MIMM 413_ Midterm

Human parasites are composed of

- protists and nematodes
- not bacteria, fungi or viruses

What are protists

- single-celled eukaryotes, make up 4% of the Earth's biomass

What are nematodes

- multicellular worm-like organisms, make up 0.02% of Earth's biomass

Explain the origin of human infectious diseases

- humans started to change from hunter/gatherer pops to agriculture based 15,000 years ago (more cals \= more pop), started living in communities and began to domesticate animals
- most of the common infectious diseases of today had to have emerged 10-12000 years ago after the beginning of agricultural-based populations
- before this, humans were too spread out

Animals domesticated in the old world

- cows, sheep, goats, pigs, chickens, horses and camels
- these animals transmitted viruses like mumps, measles, pertussis
- only the llama was domesticated in the New World, did not transmit any disease

Old world to new world contagion

- EU conquest of the New World, migration brought many pathogens from the OW to the NW, decimating native pops
- more people died from smallpox and influenza than bullets
- almost all diseases developed in the OW, only chagas is known to develop in the NW

5 stages in the evolution of animal pathogens into human pathogens

Stage 1) only lives in animals

Stage 2) can infect humans or animals, but does not get transmitted from human to human, only animal to human (rabies)

Stage 3) can infect humans or animals, gets transmitted from animals to humans or humans to humans, however human to human transmission is not very effective and outbreaks don't last very long (ebola)

Stage 4) can infect humans or animals, gets transmitted from humans to animals or humans to humans effectively (dengue)

Stage 5) no longer infects animals, only humans (HIV)

Zoonotic infection

- stage 2, 3, 4
- pathogen has an animal reservoir but can also infect humans

Anthroponotic infection

- stage 5
- pathogen only infects humans

R value

- rate of transmission from human to human
- 1 case will transmit to R number of cases
- R \> 1: infection will spread
- R < 1: infection will die out

what are factors supporting the transition of a pathogen from animal to human

1) high number of close encounters
- the abundance of the animal reservoir and the prevalence of encounters with humans
- ex. avian influenza virus where infected chickens are in close contact with humans in countries throughout Asia
- SARS-CoV in 2003, originated from breeding Civet cats
2) similar physiology
- the phylogenetic distance between the animal reservoir and humans.
- easier to acquire an infection from a monkey than a horse
- ex. HIV, Dengue, Yellow Fever all originate from monkeys

SARS-CoV-2 origin

- unknown
- possibly in bats since they contain many COVID viruses
- bats can harbour so many viruses effectively because they are flying mammals
- they have high body temp and are close contact with each other

Differences between tropical and temperate infectious diseases (examples)

1) Malaria is an example of tropical disease
- absent from the northern part of the world, mosquitoes transmit malaria much better in the tropics than in temperate regions
2) Tropical diseases (include parasite)
- most are vector borne (insect), chronic, with little long-lasting immunity
- many have non domesticated animal reservoirs, except visceral leishmaniasis (dogs reservoirs)
- not many of them have vaccines available

Tropical diseases characteristics

- *all parasitic diseases* are generally tropical diseases (closer to the equator)
- most are *vector-borne* diseases transmitted by insects (Malaria)
- do not generally provide long lasting immunity, leading to multiple reinfections and *few vaccines*

temperate diseases characteristics

- diseases which are farther N and S
- *no parasites*
- over the last 200 years, humans have dealt with bacterial and viral temperate diseases such as hep b, influenza A, mumps, pertussis
- *vaccines developed for all* of them
- long lasting immunity is common for temperate diseases
- transmitted by *aerosols*
- most are *stage 5 diseases*

temperate diseases

- most *do not have animal reservoir*, except plague
- it has been possible to develop vaccines because infection provides some long lasting immunity

Comparison of temp vs tropical diseases

- *parasitic diseases are all tropical* not temperate
- tropical are much more transmitted by insects than temperate, ex. plague and typhus only temperate diseases with an insect vector
- *tropical diseases involve animal reservoirs*
- temperate diseases often have person to person transmission via aerosols compared to tropical
- temperate diseases result in long lasting immunity compared to tropical
- *more stage 5 in temperate* than tropical
- vaccines available in most temp diseases except syphillis in contrast to tropical diseases

Index case - Ebola outbreak - 2014

- first person who got the disease identified and then it is possible to follow the spread
- Dec 2: 2 year old girl who died in Guinea

Ebola progression

- March 2014: 79 deaths, disease started to spread along major road near village and infected people brought the disease to other tows
- April 2014: spread to Liberia then to Sierra Leone, causing 100s of deaths
- people did not travel N because that area is unpopulated, rather W and S to countries with close borders
- Dec 2014: 7000 deaths, half the people died from it and half recovered naturally because no vaccines or treatment
- Sept 2015: 15000 cases

Transmission of Ebola

- bodily fluid
- in contact with secretions of an infected individual
- diagnosed with PCR
- cases went down

why did ebola cases go down

- without treatment or vaccines, behavioural changes in funeral rituals and families stopped hiding cases
- in west africa, when someone dies they kiss the body and wash it, but this was stopped when people realized it was a major route of transmission
- people with ebola symptoms voluntarily go to isolation centres to protect fam, get diagnosed by PCR and if positive, go to hospital for treatment and avoid direct contact
- public buy in was essential

Why can we control ebola but not COVID

- ebola transmitted via physical contact but COVID is a temp disease, transmitted by aerosols
- people need to have symptoms beforehand to transmit ebola whereas COVID can be transmitted by asymptomatic infection

Would isolation centres work for covid

- no
- we would have concentration transmission and isolation centres would become high sources of transmission due to high concentration of virus particles in the air

