Module 2 - Cystic Fibrosis

Question 0

What was the early characterisation of CF? (post 1938)

Answer 0

Fat and protein malabsorption
Failure to thrive
Lung disease
Abnormal electrolyte composition in sweat

Question 1

When was cystic fibrosis first identified and how?

Answer 1

1938. Identified mucous plugs in pancreas in babies dying of malnutrition.
      Condition called “cystic fibrosis of the pancreas.”
      In 1940s it became apparent that CF was of genetic inheritance.

Question 2

How was the gene responsible for CF identified?

Answer 2

1983 - abnormal cAMP-mediated regulation of chloride transport (sweat ducts)
1985 - gene associated with CF mapped to chromosome 7q31.2 by linkage in families
1989 - gene identified by positional cloning

Question 3

Describe the CFTR gene

Answer 3

“Cystic Fibrosis Transmembrane conductance Regulator”
90kb DNA
27 exons coding for 1480 amino acids
Codes for large integral glycosylated membrane-spanning protein

Question 4

Describe the CFTR protein. What type of protein is it and how is it regulated?

Answer 4

Member of ABC (ATP-binding cassette) superfamily
Chloride channel protein. It is different from other ABC transporters because the passage of ions is via passive diffusion.
Regulated by cAMP-dependent phosphorylation
Expressed in epithelial cells (apical membranes)

Question 5

What are the domains of the CFTR protein?

Answer 5

5 domains.
2 membrane spanning domains (MSD) - forma channel for passage of chloride ions
2 nucleotide-binding domains (NBD) - bind and hydrolyse ATP
1 regulatory domain (R) - phosphorylation by cAMP-dependent protein kinase)

Question 6

How does CFTR interact with other proteins?

Answer 6

Carboxy-terminal is anchored to cytoskeleton and kept close to other proteins.
These proteins influence CFTR functions such as:
- conductance
- regulation of other channels (e.g. eNAC: epithelial sodium channel)
- signal transduction
- localisation at apical plasma membrane

Question 7

Describe CFTR function in the airways and how a can mutation affect its function in a patient with CF.

Answer 7

In a normal lung: CFTR assists in Cl- secretion. Cl- ions move out of the cell down a concentration gradient, while Na+ ions and water move into the cell. CFTR inhibits ENac so that there is less absorption of Na+.
In a CF lung: Cl- ions build up within the cell resulting in an even greater movement of Na+ ions and water into the cell. –> dehydrated mucous in airways.

Question 8

Describe CFTR function in the sweat duct and how a mutation can affect its function in a CF patient.

Answer 8

In normal sweat ducts: Cl- ions move into the cell, down a concentration gradient, together with Na+ ions and water. CFTR activates ENac in the sweat duct. Sweat is secreted into the duct by the gland and both Na+ and Cl- are normally reabsorbed by the duct cells before reaching the skin surface.
In CF sweat ducts: Cl- ions cannot enter the cell and Na+ and water remain in the sweat duct. –> elevated Na+ and Cl- levels in the sweat.

Question 9

How many known mutations of CF are there? What can they cause? In which exons are the majority of mutations found?

Answer 9

More than 1900 known mutations.
These mutations are a variety of CF-causing, non CF-causing, varied penetrance and unknown significance.
Most mutations are found in exons 4, 8, 14 and 20.

Question 10

What is a Class I mutation? Provide an example of one.

Answer 10

No protein production.

E.g. Spice mutation intron 4 donor site (G -> T) in MSD1. Absent protein, normal mRNA abundance.

Question 11

What is a Class II mutation? Provide an example of one.

Answer 11

Defective processing (maturation, premature degradation)

E.g. F508del in NBD 1.

Question 12

What is a Class III mutation? Provide an example of one.

Answer 12

Defective regulation (e.g. decreased ATP binding and hydrolysis).

E.g. S1255P in NBD2.

Question 13

What is a Class IV mutation? Provide an example of one.

Answer 13

Defective/reduced ion conductance or channel gating.

E.g. R117H in MSD1. Alteration of Cl- channel.

Question 14

What is a Class V mutation? Provide an example of one.

Answer 14

Reduced protein production (e.g. promoter or splicing abnormality)

E.g. Splice mutation intron 4 donor site (G -> T) in MSD 1. Reduced number of transcripts.

Question 15

What is a Class VI mutation? Provide an example of one.

