Immunology - Vaccination

Question 1

Vaccine definition

Answer 1

Something that stimulates the immune system without causing serious harm or side effects, single most cost-effective tool for improving health, population intervention

Question 2

Aim of immunisation

Answer 2

Provoke immunological memory to protect individual against a particular pathogen if they later encounter it

Question 3

Ideal vaccine

Answer 3

Completely safe, easy to administer, single dose, needle-free, cheap, stable, active against all variants, lifelong protection

Question 4

Smallpox eradication

Answer 4

300 million dead in 20th century, 1940s heat stable vaccine, 1959 resolution on global smallpox eradication, 1966 resolution on intensified Smallpox Eradication Programme, 1979 smallpox officially eradicated (last man Ali Maow Maalin, Somalia 1977)

Question 5

How vaccines stop infection

Answer 5

• Vaccination is the generation of immune memory in absence of harmful infection
• Prevention of entry: antibody blocks entry (opsonization driven by constant region), macrophage engulfs pathogen
• Killing infected cells: CD8
• Boosting immune response: B cell makes Ig, CD4 T cells help (train CD8, related to MHC)

Question 6

R0 (Basic reproduction number)

Answer 6

• Number of cases one case generates on average over course of infectious period
• <1 infection will die out in the long run, >1 will be able to spread in a population
• Rt = real time R0

Question 7

What’s in a vaccine

Answer 7

Antigen (in one of these forms: inactivated protein (ie tetanus toxoid), recombinant protein (ie Hep B), live attenuated pathogen (ie polio/BCG), dead pathogen (ie split flu vaccine), carbohydrate (ie S. pneumoniae)), adjuvant (normally alum, sometimes something proprietary), stabilizing components (ie buffers - PBS), water

Question 8

Inactivated toxoid vaccines

Answer 8

ie tetanus toxoid, chemically inactivated toxin form, induces antibody which blocks toxin from binding to nerves, advantages: cheap, well characterized, safe, in use for many decades; disadvantages: requires good understanding of infection biology, not all organisms encode toxins; usually: toxin binds to cell surface receptor -> endocytosis of toxinoreceptor complex -> dissociation of toxin to release active chain which poisons cell; with vaccine neutralizing antibody blocks binding of toxin to cell-surface receptor

Question 9

Recombinant protein vaccines

Answer 9

ie hep b surface antigen (HbsAg), recombinant protein from pathogen, induces classic neutralizing antibodies, advantages: pure, safe; disadvantages: relatively expensive, not very immunogenic, has not proved to be answer to all pathogens

Question 10

HbsAg production

Answer 10

hep b virus -> isolated surface antigen gene -> insertion into yeast -> modified yeast cells produce HBsAg

Question 11

RSV F

Answer 11

Stalk and head change conformation pre- and postfusion with host cell, motavizumab binding site on stalk of prefusion structure

Question 11

Bacterial coats

Answer 11

Bacteria often have a capsule made of polysaccharide which is not very good at inducing a B cell response (T-independent antigen) so alternative approaches needed to prevent infection

Question 12

Conjugate vaccines

Answer 12

ie S. pneumoniae, polysaccharide coat component coupled to an immunogenic “carrier” protein, protein enlists CD4 help to boost B cell response to the polysaccharide, advantages: improves immunogenicity, highly effective at controlling bacterial infection; disadvantages: cost, carrier protein interference, very strain specific, polysaccharide alone is poorly immunogenic

Question 13

Conjugate vaccine - mechanism

Answer 13

Antigen engulfed and processed onto MHCII (major histocompatibility process II, dendritic cells and B cells with polysaccharide specific antigens) and then:

• DC: MHCII/peptide TCR (t-cell receptor) interaction
• B cell: cognate T-cell/B cell interaction -> plasma cell produces anti-polysaccharide antibodies

Question 14

Dead pathogen vaccines

Answer 14

Ie influenza split vaccine, rather than using a single antigen it is a chemically killed pathogen, induces antibody and T-cell responses, advantages: leaves antigenic components intact and in context of other antigen, immunogenic because of the inclusion of other components, cheap, quick; disadvantages: fixing/killing can alter chemical structure of antigen, quite “dirty”, requires capacity to grow pathogen (H5N1, avian flu), vaccine induced pathogenicity at risk, risk of contamination with live pathogen (polio, hasn’t happened since 1953)

