Week 2 - Viral Replication Flashcards
The growth curve is used to study the _____ ______.
virus replication
A single or one cycle of virus growth is observed, called as _____ ______ _____ experiment.
one step growth
The one step growth curve is defined as the time of a _______ process of infection by a given virus
in a _______ ____ cell until the release of _____ ______ ______.
complete, permissive, host, new viral progeny
Not all viruses can infect all cells. Based on variability of receptors, transcription/translation factors, etc.
Members of each virus _____/_____ may have unique method of replication
order, family
Starts at attachment of virus to host cell
Eclipse phase
Assembly
Release
How do you study the viral growth curve?
- Virus is an obligate parasite. Infects cell culture
- Incubate viral cells and provide required environment of CO2 and 37 degrees celsius + media
- Examine viruses under microscope daily
- Collect cell culture supernatant via suction at various intervals
- Titrate virus using block assay. Based on this assay, we can see magnitude of viral replication.
The virus growth curve occur in two main phases: _______ and _______.
eclipse, logarithmic
Eclipse phase: the time from the _____ of the virus
into the ____ cell until the assembly of ____ _____
viruses (time?)
entry, host, new progeny, 1- 20 hrs
Exponential growth phase: number of _______ viruses increase _______ until reaching a _____ thus no more virus particles will be _______?
produced, exponentially, plateau, produced.
Eclipse phase is the time from viral entry into host cell until assembly of new progeny virus. This can take anywhere from 1-20 hours and depends on virus type. Virus cna not be detected; no pathoolgoy of virus yet in host tissue. After completion of first cycle of virus, virus numbers increase exponentially –> plateaus. This plateau is where the number of viruses becomes fixed, so no more viruses will be produced. Viral infected cells will then be ruptured/lysed.
??
Bacterial growth curve
After the stationary phase, the bacteria begin to die and the number starts to decrease dramatically. In viral replication, curve looks different.
Viral one step growth curve
Viruses are ______ _______ parasites. They require _______ cells for multiplication
obligate intracellular, living (source could be cell culture, embryonated chicken eggs, lab innucleatio)n.
Viruses hijack the cellular _______ and direct them for the synthesis of ______ proteins instead of _____ proteins resulting in cell ______.
machineries, viral, cellular, damage
Despite the high variability among viruses, they replicate in standard _______ series of stages/steps.
consecutive
What are the steps of viral replication?
1- Attachment/adsorption
2- Penetration
3-Uncoating
4- Synthesis of viral proteins= translation
5- Synthesis of viral nucleic acids
6- Assembly and maturation of Virions
7- Release of the progeny virus
Viral Replication cycle
When viral NA forms, and each copy of viral genome assembles with sets of viral proteins, it is released from cell surface (rupture, lyse, bodying?).
Viral replication cycle
Viruses attach to the target cell via
- ______ on the viral surface called virus _______ proteins
- ________ on the _____ membrane of the _____ cell
- Members of same virus family may use _______ _______.
Ligands, attachment, Receptors, plasma, target, different receptors
The influenza virus uses what…
Virus ligand?
Cell receptors?
Virus ligand = hemagglutinins (RBC+clumping) Virus has spike that causes hemagllutination of RBCs.
Cell receptors = sialic acid (Virus attaches to sialic acid)
The HIV virus uses what…
Virus ligand?
Cell receptors?
Virus ligand = gp-120 (Uses this to attach)
Cell receptors = CD4++ (HIV is a virus causing immunodeficiency; this virus attacks the immune cells, particularly CD4 and CD8 cells).
Viral receptors can be the primary determinant of ?
tropism
What are some examples of tissue tropism?
e.g. measles (skin cells) vs. mumps (salivary
gland
What are some examples of species tropism?
e.g. BHV-1 (Infects cattle but not human)
Bovine herpes virus 1
Tropism is defined as the affinity of certain viruses to certain ______. This is the way the virus responds to _______ _____ in order to ______ to and _____ cells
tissues, external stimuli, attach, infect
Describe how viruses attach to and infect cells.
Attachment proteins on the cell surface binds to the host cell receptors (proteins, glycoproteins, carbohydrates, and lipids)
• Viruses use these receptors for the attachment and entry to the
host cells
What factors affect the efficacy of viral attachment to cells?
- Density of receptors on the host cells (a high number of receptors –> increased viral replication)
- Density of the ligands on the surface of the virus (too many ligands -> infect a larger number of cells)
- Ratio of the virus/host cells
- Temp, and pH
- Presence of specific ions such as Ca
True or False: Some viruses require the presence of co/receptors
True
Neurotropic viruses act on which tissues/organs?
Examples?
Neuro = from neurons which is nervous tissue in brain and spinal chord; Nervous tissue (brain, SC, etc)
Rabies virus, Avian encephalovirus? (AEV)
Pneumotropic viruses act on which tissues/organs?
Examples?
Infects Respiratory tissues (lung, trachea, bronchi, etc)
Influenza, Rhinovirus, SARSCoV-2, MERS-CoV, RSV
Viscerotropic viruses act on which tissues/organs?
Examples?