Answer 15

Accelerated turnover from cell surface.

E.g. Q1412X. Instability at cell surface.

Question 16

What is the most common mutation in CF?

Answer 16

F508del. (Class II)
Accounts for 70-75% of mutations in people from Northern European descent. Homozygosity in about 50% of patients with CF.
Misfolded protein retained in ER and targeted for degradation.

Question 17

What are some class-specific therapies?

Answer 17

Class I: Nonsense mutations corrected by compounds that allow ‘read through’ of mRNA. (e.g. aminoglycoside antibiotics)
Class II: ‘correctors’ to improve processing
Class III: ‘potentiators’ to activate protein
Class IV: flavonoid compounds to augment channel function (increase open probability
Class V: often splicing mutations. Increase levels of correctly spliced mRNA.

Question 18

What diagnostic test can be used for a F508del mutation?

Answer 18

PCR + gel electrophoresis.

Homozygotes will show a shorter band due to the deleterious nature of the mutation.

Question 19

How are mutation panels used in testing?

Answer 19

PCR +/- restriction enzymes.
Number of mutations in testing panels differs throughout the world.
Some mutations are specific to particular ethnic groups.
Previously, in VIC a total of 12 mutations were tested, together accounting for 80% of mutations. A further 7 mutations were added.

Question 20

Testing of mutations by SNP analysis

Answer 20

Multiplex PCR - single reaction, multiple primers, fluorescent tags.
Assay involves primer extension of each hybridised primer so that each product incorporates a coloured fluorescent tag (dNTP) and the colour of the peak depends on which dNTP has been added.
Analysis of each mutation performed in 2 batches on capillary gel electrophoresis.

Question 21

Most recent VIC CFTR mutation panels and testing

Answer 21

New diagnostic panel includes 38-40 mutations, accounting for 91% of mutations in VIC population.
SNP genotyping + measuring of mass of allele-specific products following PCR.
MALDI-TOF MS - Matrix assisted laser desorption/ionization-time of flight mass spectrometry.
Can genotype a large number of samples (100s-1000s) and a medium number of SNPs (10s-100s)

Question 22

What is the pattern of inheritance for CF?

Answer 22

Autosomal recessive
Carrier frequency of 1 in 25.
Often no family history.

Question 23

What ethnicity does it most commonly affect? What is the incidence rate?

Answer 23

Caucasian descent (especially Northern European). 
Incidence of approx. 1/2500-3000 live births.

Question 24

Why are there different rates of prevalence around the world?

Answer 24

Ethnic-specific mutations.

Question 25

What are the genotype-phenotype correlations in terms of class and lung disease/pancreatic function?

Answer 25

Weak genotype-phenotype correlations for lung disease, more for pancreatic disease. Classes I, II, and III (and probably VI) - severe lung disease; pancreatic insufficiency. Classes IV and V - milder lung disease; pancreatic sufficiency.

Question 26

What is one challenge of mutations with uncertain clinical significance?

Answer 26

Penetrance of mutation may also depend on another intragenic polymorphism. E.g. R117H and poly T tract in intron 8 in cis.

Question 27

Environmental and genetic modifiers both play a role in the resultant phenotype of a CF patient. What phenotypes are not well correlated with CFTR mutations?

Answer 27

Lung function, neonatal intestinal obstruction, diabetes and anthropometry (e.g. weight and height).

Question 28

It is important to identify genetic modifiers because:

Answer 28

- Identifies new targets for therapies - Increases understanding of disease variability - Expect these genes/variants to be minimally penetrant in healthy people but effects unmasked in CF patients. - These gene modifiers may contribute to development or progression of common diseases in general population

Question 29

What are 3 types of association studies that can be carried out to identify genetic modifiers of CF?

Answer 29

1. Linkage: Track genes/markers associated with specific phenotype in families with CF 2. Candidate gene expression: Genes with known function; correlate variations in gene with presence of features/phenotype in CF patients. 3. Genome wide association studies (GWAS): Examine DNA markers at many positions on multiple chromosomes in populations with and without CF phenotype of interest. Look for SNPs that are shared with much greater frequency in those with same phenotype.

Question 30

What are the limitations of genome wide association studies?

Answer 30

- When genes/SNPs are less penetrant, then need a greater sample size. - Need to replicate studies for validation. - Need to demonstrate correlation and cause (not just association) by conducting research showing specific mechanisms.