Question 15

Live attenuated vaccines

Answer 15

Ie BCG+LAIV+OPV, pathogens attenuated by serial passage which leads to a loss of virulence factors, because they replicate in situ they trigger the innate response and boost the immune response, advantages: induce strong immune response, can induce local one in site where infection might occur ie LAIV; disadvantages: can revert to virulence, can infect immunocompromised (BCG/HIV), attenuation may lose key antigens, can be competed out by other infections

Question 16

Adjuvants

Answer 16

Induce “danger signals” that activate dendritic cells to present antigen to T cells, part of licensing the response, substances used in combination with a specific antigen that produces a more robust immune response than the antigen alone, adjuvant stimulates DC -> DC uptakes antigen and moves to lymph node -> upregulates stimulatory signalling and cytokines

Question 17

Adjuvants in existing vaccines

Answer 17

Increasing use of adjuvants in vaccines, alum common, new platforms include: AS03 (GSK adjuvant in shingrix (chicken pox), mosquirix (malaria), new experimental TB and COVID), MF59 (seqirus (influenza))

Question 18

Why new vaccines?

Answer 18

Changing (aging) demographics, changing environment (dengue/other arboviruses), new diseases (COVID-19), old diseases without a cure (HIV, TB, malaria), antibiotic resistance (MRSA)

Question 19

Barriers to future vaccines

Answer 19

Scientific challenges, injection safety, logistics/cold chain, development issues (time (8yrs in 1960s, 15 now), cost of vaccine development high, cost of product), public expectation of risk-free vaccines,

Question 20

Traditional vaccines are effective at…

Answer 20

Eliciting humoral responses, are protective against invariant agents, the more diversity the less protective

Question 21

High variation of target organism

Answer 21

There are many circulating viruses, classic immune memory will only recognise one of these strains, therefore vaccine antigens need to cover all the variety

Question 22

Vaccines for COVID

Answer 22

Protein: - requires correct antigen, + good safety profile, +/- may need adjuvant, - slow manufacture
mRNA: + potentially low cost, potential for rapid production
saRNA: + potentially effective at lower doses than mRNA vaccines, - untested in humans before COVID
DNA: + relatively low cost, - poorly immunogenic
Live attenuated: - requires viral gene knowledge, - risk reverting to virulent virus, + immunogenic
Live recombinant viral vector: - potentially pre-existing immunity against the vector, + rapid production
VLPs: + can stabilise protein conformation, + immunogenic, - slower to manufacture
Inactivated: - risk of vaccine enhanced disease, + rapid production, + mature platform

Question 23

Clinical trials

Answer 23

Preclinical -> drug approved for testing in humans -> phase 1 (20-80 participants, safe?) -> phase 2 (100-300 participants, effective) -> phase 3 (1000-3000 participants) -> drug submitted for MHRA approval -> drug approved -> phase 4 (1000+ participants)

Question 24

How new vaccines are introduced into UK schedule

Answer 24

Recommendations for vaccine policy (Joint Committee on Vaccination and Immunisation (JCVI)), vaccine policy decisions (Department of Health, DH), licensing of vaccine (Medicines and Healthcare products Regulatory Agency (MHRA)), purchase of vaccine (DH from pharmaceutical companies), control of vaccine including batch release (National Institute for Biological Standards and Control (NIBSC)), post licensure assessment and changes (PHE/JCVI, epidemiology, assessment, trials)

Question 25

Scheduling - considerations

Answer 25

Aim, need, scheduling with other vaccines, availability, cost, population accessibility, cultural attitudes and practices, facilities available for delivery

