ID exam 2 ch 35 and 36, vaccination

april 10th class, slides and socrative all done, still need phage display antibody video

smallpox descr

potentially fatal, usually benign course but can cause serious scarring (65-75% of survivors have permanent facial scarring)

progress of smallpox (5)

rash - raised bumps - fluid-filled blisters - scabbing over - healing

how have the prevalences of many diseases been over time

once vaccination introduced - dropped rapidly but in recent years has risen again (due to anti-vaxxers)

numbers of whopping cough over time

decreased drastically after vaccine introduction but in recent years epidemics every 4 yrs following scare of vaccine

why is whoopinh cough so damaging to infants

have small airways - easily obstructed

vaccine types + vasic descr

live attenuated (modified, not able to cause disease)
inactivated (pathogen - “killed”)
fractions inc. conjugates (but also toxoids, protein subunits and polysaccharides)

hapten long, descr

half an antigen, combined with a carrier molecule to form a hapten-carrier conjugate, B cell recognizes the hapten but has a weak response - with the conjugate response is stronger as the TCR recognizes the carrier

options with linked recogniton

if just polysaccharide (hapten) 0 V cell cannot engage Tfh (bc sugar not allowed in MHC)
if coformulated protein and hapten (both in one compartment but separate) - B cell cannot engage Tfh (no internalization of protein)
if conjugate - C cell engages Tfh!!

should the protein be terminally (at ends) or centrally conjugated to the hapten to produce the largest antibody response?

central conjugation! terminal conjugation barely produces a response

how to make a conjugate currently

purify polysaccharide antigen from bacterial pathogen, cleave it from lipid A through chemical cleavage and purification; purify recombinant carrier protein and add it to the polysaccharide with a chemical linkage, final purification and then you have a conjugate! (can happen eg in vivo with e coli)

how are live attenuated vaccines made

attenuated by “serial passaging” - grow the pathogen in flasks on cells, keep reculturing as mutations accumulate, wait till it becomes less virulent, check by injecting into monkeys

MVA descr

modified vaccinia ankara - highly attenuated (weakened) strain of the vaccinia virus used as a safe, third-generation vaccine against smallpox and mpox, using chicken embryo fibroblasts (CEF), cripple it by multiple passaging, major deletion sites, mellowed out progeny

What is necessary in viruses to combat host innate immune defenses

5 viral proteins

why are CEF used in MVA?

the virus can enter the CEF and even if its assembly and morphogenesis is disturbed - it can exit the cell again (bc CEF lacks antiviral proteins for final virus stages) - HOWEVER all other mammalian cells do not lack the final antiviral proteins so when it tries to infect regular cells, it cannot exit them and is not that dangerous

2 reasons MVA is ideal

nonpermissive in most mammalian cells, unique ability to efficiently express viral and recombinant genes

shuttle vectors meaning

versatile plasmids that can replicate in both prokaryotic and eukaryotic cells

where is MVA replication restricted too

avian cells

MVA based vaccine production

MVA infects host cells - antigen is expressed and ends up in MHC-I or MHC-II

other usage for MVA

recominant MVA boosts Tb immunity

what do adjuvants do

greatly enhance Ab response to the same vaccine, coinjected with vaccine but not chemically connected to it; cause inflammatory response at site of injection - bigger outreach (helps recruit immune cells, enhances antigen presentation to T cells)

2 types of adjuvants

aluminium salts - induce M1, increase ROS production, metabolic reprogramming, disruption of phagosomal acidification
monophosphoryl lipid A - detoxified form of endotoxin lipopolysaccharide, recognized by TLR 4 but not deadly - causes greater inflammatory response

general how to test for vaccine efficacy

immune markers detectable in the blood that are associated with protein against infection/disease

vaccine efficacy tests (7)

toxin neutralization in vivo - schick test, toxin neutralization in vitro, heamagglutinin inhibition assay, serum neutralization assay, immunoprecipitation, indirect ELISA, opsonophagocytic killing assay

Schtick test descr

inject minimal lethal dose and check reaction after 2-4 days, now obsolete; assesses susceptability to diptheria

toxin neutralization in vitro descr

much safer than the schtick test, using dyes and dilution in multiple well plates, measure the ability of abx to neutralize a toxin secreted by a pathogen (eg diptheria, tetanus, anthrax)

heamagglutinin inhibition assay

checks inhibition when serum is diluted at least 40x, if haemagglutination is inhibited

serum neutralization assay

measures the ability of abx in serum to neutralize viruses or live pathogens, preventing them from infecting host cells - checks for plaques (presence of infectious virions) when serum is diluted 4-8x

immunoprecipitation

laboratory technique for the small-scale affinity purification of specific antigens (proteins or other biomolecules) from complex cell or tissue lysates

indirect ELISA

particularly favored for detecting and quantifying ab; covid recombinant protein on bottom then vaccine induced ab patient serum, then secondary ab (goat peroxidase labelled ab to human IgG) conjugate connected to an enzyme and substrate; dilution series, how much light of 450nm gets absorbed by the due

opsonophagocytic killing assay

dilution series - counting colonies, if all colonies present - no phagocytosis, heat inactivated patient serum, antibodies also complement added

3 killer diseases in need for effective vaccine

HIV, mtb, malaria

passive immunity 2 types

natural - maternal ab
artificial - monoclonal ab

sources of antibodies in mab (2)

human, horse

monoclonal ab production steps

immunize mouse w/ antigen, take spleen cells and fuse with myeloma cells (from cell culture) in polyethylene glycon, select and grow hybrid cells, select cells marking ab of desired specificity → monoclonal ab

scFv descr

short chain variable fragment, combined, so much variety - helps w/ production of mab - also for self molecules when being harmful cus normally expelled from repertoire so this way can be added back in

What is the main objective of the serum neutralization test in assessing vaccine efficacy?