Infects GIT tissues (cecum, etc).
Rotavirus, NDV, Enterovirus
Dermotropic viruses act on which tissues/organs?
Examples?
Skin
Poxvirus, LSDV, HPV, Cattle papillomavirus
Pantropic viruses act on which tissues/organs?
Examples?
Many tissues and organs
SARS-CoV-2 (May cause kidney failure), NDV, YFV
Rabies virus has a ______ host range
broad
HBV: ______ host range (virus infects certain cell types such as human hepatitis b virus) : _______
narrow, hepatocytes
Picornaviruses create a _____ either at ______ or ________ membrane to inject their genomic _____
pore, plasma, endosomal, RNA
Virus here inside uses positive pressure to make a hole in the cell membrane
Picornaviruses
Virus attaches to receptors on cell surface
virus injects genetic material into cytoplasm of cell
completes replication cycle
Saliac receptors
??
Removal of viral receptors
Application of monoclonal antibodies (MAb) for cellular receptors
MAb used as a sera for COVID-19
Virus can not replicate the cell
Describe the mechanism of virus entry (penetration)
- Binding of the virus to the host cell receptors
- Invagination of the membrane
- Formulation of an endocytic vesicle
- Release of the Virion into the cytoplasm
Mechanisms of enveloped virus entry (penetration) into the cells
–> Receptor mediated fusion with host cell plasma membrane
MHV is absorbed on the host cell surface and
enter the cell through ?
receptor mediated endocytosis
Most naked viruses enter the cells through ?
receptor-mediated endocytosis
Describe the process of naked virus entry
- Viral ligands-cell surface interaction causes a
clathrin-coated pit formation/invagination at the cell
surface - Clathrin-coated pit (CCP) bud off to form vesicles
- CCP shed, and the virus contains a vesicle fuse with lysosomes
- Low pH and protease in endocytic vesicle
- dissociate the capsid proteins
-and release viral NA
The objective of viral uncoating is to make the viral _______ ______ accessible to the host cellular ________ and _______ machineries to initiate the downstream viral ______ steps
genetic materials, transcription, translation, replication
There are two methods of viral uncoating based on the ?
type of the virus
Enveloped viruses undergo ?
complete uncoating
Viral uncoating of enveloped viruses
- Virus ______ with the _______ ______ of the host cell or to the plasma membrane
- The viral _______ released directly into the cell ________
- ________ starts immediately
fuses, plasma membrane, nucleocapsid, cytoplasm,Transcription
Describe the viral uncoating of naked viruses
- Removal of some viral _______ ______
- Viral genome perform all functions without being released from the _____ ____
capsid proteins, viral core
I-Membrane fusion
Ex: Paramyxoviruses: _____ uncoating
Ex: HIV: ______ uncoating
direct, partial
II- Receptors mediated endocytosis
Fusion in endosome
Ex: ?
Receptors mediated endocytosis
Lysis of endosome
Ex: ?
Influenza virus, Adenovirus
Mechanism of the viral fusion with the host cell
Once a virus enters the host cell, what happens?
- Production of viral structural proteins & enzymes
- Production of the viral genome
The viral genome may be either?
- Monopartite = linear genomes
- Multipartite = segmented genomes
Viral genome replication creates _____ genomic DNA or RNA for the ______ _____.
more, progeny virions
General criteria of the DNA virus replication strategies:
Most DNA viruses replicate in the ______ and use the ____ cell’s _____- dependent ____ polymerase. The exceptions to this are _______ because they replicate in the cytoplasm and has its own DNA polymerase.
nucleus, host, DNA, DNA, poxviruses
General criteria of the RNA virus replication strategies
Most RNA viruses replicate in the ______. The exceptions to this rule are ____ and _____ viruses.
cytoplasm, Retro, Influenza
RNA viruses have their own _____ dependent ____ polymerase
RNA, RNA
Negative sense ? ________ with the genome in a ______
Positive sense ? ______ ______ acts as mRNA. __ steps; _______ translated
The Retrovirus (______ sense)- _____ transcriptase; Also prepackaged with the genome in a _____
??
prepackaged, capsid, Viral genome, 6, Directly, positive, Reverse, capsid
Viruses are divided into ____/____ classes based on their _______ structure and the pathways they use to produce their ______.
6/7, genome, mRNAs
1-double stranded-DNA
Virus makes _____ of genome which acts as _______ for mRNA. One copy from proteins assembles together with ds-DNA to form ______ virus particle.
Example of ds viral replication in Nucleus = ?
Exmaple of ds viral replication in Cytoplasm = ?
copy, template, mature
Example of ds viral replication in Nucleus: Adenovirus and Herpesvirus
Exmaple of ds viral replication in Cytoplasm: Poxvirus
2-ss-DNA
single stranded + _____ ________ = double strand.
one strand is _______, the other strand acts as _____ strand. ____ strand assembles with protein to form _____ virus particle.
degraded, single, Single, mature
3-(+Ve) SS-strand RNA viruses
The viral genome itself act as mRNA
• It is translated directly into proteins
• Ex: Toga, Picorna, corona, Calici, Flavi)
4- (-Ve) SS-strand RNA viruses
Virus utilize their ____ transcriptase (____
dependant _____ polymerase to transcribe ____)
own, RNA, RNA, mRNA
5- ds-RNA viruses have segments of RNA and use its ____ enzyme RNA polymerase, which transcribes
each ______ into ____. Examples?
own, segment, mRNA, Reoviridae, Birnaviridae
6-Ambisense viral genomes
Examples?