Question 26

Routine immunisation schedule

Answer 26

• 8 weeks: diphtheria, tetanus, pertussis (whooping cough), polio, haemophilus influenzae type b, HepB (vaccine given and trade name: DTap/IPV/Hib/HepB, infanrix hexa; usual site: thigh), meningococcal group B (MenB, Bexsero; left thigh), rotavirus gastroenteritis (rotavirus, rotarix, by mouth)
• 12 weeks: diphtheria, tetanus, pertussis, polio, Hib, HepB (DTaP/IPV/Hib/HepB, infanrix hexa; thigh), pneumococcal 13 serotypes (pneumococcal conjugate vaccine (PCV), prevenar 13; thigh), rotavirus (rotavirus, rotarix; by mouth)
• 16 weeks: diphtheria, tetanus, pertussis, polio, Hib, HepB (DTap/IPV/Hib/HepB, infanrix hexa; thigh), MenB (MenB, Bexsero; left thigh)
• 1 year: Hib, MenC (Hib/MenC, menitorix; upper arm/thigh), pneumococcal (PCV booster, prevenar 13; upper arm/thigh), MMR German measles (MMR, VaxPRO or Priorix; upper arm/thigh), MenB (MenB booster, Bexsero; left thigh)
• Eligible paediatric age groups: influenza each year from September (live attenuated influenza vaccine LAIV, Fluenz Tetra; both nostrils)
• 3 yrs 4 mo: diphtheria, tetanus, pertussis, polio (dTaP/IPV, Repevax or Boostrix-IPV; upper arm), MMR (MMR check dose 1, VaxPRO or Priorix; upper arm)
• 12-13: cancers caused by human papilloma virus (HPV) types 16 and 18 and genital warts caused by type 6 and 11 (HPV 2 doses 6-24 months apart, Gardasil; upper arm)
• 14: tetanus, diphtheria, polio (Td/IPV check MMR status, Revaxis; upper arm), Meningococcal groups ACWY disease (MenACWY, Nimenrix or Menveo; upper arm)
• 65: pneumococcal 23 serotypes (pneumococcal polysaccharide vaccine PPV, pneumococcal polysaccharide vaccine; upper arm)
• 65+: influenza each year from September (inactivated influenza vaccine, multiple; upper arm)
• 70: shingles (shingles, zostavax, upper arm)

Question 27

Who invented vaccination?

Answer 27

Jenner (smallpox)

Question 28

Vaccines against viruses are usually

Answer 28

either live-attenuated or killed

Question 29

For which illness is the current vaccine most effective?

Answer 29

Tetanus

Question 30

Successful immunisation can be impaired by

Answer 30

Maternal antibodies

Question 31

Classification: vaccine for corynebacterium diphtheriae

Answer 31

Toxin-based

Question 32

Classification: vaccine Haemophilus influenzae type B

Answer 32

Conjugate polysaccharide

Question 33

Classification: vaccine MMR

Answer 33

Live attenuated

Question 34

Classification: vaccine Bacille Calmette-Guérin (BCG)

Answer 34

Live attenuated

Question 35

Classification: vaccine Influenza A virus

Answer 35

Live attenuated, killed

Question 37

Classification: vaccine Sabin polio

Answer 37

Live attenuated

Question 38

2 most important contributions to public health in past 100 years

Answer 38

sanitation and vaccination

Question 39

modern immunology grew from the success of…

Answer 39

Edward Jenner’s smallpox and Louis Pasteur’s chicken cholera vaccines

Question 40

greatest modern immunology achievement

Answer 40

global eradication of smallpox (1979)

Question 41

goal

Answer 41

long-lasting and protective immunity

Question 42

Peloponnesian War -> Thucydides

Answer 42

2 successive plague outbreaks in Athens, ppl that had it first time were immune second

Question 43

variolation

Answer 43

inoculation of small amount of dried material from a smallpox pustule which produced mild infection (but 3% of cases = fatal smallpox)

Question 44

cowpox to protect from smallpox

Answer 44

humans are not a natural host of cowpox which establishes only a brief and limited subcutaneous infection while containing antigens that stimulate an immune response that cross-reacts with smallpox antigens

Question 45

issues that face modern vaccine scientists

Answer 45

the elimination of certain major pathogens requires additional effector activities such as the generation of strong and durable cell-mediated immunity which are not efficiently generated by current vaccine technologies

Question 46

vaccines based on attenuated pathogens

Answer 46

reduced pathogenicity, stimulate protective immunity but
don’t cause disease, design
of genetically attenuated pathogens in which desirable mutations are introduced into the organism by recombinant DNA technologies

Question 47

vaccines based on killed organisms

Answer 47

purified components of organisms that
would be as effective as live whole organisms, any live vaccine can cause lethal systemic
infection in immunosuppressed people, conjugation of purifed antigens (Haemophilus infuenzae), reverse immunogenetics to identify candidate
peptide antigens for T cells and with strategies to use ligands that activate TLRs (toll like receptors)
or other innate sensors as adjuvants to enhance responses to simple antigens