Question 31

What are 3 genetic modifiers of lung disease in CF?

Answer 31

EDNRA (encodes endothelin receptor type A) - variants alter smooth muscle tone in airways and/or vasculature MBL2 (encodes mannose binding lecting) - role in innate immunity. MBL2 deficiency predisposes to early infection with P. Aeruginosa. TGFB1 (encodes transforming growth factor beta) - role in regulating inflammation and tissue remodeling. Increased expression causes worse lung function.

Question 32

What is a genetic modifier of intestinal obstruction?

Answer 32

MSRA (encodes methionine sulphoxide reductase) - role in modifying intestinal enzymes such as alpha 1-antitrypsin. Variants may alter digestion of intestinal contents and contribute to formation of viscous meconium.

Question 33

What is a genetic modifier of diabetes?

Answer 33

TCF7L2 (encodes transcription factor 7-like 2) - role in proliferation and function of beta cells of pancreatic islets. Modify risk for diabetes in CF patients who have not had recent or prolonged exposure to systemic steroids.

Question 34

What are some environmental modifiers that are associated with poorer health outcomes?

Answer 34

- being female - lower socio-economic status - exposure to tobacco smoke - active and passive - infectious exposures - disease self-management

Question 35

What are 3 purposes of screening for CF?

Answer 35

- Identify babies at risk of developing CF (newborn screening) - Identify carriers who are relatives of a diagnosed baby (cascade testing) - Identify carriers from the general population (population carrier screening)

Question 36

Describe newborn screening for CF

Answer 36

- Immunoreactive trypsinogen (IRT) on newborn screening (Guthrie cards) - Heel prick at 2-3 days - Elevated samples undergo further testing - DNA testing for initial 12 mutation panel - if necessary include extended panel. (2 mutations. Homozygotes and compound heterozygotes have CF) - Heterozygotes brought in for sweat test and maybe gene sequencing.

Question 37

What is a challenge with cascade testing?

Answer 37

Lack of communication within families may lead to poor uptake. Only 12% of eligible relatives underwent testing even when test was at no charge.

Question 38

What are some reproductive options for carrier couples?

Answer 38

No children, adoption, pregnancy with no testing, pre-implantation genetic diagnosis (PGD) - test and implant unaffected embryos.

Question 39

What are the consecutive branches of a lung?

Answer 39

``` Upper airways Trachea Bronchi Bronchioles Terminal Bronchioles Respiratory Bronchioles Alveolar Ducts Alveoli ```

Question 40

What are the stages of lung development and the corresponding weeks of gestation?

Answer 40

``` Embryonic - 0-6 weeks gestation Pseudoglandular - 6-16 weeks gestation Canalicular - 16-26 weeks gestation Sacular/Alveolar - 26-36 weeks gestation Postnatal - birth to ?? (2-adult) ```

Question 41

What occurs in the embryonic stage of lung development?

Answer 41

Lung bud appears day 26-28. Ventral outpouching of the primitive foregut. Endodermal origin. Proximal branching of the airways into surrounding mesoderm.

Question 42

What happens in the pseudoglandular stage of lung development?

Answer 42

Weeks 6-16. Airways develop (branch) to the level of terminal bronchioles (pre-acinar bronchi) 16 generations of airways.

Question 43

What occurs in the canalicular stage of lung development?

Answer 43

Weeks 16-26. Acinar region develops Thinning of peripheral epithelium Type I and II pneumonocytes develop.

Question 44

What occurs in the sacular/alveolar stage of lung development?

Answer 44

Weeks 26-36. Sacules form into the alveolar ducts and alveoli Marked decrease in interstitial tissue Sacules become thin walled Sacules and alveoli form further generations of alveoli by septation Alveoli at birth 100 x 10^6; Adult 300 x 10^6 Surface area at birth 4 sqm; adult 10 sqm

Question 45

What are the functions of the lung?