Retroviruses
Part of genome that is positive sense = positive sense genome, negative = negative.
positive packaged with proteins to make mature virus.
RNA dependent DNA polymerase enzyme (unique phenomena of retrovirus b/c usually DNA dependent).
Summary of Virus replication strategies
dont need to know chart
Assembly of the viral Nucleocapsid occurs at the site of the _______ _____ replication (___ or ___)
Nucleic Acid, IC, IN
Individual structural units of the viral nucleocapsid is formed from ?
One or several proteins.
The capsid encloses the ___ to form the ___
NA, NC
How is the viral envelope acquired?
Viral glycoproteins migrates from the ribosomes via ER towered the cell membranes and grouped into cylindrical spikes.
The NC located underneath the plasma or nuclear membranes
• The matrix or the inner protein coat become incorporated above the NC
which is underneath the lipid membranes to immobilize the glycoprotein
spikes
??
Viruses are released from the infected cells by various mechanisms (2)?
Cell lysis (cytopathic viruses (CPE) and Budding
Cell lysis usually occurs in cases of?
Non-enveloped viruses
Cell lysis involves the accumulation of the viral particles either in the ______ or in the ______.
nucleus, cytoplasm
Some viral infections trigger _______ through activation of some signal pathways
apoptosis
During the process of cell lysis, cell _____ and ______ due to the release of lysosomal ______ can occur.
necrosis, death, enzymes
Most _______ viruses are released from the cell membrane of the infected cells by budding
enveloped
In the process of budding, viruses bud to the ________ ____ to start the _____ replication
cycle in a new cell.
extracellular fluid, second
TEM release of
Measles virus by
budding
Release of Retrovirus by budding
HIV released from a cell
Virus leaving host cell enveloped by host cell membrane
Example of viral replication
Type of genome: DNA
Family: Adenovirdae
Virus: Adenovirus
Site of replication: Nucleus
General rules here:
virus attaches to receptors
reploccation cycle
release by cell lysis or rupture
Example of viral replication
Type of genome: RNA
Family: Picornaviridae
Virus: Poliovirus / Foot and mouth disease
Site of replication: Cytoplasm
Example of viral replication
Type of genome: RNA
Family: Rhabdoviridae
Virus: Rabies virus
Site of replication: Cytoplasm
Which of the following is not true regarding viruses?
A. May be DNA, RNA, Single, or Double-stranded
B. All are obligate intracellular parasites
C. All have a protein capsid and envelope
D. All are true
- Viruses are different from bacteria because:
A. Viruses do not replicate by binary fission
B. Viruses are obligate intracellularly parasites
C. Viruses are sensitive to antibiotics
D. A , B
Describe the innate immune response of the body to a viral infection.
Natural barriers are nonspecific but play important roles in blocking viral entry to host. (eyes, nose, mouth, open wound, insects, blood transfusion, etc).
Describe the adaptive immune response of the body to a viral infection.
Antiviral antibodies = most famous strategy for antiviral response
Cytotoxic T cell
Helper T cell
What are the organs of the immune system in humans?
Tonsils = when you get tonsilitis or sore throat, tonsils are enlarged b/c considered one of the first mechanisms of defense in the body. Swelling due to inflammation, edema, and congestion.
Thymus
Bone marrow
Lymphatic vessels
Appendix
What are the organs of the immune system in avian species?
Harder gland
Thymus
Spleen
**Burse of Fabricus = factory of p cells in chickens; IBDV = ds RNA virus
What are the organs of the immune system in other mammalian species?
bone marrow
spleen
______ is an immune response of reflex action.
Alveoli = narrow pore (particles ___ micrometer in size can reach this)
Nasal mucosa is rich in ____.
Sneezing, 1, IgA
Cells of the immune system
Plasma cell = produces IG
MQ = important role in phagocytosis
Structure of immunoglobulins
Composed of heavy chains and two light chains
protease cleaves in Fab and FC regions.
What is the process and stages of phagocytosis
Process of phagocytosis
Chemotaxis = the phagocytes are attracted to site of infection or invasion
Adherence, Ingestion, Destruction = Phagocytes approach antigen, engulfs it, Within phagocyte there are enzymes that destroy pathogen
The virus itself does not secrete IFN. The IFN is secreted from viral infected cell.
