Microbio FINAL: Vaccines, Immunological Applications, West Nile Virus

-some test require no special equipments and can be performed easily in a clinical setting.

-"Antibody titer": the level of antibodies in serum; can be measured-serial dilutions of serum need to determine antibody titer quantitatively.

-"Monocolonal antibodies": antibodies with the same specificity (same variable region to attach to same antigen);came from one B-cell fused with cancer cell to produce quickly in a lab; typically produced by a company for use in research and diagnosistics; occasionally used for treatment of disease

visible clumping of solids in a liquid; two kinds: direct and indirect

A.direct agglutination: blood typing lab; soluble antibody protein dissolves in liquid  interacts with antigen like "A" or "B" on cell    ex: hemagglutination of RBC to detect blood type (lab)

• B.indirect/passive agglutination: solube antigen or antibody is coated onto a latex bead;(take a bead and attach constant region of antibody specific to a microbe, mix beads with sample and if you see clumping then the microbe is there)    
• ex: "Rapid Strep Test" use small latex beads coated with antibody against Staph aureus & Strep pyogenes plus patient sample

soluble antigen so it's not a cell and soluble antibody together form area of precipitate (visible reaction); both are soluble, whereas in agglutination, the cell or bead is not soluble; THE LEASE SENSITIVE OF ALL THE METHODS

• antibody-antigen interaction will inhibit a biological activity; cells to detox: antibodies may inhibit the ability of a virus to infect a cell; antibody bind to antigens and stop them from infectiing other cells.
• --> Neutralization assay: control: virus and cells--> cells are dead. To see if someone has antibodies against something, you add antibodies to the control and if the cells are surviving then you know that antibodies are working and neutralizing the virus.

fluorescently labeled monoclonal antibodies are incubated with cells to detect microbial antigen or normal proteins; usually fluorescent red/yellow-green molecules are used; detected by microscopy using a special microscoby (ex: Giardia lamblia intestinalis); detected by flow cytometry using a FACS machine -flow cytometer (ex: detection of CD4+ T cells and CD8+ T cells in HIV patients)

(monoclonal antibody w/ attached flourescent molecule to bind  to specific antigen; then use flurescent molecule to observe it; can also be used to differentiate T and B cells)

• --> direct method: primary antibody is attached to a fluroescent molecule
• --> indirect method: secondary antibody is attached to a fluorescent molecule

proteins (antigens) are separated by size or electrophoresis gel  and detected by antibody "sandwich" using radioactivity or visible light detection (ex: used to confirm a positive HIV antibody ELISA test);  present antibodies will stick... color response. End result: bands. Does the patient respond to one, two or three proteins from microbe. ex: HIV has three proteins..

(other notes: The basic technique of a Western blot involves sorting proteins by length on a gel and then probing the gel with antibodies that react to the proteins that are being searched for. However, when Western blots are used for HIV testing, the process is actually performed in reverse. Instead of unknown proteins being tested for with known antibodies, labs work with prepared protein samples, and look to see if there are any antibodies in a person's blood that stick to them.)

• **STEPS**
• 1.Proteins from a known bacterium or virus are separated by an electric current in electrophoresis
• 2. The proteins are then transferred to a filter by blotting.
• 3. Patients serum is washed over the filter. IF the patient has antibodies to one of the proteins in the filter then antiboidies and protein will combine. Anti human serum linked to an enzyme is then washed over the filter.
• 4. THis will be made visible as a colored band on the filter after addition of the enzyme's substrate

enzyme-linked immunosorbent assay

• --> Purpose:determine whether a particular antibody is present in a patient's blood sample; can also detect antigens
• --> uses specificity of antibodies to captures and stick to antigen

--> detection method: using enzyme

• -->Limitations of test: people can be poor produces of antibodies and won't be detected, false positives are possible: unrelated antibody reacts w/ antigent nospecifically
• -->AVOID SOURCES of error: use replicates, wash properly, use positive & neg controls and follow instructions carefully.

• Centrifuge for serum
• second antibody is added and antigen antibody complex signsals enzyme attached to second antibody that converts a added chemical to another color....
• "Detection becomes possible when a second antibody is added. This antibody is prepared from the serum of an animal injected previously with human antibody; the human antibody in this case serves as an antigen and the animal thus produces an antibody against the human antibody. Once isolated, the second antibody can be chemically linked to a system that can produce a detectable signal."
• Finally, because the first antibody binds to antigen, the more antigen that is accessible, the more first antibody will be retained. The measure of color, therefore, reflects the amount of antigen initially present.

To Prevent False positive...