Answer 45

``` Gas exchange defence acid-base exchange metabolic heat exchange water balance phonation ```

Question 46

What are the physical and cellular defence mechanisms of the lung?

Answer 46

Physical - upper airway filter; reflexes (sneeze, cough); mucociliary escalator. Cellular - phagocytes (e.g. alveolar macrophages), immunological

Question 47

What is the mucociliary escalator?

Answer 47

Airway surface liquid (ASL) has 2 layers - periciliary layer + mucus gel layer Mucus produced by secretory cells including goblet cells. Mucus propelled by cilia - 12-15 beats/second; 1mm/minute

Question 48

What can be measured through spirometry?

Answer 48

Forced expiratory volume (FEV1) - volume exhaled in first second Forced vital capacity (FVC) - total volume exhaled

Question 49

What are pulmonary exacerbations associated with CF? What are some other features?

Answer 49

Increased cough and sputum production Increased dyspnoea (shortness of breath/awareness of breathing) Lethargy/poor appetite Reduced lung function Progressive decline in lung function Respiratory failure Death/transplantation (30% lung function)

Question 50

On which cell types is CFTR expressed?

Answer 50

Apical plasma membrane of ciliated epithelial cells in airways Serous cells of submucosal glands Alveolar epithelial type II cells Cells of immune system (alveolar macrophages, neutrophils)

Question 51

What is the significance of CFTR dysfunction and ASL?

Answer 51

ASL volume is decreased due to water entering the cells. This has consequences on ciliary beating as the periciliary layer is decreased. Absence of lubrication also results in adherent mucus plaque. These can promote chronic infection.

Question 52

How are CFTR dysfunction and pH related to lung function?

Answer 52

CFTR mediates transport of bicarbonate and regulates pH of ASL. Deficiency of this inhibits antimicrobial function.

Question 53

What is the consequence of CFTR dysfunction in the maintenance of glutathione and thiocyanate?

Answer 53

Glutathione deficiency might contribute to lung inflammation and oxidative stress.

Question 54

How does CF create an anaerobic environment in the lung?

Answer 54

Thick mucus plaques adherent to epithelial surface + increased oxygen consumption by CF epithelia. Pseudomonas converts to anaerobic biofilm mode of growth.

Question 55

What happens when CFTR dysfunction promotes chronic airway infection?

Answer 55

Exaggerated, predominantly neutrophilic inflammation. | May also directly affect airway immunity.

Question 56

What role do neutrophils play in CF airways?

Answer 56

- role in ensuing tissue damage and disease progression - Possess an array of mediators, oxidants and proteases, critical for response against infection - Large amounts of these enzymes escape from neutrophils in cell death and during phagocytosis Antiprotease defences in the airways overwhelmed by the protease burden in the lung

Question 57

What is the relationship between CF inflammation and oxygen?

Answer 57

CF airways exposed to oxygen radicals derived from environmental oxygen and bacterial products and also from host immune response. Oxidative stress exacerbates pulmonary deterioration and advances bronchiectasis. Oxidative stress may lead to oxidation of airway proteins. May further augment cytokine release and airway inflammation.

Question 58

What are some extracellular triggers of inflammation in CF?

Answer 58

``` Bacteria Vriuses Fungi GER Protease/anti-protease balance ROS/anti-oxidants balance ```

Question 59

Explain the proinflammatory property of CF airway epithelium.

Answer 59

Increased cellular response to inflammatory triggers. | May be due to dysregulated activity of transcription factors.

Question 60

What can lead to the deposition of DNA in the airways?

Answer 60

Accumulation of abnormally folded CFTR in ER triggers 'cell stress' and apoptosis. Ceramide (breakdown product of sphingomyelin found in plasma membrane and in endolysosomal compartments) accumulation may further augment inflammation by inducing apoptosis with subsequent deposition of DNA in airways.

Question 61

What are some intracellular triggers of inflammation in CF airway cells?

Answer 61

Inherent abnormal intracellular pathways, primarily in airway epithelial cells, lead to apoptosis on the one hand, and to cell stress and excessive cytokine production on the other, perpetuating the inflammatory response in the airways.

Question 62

What is bronchiectasis?

Answer 62

Dilation and damage of airways due to infection, inflammation and obstruction of airways.

Question 63

What are some pathogens that can cause lung disease in CF?

Answer 63

``` Haemophilus influenzae Staphylococcus aureus Pseudomonas aeruginosa Burkholderia cepacia Stenotrophomonas maltophilia Non-tuberculosis mycobacteria ```

Question 64

What body systems can be affected by CF?

Answer 64

``` Chronic pulmonary disease Pancreas Nutrition Liver Gut Electrolyte disturbance ENT Muscoloskeletal Genito-urinary Renal ```

Question 65

What is the sweat test?