An interferon is an _______ substance produced by the viral ______ cells of many ______ in a response to viral infection.
antiviral, infected, vertebrates
IFNs: cell coded protein induced by _____ _____ _____ (?)
foreign nucleic acid, Viral-NA
IFNs are ___-____ to the cell but are able to inhibit the _______ of the virus in the cells of _______ species.
non-toxic, multiplication, homologues
IFNs: glycoproteins, stable at ____ pH = ? , resist heat at ___°C for ___-hr
acid, 2, 56, one
IFNs are not virus _____ but cell specific in their ______ and _____.
specific, production, effects
IFNs protect specific cells against different types of viruses
IFNs can be inactivated by treatment with ______ enzymes such as _____, ______, _____.
proteolytic, trypsin, pepsin, chymotrypsin
IFNs: are not affected by _____, _____, and ____
DNAse, RNAse, lipase
IFNs production increased with the increase of temp up to ___ °C.
40
The greater the fever, the more impactful on the immune system.
What are the types of Type I IFNs?
IFN-alpha
IFN-beta
What are the types of Type II IFNs?
IFN-gamma
Examples of IFN-alpha
Leukocyte IFN (Monocytes) B-Lymphocytes
Examples of IFN-beta
Fibroblast IFN
Epithelial cells
Examples of IFN-gamma
Immune IFN
-Some activated T-Cells
-NK cells
Produced by immune cells including T cells and NK cells
IFNs are sets of proteins released by the ____ infected cells which react with the surrounding ____-infected cells rendering
them _____ to this ____ of viral infection.
virus, non, resistant, type
Cytokines activate IFN in a +/- feedback mechanism.
alpha = 20
beta = ?
gamma = ?
Infectious or inactivated virus induce the production of _____ for ___-___ __ after exposure
IFNs, 20-50, hrs
Vaccine could be live, inactivated, etc.
Inactivated can still trigger the production of IFN against upcoming infection.
Viruses that might multiply slowly and not damage the cell or the synthesis of protein early are good ___ ______.
IFN inducers
Usually, the virus that have slow replication cycle give the cell the time to respond by producing IFN.
Cells of _____ ______, _____, and ______ are highly IFN producers.
bone marrow, spleen, macrophages
Polynucleotides (Poly-IC) are ?
good IFN inducers.
Analog of ds RNA. When we want to study effect of IFN in certain signal transduction pathways in the lab.
CPG = immune stimulator for IFN
DO NOT NEED TO KNOW THIS
Receptors for the type-I INF ligand binding
trigger ?
JAK/STAT
The transduction pathway and eventually activates the?
- ________ pathway
- _______–_________ pathway
Antiviral, Immuno-regulatory
DO NOT NEED TO KNOW THIS
IFNs antiviral mechanisms of action:
1- Block ______ _______ synthesis
2- Block the ______ of viral _____
viral mRNA, translation, mRNA
TLR0 activates IFN
Actions in cell:
1. Degrading of viral RNA
2. Prevent viral translation
3. Block viral assembly between viral NA and viral protein. Mature viral particle will not form.
4. Destroy viral protein
5. Prevents viral release.
6. ?
Results in viral replication inhibition.
Viral peptides are produced from _____ in the _____ and transported to the __ to bind to Class __ MHC complex.
proteins, cytosol, ER, I
The peptide/Class I MHC complex are transported to the ____ _____ and displayed for recognition by the ___
cell surface, CD8+
Viral peptides
Get text on this slide 13
Describe the antigen processing and display by the class II-MHC molecule.
Virus infected cell secretes IFN alpha and beta
Host sometimes triggers apoptosis to prevent virus from infecting other cells (if virus did not find permissive cell for replication, it won’t replicate).
IL12 and 18 stimulate NK cells –> lysis of infected cell and IFN gamma –> activate antigen presentaiton, promote?
nothign here
B-lymphocyte triggers plasma cells and antibody secretion. Antibodies attach to receptor on cell so virus can not infect cell.
CD4 cascade
CD8 cascade
What is the immune response to viral infection?
- Type-1 IFNs (after a few hours –> 4 days)
- NK cells (at around day 2 days –>4 days then slowly declines
Adaptive immunity by day 5 - Cytotoxic TcellsL (start at 2 –> highest at 7 then plateaus).
- First show of antibody anywhere from 5-7 days
- Virus titer is very high in beginning, with progression of infection, eventually decreases.
Antibodies and T cells are high after course of infection.
Antiviral-adaptive immune response
T cell recognize the virus infected cells then ________ in number to tackle the infection.
increase,
What is the function of CD4+ cells?
Attract other immune cells and stimulate B-
cells to produce.
What is the function of CD8+ cells?
- Slow down the viral infection
- Kill the virus infected cells
T cell receptors (TCRs): specifically _____ and ______ to some antigenic peptide bound to ___ molecules.
recognize, bind, MHC
Cytotoxic-T cells: ____ the virus-infected cells through the release of some ____ ______ _____.
Kills, toxic chemical mediators
Virus infected cells express multiple ____
indicators and viral infection _____ ______ of cell proteins
stress, inhibits, expression
NK cell’s multiple _______ and _____
receptors are bound.
activating, inhibiting
When activation stimuli overcome inhibition,
cell _______ is initiated.
death
_______ granules orient to the cell junction and are released.
Cytotoxic
________ creates access to the target cytosol delivering _____ (serine proteases that mediates cell _____)
Perforin, Granzymes, death
Describe Cytotoxic T Lymphocyte (CTL) killing of a virus infected cell
Granzyme A and B
Perforin = makes pore/perofration of cell –> cell contents leaks out –> cell dies
Ends with cell death = prevents virus access to permissive cell
What are the antiviral effects of antibodies?