To evaluate the protective effect of vaccine-induced antibodies against viruses

What is the primary goal of serial passaging of pathogens in the development of live attenuated vaccines?

To reduce the pathogen's viulence while retaining its antigenicity

How does immunoprecipitation contribute to understanding the specificity of the immune response elicited by a vaccine?

By isolating and identifying specific antigens that are recognized by vaccine-induced antibodies

What is the primary goal of using immunoprecipitation tests in the assessment of vaccine efficacy?

To identify the presence of vaccine-induced antibodies that specifically bind to antigens

What type of vaccine contains a piece of the pathogen (like its protein, sugar, or capsid) to stimulate an immune response?

Subunit, recombinant, conjugate, and polysacchaide vaccine

This vaccine type contains a toxin produced by the pathogen that has been made harmless and is used to elicit an immune response against the toxin, not the gem itself

Toxoid vaccine

In the context of developing passive immunization strategies, what advantage does the phage display antibody library offer?

Rapid screening and selection of specific antibodies against vitually any antigen

In the context of vaccine efficacy assessment, what does a positive hemagglutination inhibition test indicate?

The presence of antibodies capable of inhibiting the agglutination of red blood cells

How do adjuvants enhance the immune response to a vaccine antigen?

By stimulating the immune system to respond more vigorously

How does the MVA platform ensure safety in vaccine recipients?

By utilizing a viral vector that cannot replicate in normal mammalian cells, reducing the risk of infection

How is the toxin neutralization test typically conducted to assess vaccine efficacy?

By mixing serum from vaccinated individuals with a known amount of toxin and then assessing survival of cell exposed to the mixture

In the context of an indirect ELISA, what does a positive result indicate about the vaccine's impact?

The individual has developed antibodies against the vaccine antigen

Which vaccine type uses a harmless piece of the pathogen's DNA or RNA to generate an immune response?

Messenger RNA (mRNA) vaccine

The Schick test is designed to

Detemine susceptibility or immunity to diphtheia toxin in an individual

What primary role do adjuvants play in vaccines?

To boost the body’s immune response to the vaccine

What is the primary purpose of the toxin neutralization test in the context of vaccine efficacy

To detemine the ability of vaccine-induced antibodies to neutralize toxins

What indicates a positive Schick test result, implying susceptibility to the toxin?

A local inflammatoy reaction at the site of injection

In the context of vaccine research, what does the opsonophagocytosis test measure?

The capability of antibodies generated by vaccination to promote phagocytosis of pathogens

Phage display technology is primarily used in passive immunization strategies to:

Identify antibodies with high specificity and affinity for target antigens

What is a key characteristic of the Modified Vaccinia Ankara MVA platform used in vaccine development?

It employs a highly attenuated, non-replicating poxvius vector.

What is a critical function of adjuvants in subunit vaccines

They compensate for the absence of whole pathogen components by enhancing the immune response.

Which of the following is a commonly used adjuvant in vaccine formulations?

Aluminum salts

Which of the following strategies is most effective in countering anti-vaccination misinformation?

Providing evidence-based responses and engaging in respectful dialogue

Which of the following is a common source of antibodies used in passive immunization?

Human or animal donors who have recovered from an infection or have been immunized

How does the serum neutralization test measure the efficacy of a vaccine?

By mixing serum from vaccinated individuals with a virus and assessing the prevention of viral cytopathic effects in cell cultures

The selection process in a phage display library to find suitable antibodies for passive immunization is characterized by:

Binding affinity and specificity to the target antigen, followed by amplification of successful phages

In the context of vaccine development, what makes the MVA platform particularly versatile and adaptable?

Its suitability as a vector for expressing foreign antigens from different pathogens.

Which type of vaccine uses a weakened form of the germ that causes a disease to elicit an immune response without causing the disease itself?

Live attenuated vaccine

What is the significance of a positive opsonophagocytosis assay result in vaccine efficacy studies?

It shows that the vaccine can induce antibodies that assist in the clearance of pathogens by phagocytes

What is the principle behind the hemagglutination inhibition HI test used to evaluate vaccine efficacy?

To assess the ability of serum antibodies to prevent the clumping of red blood cells caused by the pathogen

A critical step in utilizing phage display technology for passive immunization involves:

Panning the phage display library against the target antigen to isolate specific binding antibodies

What is the key purpose of pefoming an indirect ELISA assay in vaccine efficacy studies?

To detect and quantify specific antibodies produced in response to vaccination

Why does phage display allow combinations that are normally eliminated in vivo?

Self-reactive and stucturally faulty combinations are not negatively selected

What is the functional significance of the pIII coat protein in M13 phage display systems?

It mediates infection and serves as the fusion partner for displayed scFv (single chain variable fragment)

What is the primary objective of phage display antibody library technology?

To generate and screen vast numbers of antibody vaiants for specific binding

What is a key difference between natural antibody repetoires and phage display libraries?

Phage libraies allow combinations not nomally present in vivo

Which property of the M13 bacteriophage enables continuous production of antibody- displaying particles without killing the host?

Non-lytic life cycle with clean extrusion through the bacterial membrane