Answer 65

Gold standard for diagnosing CF Transdermal administration of pilocarpine by iontophoresis Collection and quantitation of sweat onto gauze or filter paper or into a macroduct coil. Normal value Cl < 40mmol/L

Question 66

What are the clinical features of high salt sweat?

Answer 66

``` Hyponatremic/hypochloremic dehydration Hypokalemic metabolic alkalosis secondary to chronic salt loss -Pseudo-Bartter's Syndrome Headache or irritability Muscle cramps Nausea and vomiting Fatigue Poor Concentration ```

Question 67

What are the two functions of the pancreas?

Answer 67

Exocrine (Pancreatic acini) - lipase, amylase, protease secretion. Endocrine (Islet of langerhans) - insulin, glucagon secretion.

Question 68

What are the features of pancreatic dysfunction in CF?

Answer 68

Pancreatic enzyme insufficiency in 85% of patients. Class I, II, III or VI mutations Steatorrhoea Fat malaborption Malnutrition Low levels of fat soluble vitamins - A, D, E, K Use of pancreatic enzyme replacement therapy (PERT) - enteric coated

Question 69

Explain how a mutation in CFTR can result in pancreatic disease.

Answer 69

Dysregulation of chloride secretion = reduced luminal liquid. This results in viscous pancreatic secretions and thus, pancreatic duct obstruction and progressive fibrosis and fatty infiltration can occur. Dysregulation of bicarbonate secretion = acidic pH of luminal liquid. This results in premature activation of proteolytic enzymes and therefore, inflammation and destruction of pancreas.

Question 70

What is cystic fibrosis related diabetes mellitus (CFRDM)?

Answer 70

``` Increasingly common with increasing age (rare under 10 years, 50% over 30 years) Impaired and delayed insulin secretion Insulin resistance Higher mortality rates (6 fold) Lung function decline Microvascular complications ```

Question 71

How can the liver be affected by CF?

Answer 71

25% of patients have liver disease. CFTR in epithelial cells lining intra-hepatic bile ducts. Dysfunction increases viscosity of bile and plugging of intra-hepatic bile ducts. Cirrhosis (replacement of liver tissue by scar tissue and regenerative nodules) in minority Most cirrhosis evident by 20 yo.

Question 72

What are the clinical features of liver disease?

Answer 72

Prolonged neonatal jaundice Cirrhosis and portal hypertension Hepatocellular failure is uncommon, 2-3%

Question 73

What are the ways CF can affect the GIT?