Question 48

issues in vaccinating developing countries

Answer 48

technical and economic problems, simple costs of storage and deployment can be signifcant barriers to the use of existing vaccines in poorer countries

Question 49

antibody generation

Answer 49

induced by vaccines, some require
additional cell-mediated immune responses such as those mediated by CD8
T cells, effective protective immunity against some microorganisms requires the
presence of preexisting antibody at the time of infection (either to prevent
the damage caused by the pathogen (tetanus and diphtheria exotoxins, even survivors require vaccination to be protected against the risk
of subsequent attack) or to prevent reinfection by the pathogen altogether)

Question 50

neutralization

Answer 50

While CD8 T cells are able to kill already virally infected cells during an infection antibodies are able to prevent infection of cells by the virus in the first
place, ability of antibody to neutralize a pathogen may depend on its affinity + isotype subclass + complement + activity of phagocytic cells, preexisting antibodies
are required to protect against the polio virus which infects critical host cells
within a short period after entering the body and is not easily controlled by
T lymphocytes once intracellular infection has been established, vaccines
against seasonal infuenza virus provide protection by
inducing antibodies that reduce the chance of a second infection by the same
strain of infuenza, for many viruses antibodies produced by an infection or
by vaccination can neutralize the virus and prevent further spread of infection, in HIV despite the generation of antibodies that can bind to surface viral epitopes most fail to neutralize the virus + HIV has many diferent strains (clades) but a
recent clinical trial suggests that boosting previously vaccinated subjects with
protein 5–7 years after immunization may induce some antibodies with crossclade activity

Question 51

linked recognition

Answer 51

Immune responses to infectious agents usually involve antibodies directed at
multiple epitopes and only some of these antibodies if any confer protection, the particular T-cell epitopes recognized can affect the nature of the
response, antigen specific B cells and T cells provide mutually activating signals leading to affinity
maturation and isotype switching that may be required for neutralization, requires that an appropriate peptide epitope for T cells be presented by
the B cells and typically that the T-cell epitope be contained within the region
of protein epitope recognized by the B cell (must be considered
in modern vaccine design)

Question 52

Effective vaccines must induce ___-lasting protection while

being ____ and _________

Answer 52

long, safe, inexpensive

Question 53

herd immunity

Answer 53

By lowering the number of susceptible members of a population vaccination decreases the natural reservoir
of infected individuals in that population and so reduces the probability of
transmission of infection, even unvaccinated members will be protected
because their individual chance of encountering the pathogen is decreased.

Question 54

Live-attenuated viral vaccines are usually ____ potent
than ‘killed’ vaccines and can be made safer by the use of
________ ___ technology

Answer 54

more, recombinant DNA

Question 55

Inactivated/”killed” viral vaccines

Answer 55

viruses treated so that they are unable to replicate, cannot produce proteins in the cytosol of infected cells -> peptides from the
viral antigens are not presented by MHC class I molecules -> CD8 T cells
are neither efficiently generated nor needed

Question 56

Live attenuated viral vaccines

Answer 56

generally far more potent, elicit a greater
number of effector mechanisms including activation of CD4 T cells and
cytotoxic CD8 T cells, CD4 T cells help in shaping the antibody response which is important for a vaccine’s subsequent protective effect, cytotoxic CD8
T cells provide protection while infection by the virus itself is under way and
if maintained may contribute to protective memory, routine childhood vaccines for polio + measles + mumps +
rubella + varicella, other vaccines that are licensed for
special circumstances or for use in high-risk populations ie infuenza + poxvirus (vaccinia) + yellow fever virus

Question 57

how is attenuation achieved traditionally

Answer 57

growing the virus in cultured cells, viruses usually selected for preferential growth in nonhuman cells and in
the course of selection become less able to grow in human cells,
because these attenuated strains replicate poorly in human hosts they induce
immunity but not disease

Question 58

possible live attenuated side effects

Answer 58

although attenuated virus strains contain multiple
mutations in genes encoding several of their proteins it might be possible for a
pathogenic virus strain to reemerge by a further series of mutations

Question 59

isolation and in vitro mutagenesis of specific viral genes

Answer 59

the mutated genes are
used to replace the wild-type genes in a reconstituted virus genome, mutations can be engineered so that reversion to wild type is virtually impossible