What are the strategies of the immune system protection against viruses
promote phagocytosis or block viral adherence by saturating receptors. no receptor = no entry
core receptors work on attachment of virus to the cell, so there are some meds that work on that mechanism; must take within few hours of testing positive to covid
All of these strategies work synergistically
Describe the immunity in new born animals
What are the types of the placenta and Ig transfer
________ intake is required to protect young animals against septicemic disease
Colostrum
Colostrum must drink during the first few hours after birth otherwise will be immunocompromised
Rich with proteins and the intestinal pores during the first few hours are bigger in order for Ig to pass through. After few hours, pores of intestine narrow and IG can not pass.
Colostrum has a cocktail of IG to pass to newborn.
Relationship between Ab titer of the mother and the age at
which to vaccinate her offspring with modified live virus
vaccine
Blue eye diseases in dogs is an _____ response to viruses could be on some occasions, a ______.
immune, disadvantage
Blue eye diseases in dogs can develop in animals infected or vaccinated with the ______ ____ vaccine.
ICHV-1 live
Dogs with blue eye diseases develop ?
anterior uveitis leading to corneal edema and
opacity.
In cases of blue eye disease in dogs, the cornea is ?
infiltrated by neutrophils, attracted by virus antibody complexes deposited in tissue
In cases of blue eye disease, there is an accumulation of ______ which release ______ that damage _______ _____ cells resulting in _________ and _____, then _______ _____ in dogs
neutrophils, enzymes, corneal epithelial, inflammation, edema, corneal opacity
Blue-eye disease develops about __ to __ _____ after the _____ of infection and usually resolves _______ once the virus is eliminated.
1, 3, weeks, onset, spontaneously
Blue-eye disease is an example of?
Type III hypersensitivity
Aleutian diseases in the mink is a persistent ________ infection first recognized in ______ coat color. Animals develop complex immune _____ including ________ and _____.
Parvovirus, Aleutian, lesions, glomerulonephritis, arteritis
Usually find kidney failure due to glomerulonephritis
The serum of animals infected with aleutian disease demonstrates ______ _________ patterns as well as _______ ______. The _____ globulin accounts for ___% of serum proteins (The normal level is ___%)
protein electrophoretic, polyclonal
gammopathy, gamma, 62.4, 14.3
What are Some Strategies of viral evasion to the host immune destruction
- Inhibition of ______ presentation
- Modulation of MHC Class ___ presentation
- ______
- _______ variation
- Prevention of _______
- _______ targeting
- Destruction of ______ cells
antigen, I, Latency, Antigenic, apoptosis, Cytokine, immune
Immune evasion strategies is different from between viral classes. E.g. influenza viruses, there are some common influenza viruses called antigenic drift. When the host cell recognizes HA spikes, when the virus invades the host again the host is ready to counteract. The virus, however, will mutate to outsmart the host.
Start to see viral detection post eclipse phase.
Who is the father of immunology?
What did he do?
Edward Jenner
Collected specimen from cowpox sores, injected cowpox specimens into a boy named James Phipps, the injected james with specimens from smallpox sores. James Phipps did not get infected with smallpox.
IFN is a ____ specific not ____ specific, so it can work on viruses of most ______ species. This is one strategy if we do not know anything about the virus.
cell, virus, homologous
The first generation of conventional vaccines included?
Live attenuated vaccines, heterologous viral vaccines (e.g. cowpox, smallpox), inactivated viral vaccines.
Viral vaccines are classified into?
First generation, second generation, third generation
The second generation of conventional vaccines included?
subunit vaccines, genetic engineering vaccines
chemotherapeutic agents = topical creams, etc
The third generation of conventional vaccines included?
DNA vaccines
What is the main purpose of a vaccine?
To render the virus non infectious without destroying its antigenicity/immunogenicity
Prophylaxis is defined as when a vaccine is given to animals to ?
protect them against some expected viral diseases.
You are a step ahead of the virus BEFORE it infects you.
Pregnant animals are vaccinated to transfer the ______ immunity to their offspring
passive
Vaccinate the mother so that antibodies can be transferred via colostrum.
Vaccines must be given in _____ before _____ infections with reasonable time to allow the _____ to develop.
advance, expected, immunity
Vaccines may be administered during an ______ with some viral infections in an attempt to protect the ____-_____ animals at risk
outbreak, non infected
emergency vaccine; you have a viral infection in your herd/flock and you vaccinate entire herd/flock.
What criteria could an ideal vaccine have?
- Produce some kinds of solid immunity (long lasting)
- Produce an early protective immunity
- Provides protection against pathogen variants
- Produce a life long immunity preferably in a single dose
- Prevents infection
- Do not produce any carrier state in vaccinated animals
- Can be administered by mass immunization
- Safe and stable
- None or minimal side effects in the vaccinated animals
- To be fit for a long term storage vaccine banks
- Thermostable to avoid cold chain
- Differentiation between infected and vaccinated individuals (DIVA) = if you take a blood (serum) sample, find antibody agonist, how do you know if the antibody is due to vaccine or natural infection? Medical history - are they vaccinated or did they suffer from an infection before? OR markers = some ? are expressed in vaccine treatment.
