNA was constructed
using extraction, amplification (with RT-PCR),
digestion, insertion to vector and automated DNA
sequencing
2. Western blot analysis was used to confirm expression of
proteins
3. The pCBWN DNA was then injected in disease-
challenged mice and horses which resulted to their
induced protective immunity against WN virus infection
b. Results
● Methods used were similar to constructed recombinant
plasmid of JE virus prM and E proteins
● Proved that single IM injection can induce a long-lasting
protective immunity on injected horses and mice. WN
virus plasmid encoding extracellular subviral particles
like prM and E proteins have excellent vaccine potential
in horses and mice
● IM electrotransfer method has potential to improve
immunogenicity of DNA vaccine
2.) Ledgerwood et al. (2010)
a. Methodology
Design: Protocol VRC 303
 To examine safety, tolerability, and immune response to
an investigational recombinant DNA WNV vaccine
 Study includes healthy adult subjects negative for WNV
IgG in age groups 18-50 y/o and 51-65 y/o
 Antibody responses were measured by ELISA, Reporter
Virus Particles, T Cell Responses by ELISpot, and T
Cell Responses by Intracellular Cytokine Staining
b. Results
 Vaccine caused no serious adverse events and only
mild local & systemic reactogenicity
 With vaccine-induced antibody production; antibody
responses peaked at week 12
 With vaccine-specific T cell responses against WNV
virion Envelope protein and WNV virion-associated
membrane peptide
c. Discussion
Increased immune response in older adults may be due
to:
 Older cells may be less resistant to the uptake of
random DNA
 Features of antigen or delivery approach exceeded the
threshold of response in both young and old
 Use of CMV/R promoter - more efficiently stimulate
antigen-presenting cells to help promote an antigen-
specific T cell response
 Use of the Biojector – may be more effective antigen
delivery method in aged skin.
F. Study Updates
 A Phase 1 clinical trial (NCT02840487) of VRC5288 has
already been launched to test a variety of regimens and
doses for safety and immunogenicity
Page 7 of 9
 ➢ This study has, as one of its objectives, to define the
level of vaccine-induced NAbs required for prevention of
ZIKV viremia
➢ These trials are being designed in parallel with other
groups who will evaluate a purified, protein-based,
whole-inactivated ZIKV vaccine (ZPIV) or li ve-attenuated
vaccine approaches
 This will provide safety and immunogenicity data in humans
that will inform the next steps of vaccine development and
provide options for achieving both the short-term goal of
identifying an intervention to protect.
G. Updates on Zika Virus Vaccine
 The National Institute of Allergy and Infectious Diseases
(NIAID), part of the National Institutes of Health, has
launched a clinical trial of a vaccine candidate intended to
prevent Zika virus infection. At least 80 healthy volunteers
ages 18-35 years at three study sites in the United States
are expected to participate in the trial.
 Initial safety and immunogenicity data from the Phase 1 trial
are expected by January 2017. If results show a favorable
safety profile and immune response, NIAID plans to initiate a
Phase 2 trial in Zika-endemic countries in early 2017.
H. Open Forum (Question and Answer Portion)
1. Is this type of vaccine (DNA vaccine) applicable in the
Philippines?
Ans.: Yes. There has already been a reported case of the
utilization of the vaccine here in the Philippines. Consideration
may be on the cost of the vaccine, meaning its accessibility to the
people especially among the poor. Note that it is easier to
produce and way more stable.
2. Will there be a difference if you will give an RNA vaccine
from a DNA vaccine?
Ans.: None.
Dr. Dimamay: It doesn‟t matter if your constructs is an RNA or
DNA as long as it can produce your antigen.
3. Based on Figure 2, which among the constructs will you
prefer once you start producing your vaccine?
Ans.: VRC5288 group, since it has more SVP secreted based on
the figures in the study.
Dr. Dimamay: When you consider producing the vaccine, you
would want the group which has a higher expression. Despite the
longer size and larger protein of VRC 5288 group, it has a higher