Question 60

heterosubtypic immunity

Answer 60

the infuenza virus can reinfect the same host several times because it undergoes antigenic shift and thus predominantly
escapes the original immune response, a weak protection conferred by previous infections with a diferent subtype of infuenza is observed in adults and is called heterosubtypic immunity, the current
approach to vaccination against influenza is to use a killed virus vaccine that
is reformulated annually on the basis of the prevalent strains of virus, the ideal influenza vaccine would be an attenuated live
organism that matched the prevalent virus strain which could be created by
first introducing a series of attenuating mutations into the gene encoding a
viral polymerase protein PB2 -> the mutated gene segment from the attenuated
virus could then be substituted for the wild-type gene in a virus carrying the
relevant hemagglutinin and neuraminidase antigen variants of the current
epidemic or pandemic strain, alternatively broadly neutralizing antibodies
that block the receptor-binding domain of the hemagglutinin can be generated in humans and could be used as a universal vaccine.

Question 61

Live-attenuated vaccines can be developed by…

Answer 61

selecting
nonpathogenic or disabled bacteria or by creating
genetically attenuated parasites (GAPs)

Question 62

routes of vaccination

Answer 62

mucosal (ie through nose), injection, oral route

Question 63

conjugate vaccines

Answer 63

have been developed as a result of linked
recognition between T and B cells, many bacteria ie Neisseria meningitidis (meningococcus) + Streptococcus pneumoniae (pneumococcus) + H. infuenzae have an outer capsule composed of polysaccharides that are species- and type-specific for
particular strains of the bacterium, the most effective defense against these
microorganisms is opsonization of the polysaccharide coat with antibody, however effective acellular vaccines cannot be made from a single isolated constituent of a microorganism
since generation of an effective antibody response requires the participation
of several types of cells which has led to the development of conjugate
vaccines, capsular polysaccharides can be harvested from bacterial growth medium
and because they are T-cell-independent antigens they
can be used on their own as vaccines, however young children under the age of 2 years cannot make good T-cell-independent antibody responses and cannot be vaccinated effectively with polysaccharide (PS) vaccines so conjugate bacterial polysaccharides
chemically to protein carriers that provide
peptides that can be recognized by antigen-specifc T cells converting a
T-cell-independent response into a T-cell-dependent antipolysaccharide antibody response

Question 64

Endemic meningitis B

Answer 64

due to diverse serogroup B strains so an ideal vaccine would target the group B capsular polysaccharide but group B
polysaccharide is identical to some polysialyl polysaccharides on human cells
and is poorly immunogenic due to tolerance of these self antigens

Question 65

Peptide-based vaccines

Answer 65

can elicit protective immunity but require adjuvants and must be targeted to the
appropriate cells and cell compartment to be effective, overlapping peptides from immunogenic proteins are systematically synthesized and their ability to stimulate protective immunity is tested or a reverse immunogenetic approach can be
used to predict potential peptide epitopes from a genome sequence (ie malaria by using the complete sequence of
the Plasmodium falciparum genome), a particular peptide may not bind to all the MHC molecules present in the
population, some direct exchange of short peptides on MHC molecules can
occur without physiological antigen processing: if the required antigenic peptides load directly onto MHC molecules on cells other than dendritic cells this
may induce tolerance in T cells rather than stimulating immunity, exogenous proteins and peptides delivered by a synthetic vaccine are efficiently
processed for presentation by MHC class II molecules but require cross-presentation in specific types of dendritic cells to be loaded onto MHC class I molecules and directing peptide-based vaccines to such cells may
enhance vaccine efficacy

Question 66

Adjuvants

Answer 66

are important for enhancing the immunogenicity of
vaccines, few are approved for use in humans, vaccines based on peptides or purifed proteins require additional components to mimic how real infections activate immunity, substances that
enhance the immunogenicity of antigens (ie tetanus toxoid is not immunogenic in the absence of adjuvants so vaccines contain inorganic aluminum salts (alum) in
the form of noncrystalline gels that bind polyvalently to the toxoid by ionic
interactions, pertussis toxin has adjuvant properties in its own right and when
given mixed as a toxoid with tetanus and diphtheria toxoids not only protects against whooping cough but also acts as an additional adjuvant for the other
two toxoids (DTP triple vaccine)), alum seems to act as an adjuvant by stimulating
one of the innate immune system’s bacterial sensor mechanisms NLRP3 thus
activating the inflammasome and the inflammatory reactions, many adjuvants seem to work by triggering the innate viral and bacterial sensor pathways in APCs + TLRs + proteins of the NOD(nucleotide-binding oligomerization domain)-like receptor family such as NLRP3 activating them to initiate an
adaptive immune response