- Cost Effective
- Produced inexpensively and in large quantities
What are the general strategies for making viral vaccines?
Attenuation = weakened virus, mimics live virus b/c you are just making it slightly weaker. Most ideal vaccine
Inactivation = we killed virus, but its protein immunogenic part is active so it stimulates immune response to produce antibodies.
Fractionation
Cloning = copies of certain genes and use prokaryotic expression system (bacteria like e.coli), etc.
What are examples of active immunity?
natural infection, vaccination
Must be stimulated and produce its own material
What are examples of passive immunity?
Maternal immunity, hyperimmune sera
IgG is a viral antigen- processed/presented in the context of MHC-__ by antigen presenting cells (_____ cells, ____, etc.
- _______ virus, proteins
- No need of virus ______ and/or viral protein ______ in cell
II, dendritic, macrophages, Inactivated, replication, synthesis
IgA is a viral antigen that must contact ______ lymphocytes
- ____ virus introduced via _____ route
submucosal, Live, mucosal
This is in your body secretions: Vaginal tract, GI tract, saliva, etc.
Cytotoxic T cells (CTL) are viral proteins that are synthesized in the ______ cells, antigens processed and presented at cell ______ in the context of MHC - __
- ____ virus
- _____ vaccines (protein synthesized in the cell).
infected, surface, I, Live, DNA
*, ****= magnitude or concentration of the immune markers
Sometimes, some vaccines are based on intranasal vaccine. By vaccinating here, IgA produced in excessive amounts to prevent virus from attaching.
What design parameters must be taken into account when designing and preparing a vaccine?
- Clear understanding of _________ of the target virus
- Consider characteristics of the virus for selection of vaccine ____ and _______ ______
- _______ vs ______ immunity, or _____
- _______ vs ______ vaccination or both
- 90% of all viruses enter through ______ surfaces (point of entry - mouth, eyes, nose, etc.)
pathogenesis, type, delivery route, Cellular, humoral, both, Mucosal, parenteral, mucosal
What safety and efficacy must be taken into account when designing and preparing a vaccine?
- Effective in _______ animals
- Minimal ______ reactions
- Minimal _____ damage
- Safety in ________ animals (no _______)
- Induction of both ______ & ______ immunity
- ______ immunity
- ______-term memory
newborn, adverse, tissue, pregnant, abortion, humoral, cellular, Mucosal, Long
What practical considerations must be taken into account when designing and preparing a vaccine?
_______/___ in handling
• ________,____-shot
• Low _____ of production
• ______
• _____-free delivery
Economical, Ease, Multivalent, one, cost, Stable, Needle
What are the principles of a live, attenuated vaccine?
• Weakened virus/es under laboratory conditions
- isolate virus, weaken in lab
• They will grow in a vaccinated individual, but because they are weak, they will cause no or very mild form of the diseases
- this is ideal
What are some examples of some current live attenuated vaccines
- Oral polio vaccine (OPV)
- Measles
- Rotavirus
- Yellow fever
What is the immune response after a live attenuated vaccine is given?
- Stimulates the production of excellent immune response (humoral + CMI) similar to the wild type virus
- Stimulates the production of memory cells
What are the side effects of a live, attenuated vaccine?
• Revert to _______ and causing the ______ form of diseases of the _____ type virus in some cases
• Potential harms to the __________ personnel
• Contaminated ____ ______ vaccines: Rota virus and Measles virus if
grow in cell culture
• Not recommended in case of ______
virulence, original, wild, immunocompromised, cell culture, pregnancy
What are some preparation methods for a live attenuated vaccine?
- Passage the wild type virus in unusual host
- Passage of the wild type virus in unusual conditions
Live Attenuated Vaccine - Passage of the wild type virus in unnatural host
How can we prepare a LAV? Either by continued passage of the wild type virus in ______ _____ - progressive adaptive mutations - virus adapts to new host- retain their capacity for transient growth within an inoculated natural host.
unnatural host
Infect virus in embryonated egg or cell culture → continuous passage in unusual host → several mutations occur → virus becomes attenuated.
LAV-Passage of wild type virus under abnormal culture conditions
By growing a pathogenic virus for prolonged period under ______ culture condition → ____ mutants better suited to growth in the ______ culture conditions and ____ capable of growth in the ____ host.
Provide an example.
abnormal, select, abnormal, less, natural
Each virus has a specific environment that it can replicate in.
E.g. If we grow an influenza virus in lower temp in culture → let it go through several rounds of replication → virus become adapted to this environment. Cold adapted vaccines grows well in the upper respiratory tract of nose and is an efficient way of immunization. If we swallow it, it will be inactivated?
What are the advantages of a live, attenuated viral vaccine?
- Core antigen is still the same so immune response is similar to that if infected with wild type virus.
What are the disadvantages of a live, attenuated viral vaccine?
- Revert to virulence is a CORE disadvantage.