Answer 73

``` Dehydration of luminal contents Meconium ileus Constipation Distal intestinal obstruction syndrome Gastro-oesophageal reflux Rectal prolapse (pancreatic insufficient, frequent stools, diminished muscle tone, coughing) Malignancy Coeliac disease Fibrosing colonopathy ```

Question 74

What is meconium ilieus?

Answer 74

Inspissated intraluminal meconium causing bowel obstruction associated with pancreatic insufficiency Incidence: 10-15% of CF neonates. Late complications (increased DIOS risk, adhesive small bowel obstruction)

Question 75

What is distal intestinal obstruction syndrome?

Answer 75

Abdominal pain, crampy in right iliac fossa (RIF) Palpable mass in RIF Partial or complete bowel obstruction intussusception AXR: dilated small bowel with bubbly ileocaecal mass Constipation also common

Question 76

What is gastro-oesophageal reflux?

Answer 76

Very common in CF Aetiology is multifactorial Exacerbated lung disease due to aspiration and reflex bronchospasm Delayed gastric emptying and small bowel transit also common.

Question 77

How does CF affect bone?

Answer 77

Osteoporosis in CF patients. Low bone mineral density very common. Risk factors include: poor lung function, delayed puberty, nutritional failure, steroid use, CFRDM, hypovitaminosis D and K, physical inactivity 2-fold increase in fracture rates in young adults.

Question 78

How does CF affect the genito-urinary system?

Answer 78

Pubertal delay and growth delay Male infertility (99%, absent vas deferens results in azoospermia) Reduced female fertility Urinary incontinence No clinical effect of CFTR in renal tubes. Increased renal clearance of antibiotics (e.g. nephrotoxic aminoglygosides)

Question 79

What are the goals of treatment of CF?

Answer 79

Maintaining lung function - treat infections; airway clearance Adequate growth - diet; supplementation Managing complications - e.g. CFRDM, Liver disease

Question 80

What type of professionals can help with CF?

Answer 80

Physicians, nurses, physiotherapists, dieticians, geneticists, pyschologists

Question 81

What types of physiotherapy techniques can be used?

Answer 81

Gravity assisted drainage and manual techniques. - modified 5 position technique in infants - active cycle of breathing techniques (ACBT) (introduced as blowing games) - Autogenic drainage (AD) - Assisted AD - Positive expiratory pressure (PEP) (mask, mouthpiece - MoPEP, Combination, bubble) - Activity and exercise

Question 82

In what 2 ways can antimicrobials be useful?

Answer 82

prophylaxis and exacerbations

Question 83

What things have to be considered when developing antibiotics?

Answer 83

Route of administration - oral,intravenous (home/hospital), nebulised Susceptibility testing Resistance Pharmacokinetics

Question 84

What patterns are there with antimicrobials and CF?

Answer 84

Acquisition of pathogens occurs age-wise | Majority eventually acquire pseudomonas

Question 85

How measures are taken to control infection?

Answer 85

Cohorting. Patient to patient spread of infection of pseudomonas aeruginosa, burkholderia cepacia, MRSA Segregation of patients - in-patient, out-patient, social activities (can have implications on support community)

Question 86

What are some other respiratory therapies?

Answer 86

Mucolytics - recombinant human deoxyribonuclease-1 (Pulmozyme), hypertonic saline, mannitol Anti-inflammatory agents - azithromycin, prednisolone, inhaled steroids, ibuprofen

Question 87

What measures can be taken with regards to nutrition?

Answer 87

- Close monitoring - Specialist dietician - Energy requirements 120-150% normal - wide variation, increased expenditure, increased losses, reduced intake - high fat (35-40%), high protein diet - supplemental feeds - oral, nasogastric, gastrostomy (PEG) - Improved growth improves lung function

Question 88

Vitamins and electrolytes

Answer 88

Fat-soluble vitamins - A, D, E, K | Salt replacement

Question 89

Pancreatic enzymes

Answer 89

- Enteric coated pancreatic enzyme microspheres (e.g. Creon) - Derived from porcine extract - Taken with all fat, protein and complex carb containing foods - contain lipase, amylase, protease - 500-2000 units lipase/kg/meal - Maximum 10,000 units lipase/kg/day

Question 90

Future therapies

Answer 90

- Novel inhaled antibacterials - Anti-inflammatory agents - correction of the underlying gene effect - protein rescue therapy

Question 91

What are the phases of clinical trials?

Answer 91

``` Phase 1: - Safety and pharmacology - Start with low doses and increase - Healthy volunteers Phase 2: - Examine effectiveness (dose, delivery method, dosing interval) - Confirm safety - 100-300 patients Phase 3: - Confirm previous findings - Demonstrate safety and efficacy - Determine best dosage - 1000+ patients ```

Question 92

What are some examples of novel antibacterials?

Answer 92

Dry powder inhalation: colistin, tobramycin, vancomycin | Nebulised: Aztreonam, amikacin, levofloxacin

Question 93

What are some anti-inflammatories?

Answer 93

Glutathione (antioxidant) Sildenafil (phosphodiesterase inhibitor) KB001A (humanised monoclonal Fab fragment; targets pseudomonas virulence factor and decreases local inflammation) alpha1-antitrypsin (protease inhibitor; phase 2 study of inhaled version)

Question 94

Gene therapy

Answer 94

Introduce a normal copy of the CFTR gene into the cells of the conducting airways. Likely to need repeated application. Lung efficient barrier to foreign material (avoid being cleared from lungs by mucociliary elevator, penetrate mucus and then cell membrane, cross cytoplasm and DNA must enter nucleus, avoid host immune system)

Question 95

Gene therapy vectors

Answer 95

- Viral vectors (adenovirus, retrovirus, adeno-associated virus); Multiple clinical trials in 1990s, repeated administration not efficacious - Non viral vectors: cationic liposomes complexed with plasmid DNA, short duration of efficacy (viral promotor exchanged for humanized promoter capable of sustaining prolonged gene expression; mild flu-like symptoms (presence of unmethylated CpG groups on bacterially derived DNA)