amount of expression meaning it can produce your antigen more.
Spherical capsids are form by VRC 5288 because of this.
4. Is the production of the DNA vaccine infinite?
Ans.: No. In the paper the graphs showed a peak with a
succeeding decline in number of antigens produced.
5. Can the immunity given by the DNA vaccine inherited or
passed on?
Ans.: No, since somatic cells are the target cells, not your germ
line cells, which are usually found in the muscles.
Ii. Biology of Zika Virus Infection in Human Skin Cells
A. Introduction
 Mosquito-borne flavivirus (arbovirus)
 Has a positive, single-stranded genomic RNA encoding a
polyprotein that is processed into
 3 structural proteins
 7 non-structural proteins
 Emergence in Americas and Caribbean
 Mosquito-borne viral diseases
 WHO: Global health emergency (Feb. 2016)
 Also likely to occur :
 Significant decline in 1-2 years
 May become endemic (tropical and subtropical regions)
 Similar to arboviruses (ex. West Nile, Chikungunya)
 Can be sexually transmitted
 Can be found in semen for several months following a
clinically unapparent infection
 High-risk groups:
 Pregnant women and their fetus
 If immunity is not established before child bearing age
 Women in non-endemic regions
 If e xposed to men who have travelled to endemic
regions
BASIC SCIENCE RESEARCH | Block 2
Zika Virus Disease
▪ Transmitted by mosquitoes in the Aedes genus
o Aedes aegypti – considered main vector of the vi rus in the
South a nd Southeast Asia
▪ Classic manifestations
✓ Fever
✓ Rash
✓ Joint Pain
 During the outbreak in French Polynesia, ZIKV infection-
related neurological disorders have been described and the
incidence of the lethal Guillain-Barre Syndrome increased.
 “Because ZIKV has received far less attention than other
emerging arboviruses, the pathogenesis of ZIKV infection
remains poorly understood”
o No information is available either on the nature of the skin
cells that are permissive to infection with ZIKV or on the
entry receptor used by this flavivirus
o Mechanisms of ZIKV infection and the signaling pathways
and antiviral immune respone of the host elicited by this
virus remains to be determined
B. Objectives
 Provide general insights into the interaction between this
virus and its human host
 Specifically, to describe the entry receptors and cellular
targets of the most recent ZIKV isolate responsible for the
recent epidemic in French Polynesia
C. Methodology
1. Experimental Design
Permissive Cells
 human skin fibroblasts
 skin keratinocytes
 dendritic cells
Tests Performed
 Immunolabeling  determine the presence of viral envelope
by fluorescence, observe autophagy
 Electron Microscopy  observe autophagosome formation
 ZIKV Plaque Assay  measure the viral titer
 Western Blotting  determine proteins present in infected
cells
 Flow Cytometry Analysis  assess infection
 Inhibition of Infection  determine antibodies against
receptors
 RNA Interference  used specific siRNA to silence DNAs
encoding for the receptors and observe its effect on
infection
Brief Background of Methods Used
1. Plaque Assay
 The plaque assay is performed based on the ability of
infectious virus particles to form small areas of lysis or foci
of infection on the cell monolayer
 Procedure:
1.) First, the virus is adsorbed onto a confluent cell
monolayer
 to initiate binding to cells
2.) The monolayer is overlayed with agar
 the overlay medium restricts the spread of secondary
infection so that only areas of the cell monolayer
adjacent to the initially affected cells will become
infected and form plaques
3.) These plaques can then be counted a nd the viral
titer calculated
2. Real Time Re verse Transcription Polymerase Chain
Reaction
 A real-time polymerase chain reaction is a laboratory
technique of molecular biology based on the polymerase
chain reaction (PCR).
 It monitors the amplification of a targeted DNA/RNA
molecule during the PCR, i.e. in real-time, and not at its
end, as in conventional PCR.
 “Amplify” small segments of RNA through production of
cDNA
 Allows clinicians to diagnose and monitor diseases using a
minimal amount of sample, such as blood or tissue.
Page 8 of 9
 D. Results and Discussion 2. Explain the role of autophagy in the study.