Question 67

DNA-based

vaccination

Answer 67

DNA encoding a viral immunogen when injected intramuscularly in mice induced antibody responses and cytotoxic T cells that could
protect against subsequent infection from the live virus, DNA
coated onto minute metal particles can be administered by a gene gun so that
particles penetrate the skin and potentially some underlying muscle, because of DNA’s stability it is suitable for mass immunization, comparatively weak (mixing in
plasmids that encode cytokines such as IL-12, IL-23, or GM-CSF makes immunization with genes encoding protective antigens much more effective), the antigen is produced by cells that are directly transfected such as skin or muscle but CD8 T-cell activation requires cross-presentation
of the antigen by dendritic cells, tests for malaria + infuenza + HIV infection + breast cancer

Question 68

Vaccination and checkpoint blockade

Answer 68

may be useful in
controlling existing chronic infections of 2 types: an obvious immune response that fails
to eliminate the organism or seem to be invisible to the immune
system and evoke a barely detectable immune response, in first category the immune response is often partly responsible for the
pathogenic effects (ie infection by the helminth Schistosoma mansoni is associated with a powerful TH2-type response characterized by high levels of IgE +
circulating and tissue eosinophilia + a harmful fibrotic response to schistosome ova in the liver leading to hepatic fbrosis, the mycobacterial agents of tuberculosis and leprosy cause a persistent intracellular infection (a TH1 response helps to contain these infections but also
causes granuloma formation and tissue necrosis), hepatitis B and hepatitis C infections are commonly followed
by a persistent viral burden and hepatic injury resulting in eventual death
from hepatitis or from hepatocellular carcinoma), the second is predominantly viral (herpes
simplex virus type 2)

Question 69

Vaccine developments

Answer 69

• Whole Organism
  (Edward Jenner, 1796, crude isolate of cow pox virus prevented infection with potentially fatal and disfiguring smallpox)
• Toxoid (several pathogens that depend on a toxin in order to cause disease ie tetanus and diphtheria, toxin can be inactivated and formulated in a vaccine preparation to stimulate the production of antibodies)
• Killed/inactivated
  (if the chemically or physically inactivated form of the organism resembled the viable organism could be used to vaccinate ie inactivated Polio vaccine formulated by Jonas Salk in 1955)
• Subunit (a defined antigenic component of a pathogen against which the immune response is stimulated, the subunits can be purified from the original pathogen but increasingly are made as recombinant proteins, excellent safety profile but generally are not as immunogenic as attenuated or inactivated vaccines and a need a stronger adjuvant, ie HPV vaccine)
• Live Attenuated (by growing the pathogen outside its host it becomes adapted to these new conditions rendering it less able to infect the original host, enables the immune system to mount a protective immune response, Maurice Hilleman MMR)
• Conjugate Vaccines (many bacteria are protected by an outer polysaccharide coat, due to their presence on the surface of the bacteria they are attractive targets for the immune system but are usually poorly immunogenic, by synthesizing polysaccharides and conjugating them to an immunogenic carrier protein they can act as potent immunogens, ie HiB + meningitis C + pneumococcal vaccines)

Question 70

Future of vaccines

Answer 70

several diseases for which there are currently no vaccines (ie HIV, malaria, TB), new strategies inducing the cellular and humoral arms of the immune system may need to be employed, DNA Vaccines (early gene therapy attempts discovered immune responses directed against injected DNA and its transcripts, vaccines can contain multiple antigens + are cheap and quick to develop), Vectored Vaccines (Several organisms such as bacteria and viruses can infect cells and induce an immune response which is similar to that required to control infection, vaccine antigens can be vectored into host cells by replication deficient viruses such as Adenovirus and modified Vaccinia Ankara or bacteria such as Salmonella inducing both B- and T-cell responses), Reverse Vaccinology (Using modern genome sequencing prospective vaccine candidates can be selected based on predicted immunogenicity)