- Heat labile aka heat sensitive. Some vaccines are freeze dried so when you reconstitute it, it can lose some of its potency.
What are some examples of modified live attenuated vaccines?
- Canine parvovirus (CPV) propagated in feline kidney cells
- Feline herpesvirus (FHV) propagated in feline kidney cells
- Infectious laryngotracheitis virus (ILT) propagated in chicken kidney cells, embryonated chicken eggs
A live heterologous viral vaccine is made up of ____ viruses that are _______ _____ to each others thus protection arise from the presence of the _____ ______ _____.
two, antigenically, related, cross reacting antigens
E.g. cowpox and small pox
The heterologous vaccines are ?
naturally attenuated
What are examples of heterologous vaccines?
- Pigeon poxvirus protects against ?
- Shop fibroma virus rabbit protects against ?
- Sheep-pox virus protects against ?
- Herpesvirus of turkeys (HVT) protects against?
-Rinderpest virus (RPV) [aka?] and measles virus protects against ?
Fowl poxvirus, Rabbit myxomatosis, Lumpy skin diseases virus (LSDV), Marek’s diseases virus (MDV), Cattle plague, Canine distemper virus (CDV)
An inactivated (killed) viral vaccine is made by taking a ______ virus, _________ and ________ either by ______ methods or ______ methods.
whole, propagating, inactivating, physical, chemical
What are the physical methods used to form an inactivated viral vaccine?
- Formalin
- Heat
- Beta propiolactone
What are the chemical methods used to form an inactivated viral vaccine?
- UV
- Irradiation
usually targets viral NA → inactivates it
One major inactivated viral vaccines is that the virus does not ______ in the vaccinated animals requires a _____ antigenic mass.
Also, the Vaccine may be _______ to produce and requires an ______ to enhance the magnitude of the immune response.
replicate, larger, expensive, adjuvant
Inactivated (killed) viral vaccines ______ the viral nucleic acids without affecting the _______ or _______ proteins against them the antibodies are produced.
inactivate, capsid, envelope,
What are the advantages of inactivated (killed) viral vaccines?
What are the disadvantages of inactivated (killed) viral vaccines?
Short term only because it is a killed virus
Adjuvant means?
to add or to help.
Depot adjuvant: Causing _______ formation at the site of ______
- For example: ?
depot, injection, mineral compounds, oil-based adjuvants, liposomes
Takes awhile to disperse into body, then immune system has continuously supply of antigen → long lasting.
Particulate adjuvant acts as adelivery vehicle for the ______, which may help in targeting _____ to immune.
antigens, antigens
Immuno-stimulatory adjuvant or ______ are ___ nm large particles made up of ______ (Quil __), _____, _____ and ____, held together by _____ interactions between the first three components.
ISCOMs 40, saponins, A, lipids, cholesterol, antigen, hydrophobic
Make questions based on this chart
In 1937, Freund demonstrated the effect of ______ (_______) ____ mixed with killed _______, referred to as Freund’s complete adjuvant (FCA).
mineral, paraffin, oil, Mycobacteria
The _____-in-___ emulsion without ________, known as Freund’s incomplete adjuvant (FIA), has been used in a number of veterinary vaccines.
water, oil, Mycobacteria
An adjuvant is a component that potentiates the immune response to an _____ and/or ____ it towards the desired immune response.
antigen, modulates
What are the effects of adjuvants on the immunogenicity of viral vaccines?
- Adjuvants is an additional substance added to the vaccine. It is meant to help make the vaccine more effective, especially killed vaccines.
- Makes more cost effective
- etc.
Curve of immunity without adjuvant → dropped to zero.
The vaccine with an adjuvant → immune response started earlier than one without adjuvant → long term immune response
Live vaccines can be administered in ________ surfaces (_______, ___ or _____) OR via ______ routes (?)
Immune cells produced from mucosal administration include _____ (mucosal immunity), Ig_, ______ __ cells.
Provides protective immunity for both _____ and ____ infections.
Immune cells produced from parenteral administration include ____ and but no _
mucosal, Intranasal, oral, genital, parenteral I/M, sub cut, IgA, G, cytotoxic T, systemic, local, IgG, Tc, IgA
Where are inactivated vaccines administered?
It is a killed vaccine so it has to be injected, either IM or ID.
Parenteral route
• Administered I/M or subcutaneous routes only •Generates IgG only,
• no cellular immunity –
• protective immunity for systemic infections associated with viremia; to lesser extent local infections.
Some vaccines require boosting. in order to maintain immunity, must get second injection to boost immunity. Depending on level of immunity you maintain, you may or may need anther dose.
What are some common disadvantages of the conventional viral vaccine?
- Reversion to virulence
- Incomplete inactivation
- Contaminations
- Secondary effects
- Inflammation, granuloma, fever, hypersensitivity
- Immuno-suppression
- Inability to differentiate vaccinated from infected animals
- Cold-chain requirement
DNA vaccine or RNA vaccine use depends on strategy want to use.
RNA = Insert gene into BAC or YAC. Use product of this in order to induce protein.