Aedes mosquito deposits the virus in the epidermis and Ans.: Autophagy is a multistep process responsible for the
dermis of the bitten host during a blood meal: degradation and recycling of cytoplasmic components that
● Skin (dermal/cutaneous) fibroblasts augments the replication and dissemination of several arbo
 Infection resulted in: (1) presence of high RNA copy viruses. It doesn‟t only participate in the damage of proteins but
numbers and (2) gradual increase in production of ZIKV in the host pathogenicity against infection as well.
particles over time = active viral replication in infected Dr. Dimamay: The capsids of the viruses are resistant to the
cells normal process of autophagy, thus aren‟t digested. The presence
● Epidermal keratinocytes of a double membrane of the virus also helps in this resistance.
 Infection resulted in: (1) appearance of cytoplasmic
vacuolation, (2) presence of pyknotic nuclei in the stratum 3. Explain the possible mechanism on how Zika Virus
granulosum = cells undergoing apoptosis Infection can lead to Guillain Barre Syndrome.
● Dendritic cells Ans.: As of now, there is NO KNOWN mechanism on how the
 Involvement of skin antigen-presenting cells in the disease condition happens. What is known is that it is an
replication of other fla vivirus members, particularly autoimmune disorder. But then, based on the study, there are 2
DENV, which efficiently infects Langerhans cells possible mechanisms which could lead to the condition. The Zika
virus infection could have caused a change in:
First step of fla vivirus entry into a host cells : viral envelope a. the nerves causing the body‟s immune system to attack them.
protein b. a change in the immune cells themselves making them
● Interacts with several cell surface receptors and attachment weaker, nonfunctional and possible candidates for attack b y the
factors body‟s immune system.
● At present, >12 putative entry receptors and factors, Dr. Dimamay: Viral patterns of Zika virus may have been
particularly for DENV, have been described such as: mimicked by the body‟s own immune system.
 heat shock proteins
 laminin receptor
 integrity αvβ3
 Prohibitin
 Claudin-1
 scavenger receptor class B and
 natural killer cell receptor NKp44
● Heparan sulfate: a nonspecific attachment factor of flavi virus
that concentrates viral particles on the cell surface
 Facilitates interaction with primary receptors such as
dendritic cell-specific intercellular adhesion molecule 3-
grabbing nonintegrin (DC-SIGN aka CD209)
● Other receptors may include the mannose receptor, and C-
type lectin domain family 5 member A (CLEC5 A aka MDL-
1)
● TIM and TAM proteins (Tyro-3 and AXL)
 Participate in phosphotidylserine-dependent phagocytic
engulfment
 Removal of apoptotic cells
 Also, act as DENV entr y factors that promote viral
infection

E. Conclusion<
 Human skin cells are permissive for ZIKV infection and
replication
 DC-SIGN, TIM, and TAM receptors are involved in ZIKV
infection
 ZIKV induces an innate antiviral response in primary human
skin fibroblasts
 Type I and Type II IFNs inhibit ZIKV replication
 Autophagosome formation in infected skin fibroblasts
increases ZIKV replication

F. Future Directives
Some studies that can be further done are to determine:
● Exact role in ZIKV infection played b y TIM and TAM
receptors
● Contribution of each receptors such as AXL, DC-SIGN, TIM-
1 and other entry receptors and attachment to ZIKV infection
especially their pathogenesis as they are still unknown
● Mechanism by which TLR3 contributes to control of viral
replication
● Better understanding of the role of mosquito saliva in ZIKV
infection

G. Open Forum (Question and Answer Portion)

1. What is the rationale of the use of different cells in the
study?
Ans.: Zika Virus has a vector-mediated transmission, initiated
when a blood-feeding female Aedes mosquito injects the virus
into the skin of its mammalian host, followed by infection of
permissive cells via specific receptors. Skin immune cells,
including dermal fibroblasts, epidermal keratinocytes, and
immature dendritic cells, were all found to be permissive to
Zika Virus infection.

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