Washing helps remove any antibody that did not react with the SLE antigen in the well. When the fluid is removed from the well, antibody that has reacted with antigen remains attached to the well surface. Unreacted (unbound) antibody may also remain in the well in the small amount of fluid that is left behind. This unbound antibody must be removed, because the anti-human antibody added in the next step will recognize and react with any antibody remaining in the well, regardless of whether that antibody is specific for the SLE antigen. A reaction with non-SLE antibody will produce a false-positive result.

• 1. Centrifuge blood and transfer serum sample and dilute.
• 2. Add Phosphate-Saline buffer to regulate pH of serial dilutions
• 3. Transfer dilutions to ELISA plate (pretreated with)
• 4. Add positive controls: anti-DNA primary antibody and Negative control: PBS
• 5. Incubate so that primary antibodies stick to antigens
• 6. Wash: to remove antibodies that didn't react and avoid a false positive
• 7. ADD Horse Radish Peroxidase (secondary antibody)
• 8. Incubate again:so that secondary antibody will bind to primary/human antibodies.
• 9. Wash step.
• 10. Add the clear HRP substrate to each well
• 11. Look for color change...yellow=positive

1. Antibody is captured with specificity for the protein that is used (pretreated)

2. Human blood Sample then added to well and incubated, washed off.

3. Detection of antibody: secondary antibodies which is attached to an enzyme ( ex: HRP) is added that will specifically bind the antigen in sample if antigen is present

4. Substrate for HRP added and color change is observed if HRP is prsent in wells.

• 1: antigen is coated onto plasatic wells.
• 2. Serum from patient is added to wells; plate is washed
• 3. detection antibody w/ enzyme attached is added. Wash.
• 4. Enzyme substrate is added: color change indicates patient has antibodie

use monoclonal antibodies; sample interacts with labeled antibody, then with capture antibody after moving through a lateral flow membrane (ex: canine parvovirus test, RDT or  Rapid TB test, Oraquick test for HIV (in video), west nile virus, Treponema and pregnancy test is similiar...ehh? (http://www.rapid-diagnostics.org/tech-lateral.htm)

1. Naturally acquired active immunity: "Acquired": gaining immunity; "active": patient responds to antigen; patient gets sick but recovers (as you live life)

• 2. Naturally acquired passive immunity: "passive" receives antibodies; mom passes antibodies to the babies
• --> igG thru placenta before birth and IgA:after birth; in breastmilk

3. artificially acquired active immunity-"artificial": medical intervention thru doc/nurse; active": patient responds to antigen; thru vaccination-allows body to produce it's own response: antibodies and  T-cells and get better (vaccine)

4. artificially acquired passive immunity: Passive- receive shot of antibodies; ex: Hep A immunoglobulin shot-gives you antibodies right away!

• -provide protection against disease
• -provide herd immunity in a population to protect unimmunized individuals; protects everyone including unvaccinated ppl.
• --take vaccines at set schedule

• -safety: carry some risk; no point in having smallpox vaccine since it's pretty much eliminated; allergies, pregnancys (can't receive live virus vaccinations where virus can reproduce in the host); weigh  the benefits...
• -effectiveness at generating antibodies or CTLs
• -Immunological memory and a strong immune response

• 1. inactivated or killed:  
• 2. Attenuated:  
• 3. Toxoid:  
• 4. Subunit:  
• 5. Conjugated Polysaccharid antigen:
• 6. Dendritic cell vaccine:

The bacteria is killed (Bordetella pertussis: bacterium in old DPT vaccine) or the virus is inactivated so the microbe can't cause disease Ex:Bordetella pertusis (causes whooping cough) old DPT vaccine, Salk pollio vaccine-injected;also standard flu shot.

typically used for viruses; weakened virus but still alive so it can REPRODUCE, weakened-should not cause serious illness, may be missing some genes

• ex: Sabrine oral polio vaccine
• MMR-measels, mumps, rubella
• varicella-chicken pox virus, Rotateque :diahrrheal rota virus

treated exotoxin inactivated by fomaldehyde

• EX: Diphtheria and Tetanus toxoids in DPT-not used anymore,
• DTap -inital baby shots; more antigen in them
• Tdap vaccines-booster shot,less antigen since less capitals; for teens.

Uses antigen from a piece of the microbe OR dna by using restriction enzymes and placing it in something else like yeast and making more proteins ; may be genetically engineered protein

• Ex:
• new pertussin vaccine
• DTaP-babies; Tdap for older children and adults
• Hepatitis B vaccine: using yeast, and malaria vaccine-children (clinical trials)

• capsule is linked to a carrier protein--stimulates a better response in young children since babies respond better to proteins but anyone can take it.
• ex1: Hib children's vaccine for Heamopilus influenzae-see meningitis
• ex 2: MCV4 vaccine effective against 4 strains of N. meningitidis
• ex3: PCB: Pneumococcus conjugated vaccine (aslo on meningitidis handout)

• experimental; take patience cells treat em w/ cytokines and develop dendritic cells and give antigens to dendritic cells in lab to train so that in the future the dendritic cells will present antigens better; self dendritic cells are grown in the lab and pulsed w/ antigen from a tumor/microbe and given back  to the patient..
• ex: tried against cancer w/ DC+ tumor anitgens

-live attenuated: Nasal spray "Flumist"; only healthy can recieve b/w 5-49 yrs; heat sensitive (doesn't grow well < 38* Cels); great immune response since you're infected in a more natural way;engineered to stay in the nose.