A subunit or split vaccine is prepared from viral components devoid from viral ____ such as capsid and envelop proteins. These components are responsible for the induction of the _______ _____ against the virus of concern
NA, immune response
What are the advantages of subunit vaccines?
- It is safe because it is free from ____
- It does not induce ______ reactions which induced by some proteins in the other viruses
-It is highly immunogenic in ____ doses
NA, febrile, high
Genetically engineered subunit vaccine (single gene)
- Virally vectored subunit vaccine
- Plasmid based/vectored subunit vaccine - expressed in bacteria
- expressed in yeast and mammalian cells
The DNA vaccine (third generation) are based on the introduction of a ____ ______ into the host cells. The plasmid carries a ____-coding gene that transfects cells ___ ____ at very ___ efficiency and expresses an ______ that causes an immune response.
DNA plasmid, protein, in vivo, low, antigen
Insert plasmids that carry gene of interest into bacteria. Make large amounts of target gene → use as DNA vaccine.
How are DNA vaccines administered?
• Injection of the prepared DNA vaccine in saline solution
- Intramuscular (IM)
-Intradermal (ID)
-May be assisted by the electroporation method
• Gene gun delivery approach
Describe the molecular mechanism of gene immunization
Red = gene of interest
Gene gun -→ inject in muscle → enters cell → must make sure that the vaccine will trigger immune respinbse throughbtwo pathways: humor immunity = b cell CMI = present antigen to MHC I and II → produce CD4 + and CD8+.
What are the advantages of DNA vaccines?
• Plasmids are easily manufactured in large amounts
• DNA is very stable
• DNA resists temperature extremes so storage and transport are simple and easy
• DNA sequence can be changed easily in the laboratory. Virus has been changed so we can map changed part (a,b,gamma,omicron). modify vaccine by inserting updated version
• Mixtures of plasmids could be used as a broad-spectrum
vaccine (multivalent vaccines)
• The plasmid does not replicate and encodes only the proteins of interest
• There is no protein component, so there will be no immune response against the vector itself
• Because the antigen is presented in the context of both MHC-I (a CTL response) and MHC-II (antibody), the vaccine offers a better protection against diseases.
What are the disadvantages of DNA vaccines?
- Inducing antibody production against DNA
- May have a relatively poor immunogenicity
- May induce immunologic tolerance by antigens expressed inside host body
no vaccine will ever give 100% protection
Describe maternal immunity
- passive transfer of humoral immunity via colostrum/milk/egg yolk
- protection of newborn and young animals
- prevention of enteric infections
- No cellular immunity and lacks memory immune cells.
What is an example of maternal immunity?
- Transmissible gastroenteritis virus (TGEV) infection of pigs.
- Corona and Rotavirus infections in calves.
Vaccination of young animals: Serum IgG antibodies persist for :
- up to 6 months in cattle, horses
- up to 4 months in dogs, cats, pigs
- up to 1 month in chickens, turkeys
Maternal antibodies _______ with the immunization of young animals
interfere
How to overcome the effects of the MAbs?
- To do repeated vaccinations before the predicted loss of antibody
- Using the intranasal route (mucosal) when modified live vaccines are used
What are the common causes of vaccination failure?
- Wrong strain/serotype of organisms
- Method of production destroy protective epitopes- inactivated vaccines
Failure of an effective vaccine to stimulate protective immunity may be due to:
- Unsatisfactory administration
- A live vaccine may have died
- Timing - Vaccination Schedules - Lag time 4. Animal to animal variability
What are some potential adverse effects of vaccines?
For example, for the influenza vaccine, those who are sensitive to albumin should not take vaccine. People with hypersenitivity to albumin, when get vaccine, get hypersentiivty → anaphylactic shock.
Explain the ring vaccination strategy
If you have a viral outbreak,
- The vaccination of all susceptible herds in a prescribed area around an outbreak of an infectious disease
- Ring vaccination controls an outbreak by vaccinating and monitoring a ring of herds around each infected herd
- The idea is to form a buffer zone of immune herds to prevent the spread of the disease
When is herd immunity achieved?
Herd immunity is achieved when large number of individuals become immune against circulating infectious diseases
Herd immunity restricts?
the spread from person to person
Herd immunity can be achieved when?
Large number of community either recovered from infection or vaccinated against certain infectious diseases
In some research, herd immunity may be achieved if up to 70% of the population are immune (recovered/vaccinated)
Standard Human/Veterinary vaccine pipelines
Humans: 10-15 years for vaccine development, production, approval
Veterinary: 3-6 years
SARS-CoV-2 vaccine candidates
dont have to know
SARS-CoV-2 vaccines
Protein synthesis-Central Dogma
Design of some SARS-CoV-2 mRNA vaccines
not all spike glycoprotein but the receptor binding domain.
Design of some SARS-CoV-2 mRNA vaccines
Messenger RNA vaccine
Old theory; very weak and not stable; can be degraded very easily But with encapsulating with the LNP, it is much stronger and can produce a longer immunity.
Pfizer-BioNTech COVID-19 Vaccine (BNT-162b2): Mechanism of Action
Immune Response to SARS-CoV-2 mRNA Vaccines