-inactivated/injected: standard flu vaccine: anyone b/w 6 mon to 4 yrs and adults > 50 yrs; can't get the disease from this but the protection may wear off eventually or the immune response is weaker compared to how you would experience the actual virus.

-live attenuated: Oral "sabine" vaccine; swallow it, administered the same way you get the disease; but you can get disease if you have weak immune respnse and can transmit it to others /:

-inactivated/injected: "Salk" vaccine; IPV

-live attenuated: vaccinia (skin scraping); live viral, the only one we have and military still get it; ppl w/ eczema or skin infections can't get this vaccine b/c it reacts badly

-inactivated/injected: none

• 1. Inactivated vaccine:
• -pro:can't get the disease from this.
• -con: Less protective, protection may decrease/disappear over time

• 2. Live attentuated:
• -pro: stronger immune response & administered at infection site.
• -con: can potentially get the disease  if you have weak immune system and possibly transmit it to others /:

• --> children less than 6 yrs:
• •MMR-live attentuated
• •DTaP(Diphtheria, tetanus, acelluar pertussis)
• •IPV (inactivate polio vaccine)
• •Hep B (viral liver disease that is acquired thru blood or sexually like HIV ;kids can get it from daycare providers;)
• •Hib (haemophilus inflluenzae type b-conjugated)
• •PCV (pneumococcal conjugated vaccine; form of meniningitis)
• •varicella-chicken pox
• •influenza
• •Hep A (food borne liver disease);
• •Rotavirus vaccine-diahreal illness, in US you can get IV line or drink pedialite but better in developing countries b/c there is lack of clean drinking water...

--> 11-12 yrs olds: •Tdap booster•HPV: cervical cancer and genital warts•MCV4:meningoccoccal vaccine; vulnerable for meningococal acemia, influenza

• --> adults:
• Td every 10 yrs, Influenza, Hep B (health care workers), MMR (if lacking prior immunity), Meningococcal

History: first isolated in Uganda 1937 and NY:1937

Cali info: first case wes seen in a 31 yr old man in Riverside County who was hospitalized w/ meningitis (inflammation of membranes covering the brain) and recovered

Virology: Part of flavivirus family (includes Encephalitis and dengue fever); 40-60 nm in size, enveloped, icosahedral, +ssRNA

Transmission:-vector: female culex mosquitor carries virus and infects bird-crows/jays (host: life cycle happens)  and humans are dead end.

• signs/symptoms: Usually appears up to 15 days after a bite from an infected mosquitos)
• -Most ppl are asymptomatic
• -20% of infected get "West Nile Fever": Including fever, headache, bodyaches, swollen lymph nodes, and occassional rash.
• -1 in 50 ppl exposed develop severe disease "neuroinvasive": including headache, high fever, neck stiffness, convulsions, confusion, and other symptoms of meningitis or encephalitis (45% of population that developed disease had severe form in 2004 U.S)
• -Risk factors: older age(illness and death)
• - Younger patients may end up with long term neurological problems

• Diagnosis:
• 1. RT-PCR: genetic test to detect viral RNA; covert RNA to dna (PCR)

2. MAC-ELISA: used for initial detection of IgM antibodies against West Nile virus in either serum/CSF. False Positive is possible if person has been immunized against/exposed to related virus (yellow fever). Serum IgM antibodies can exist for about a year after virus exposure.

3. PRNT (Plague reduction neutralization test): type of neutralization assay; take virus incubated w/antibodies, do neutralization assay and then spread on a dish to look for reduction of plaques in order to test for antibodies; confirm cases of West Nile virus; takes a week or more b/c involved incubating patient serum w/ a virus from lab then infecting tissue culture cells; tissue cult cells checked to see if serum was able to reduce plaque formation

**Treatment: None; IV fluids and respiratory support are provided as support in severe cases.

• **Prevention:
• -Use mosquito repellent with DEET, get rid of standing water, wear loose fitting, long sleeved shirts and paths when mosquitos are active (dawn and dusk).
• -A plant based lemon eucalyptus insect repellent (repel) is CDC's alt to DEET.
• - Picaridin: different, newly approved insect repllent

**Vaccine: No human vaccine exxists; 2 dose vaccine for horses.