EID

- new emerging infectious diseases
- *most EIDs are zoonotic*: HIV from chimps, Ebola from bats, SARS from civets, ZIKA present in monkeys and domestic animals

Spread of EIDs

- faster than previously
- globalization means more travel, spreads very fast with aerosols, larger cities mean more trade of animal products

Are medical interventions available for EIDs

- no
- drugs, vaccines and diagnostics are not generally available
- possible to rapidly develop diagnostics with PCR because we can now sequence the genome of viruses, bacteria, and parasites
- vaccine dev also involves genome sequencing
- poor countries are first to suffer from EIDs: rural pops living close to infected animals are first to infected while G20 countries appeared to be hit as hard with COVID, within each country the poor are more affected

Infectious disease burden

- 50% of deaths in the Developing World are caused by infectious diseases, whether they be bacterial, viral or parasitic
- 5% of deaths in the Developed world are from infectious diseases

COVID and its impact on both Dev and Underdev world

- comparison between US, Can, India
- 3 major waves in the US, total number of deaths in US and India are similar even if the number of deaths in India are higher than recorded
- total death toll in Can is low because of population (1/10th US)
- deaths per capita, india has fewer deaths per capita than US or Can, the US has most deaths per capita
- COVID is different from previous major emerging infectious diseases

Epidemic

an outbreak in a single country or two or three countries
- small area
- ebola, started in guinea, then SL, an epidemic because it started small with a lot of cases but it came under control

Pandemic

- large outbreak in many countries
- no specific cut off between epi and pan, more like situational and considers the speed of transmission from one country to another
- pandemic refers to level of transmission not type of disease

Endemic

- disease become established in an area and is always going to be present there
- common cold
- COVID may move from pandemic to endemic might need to get vaxxed every year similar to the flu and we will develop immunity w vax and treatments

Leishmania donovani: VL

- called British disease because VL followed colonization by the brits in N India
- originally thought to be malaria but quinine treatment did not work
- 1900: Wiliam Leishman and Charles Donovan discovered Leishmania parasites in kala azar patients
- 1920: major stinson showed leishmania was transmitted by sandfly

Life cycle of leishmania

- transmitted by sandfly, contains promastigotes
- no noticeable lesion at site, promastigotes have flagella
- *promastigotes* enter host, replicate intracellularly in *macrophages* in high quantities
- conversion from *promastigotes to amastigotes* takes place in macrophage -\> flagella lost
- macrophages burst due to high quantities of amastigotes, can hold around 50
- liver, spleen and bone marrow are eventually full of infected macrophages -\> leads to immunosuppression and breakdown of internal organs
- if untreated -\> death
- anthroponotic and telltale sign is constant fever and enlarged liver

VL: leishmania infantum

- dogs reservoirs
- zoonotic
- infection can pass from reservoir to humans when a sandfly feeds on infected dog before biting human, resulting in VL
- Brazil, Southern Europe, North Africa

Cutaneous leishmaniasis

- L. major, L. mexicana, L. braziliensis
- less adapted to humans, so they stay in the skin causing cutaneous disease
- rodents are reservoirs
- if a sandfly feeds on an infected rodent and passes infection, it only stays in the skin causing lesion, bites tend to occur on the face
- stigma associated with scars, tells everyone you come from a poor village
- lesions can last for several months

Mucocutaneous leishmaniasis

- CL that ends up in the nose (caused by L. braziliensis)
- immune system is fighting the parasite, causing destruction of tissue as collateral damage (immune system is causing the damage)
- most rare form
- found in peru, brazil and bolivia
- if not treated in time, it will destroy the nose and face
- can be misdiagnosed as nasopharyngeal cancer
- records from 2000 years ago show that individuals had missing noses in pre-Inca pottery (peru)

pathology of leishmaniasis: VL

- infection spreads to visceral organs, fatal if untreated
- caused by L. donovani in Sudan, India, Nepal, Bangladesh, Ethiopia (only anthroponotic)
- sometimes caused by L. infantum (zoonotic, dog reservoir)

pathology of leishmaniasis: CL

- most common form
- infection remains in the skin at the site of sandly bite
- L. major, etc.
- middle east, South america, central America
- all zoonotic (rodents)

pathology of leishmaniasis: MCL

- rarest
- spreads to septum
- destruction of nose and face
- L. braziliensis in Peru, Colombia, Brazil
- all zoonotic

leishmania geography

- Cutaneous leishmaniasis: 300 million cases.
- Visceral leishmaniasis: 50 million cases (Fig. 2 bottom).
1) Visceral leishmaniasis in Bihar:
- 50% of worldwide cases take place in Bihar, India, above a boundary that has not changed in over 100 years.
- It is very unusual for a disease to remain so
localized over time.
- Bihar is the poorest state of India with a massive population of 90 million people.
- Bihar has high levels of leprosy and viral
diseases and was the last location to have
smallpox.
- The Ganges River flows through the North (where the cases are) and causes flooding, which partially explains the distribution, but there are many areas of India that are very similar and have no leishmania.

leishmania promastigotes in the female sandfly

- When a female sandfly takes a bloodmeal, the blood with the parasite gets into the gut. After several hours, the sandfly secretes the fluid and maintains the cells (red blood cells and leukocytes) in the gut
- Any of the leishmania that was picked up from the blood stays in the gut. They bind using their flagella to avoid getting secreted.
- Promastigotes undergo a rapid proliferation within the gut. Additionally, they convert from *procyclics to metacyclics* (infective stage, long flagella)
- The promastigotes migrate through the thoracic midgut area to cause infection during the next bloodmeal and enter the host. *Metacyclics are infective and they are the ones that enter the host*.
- Promastigotes can't stay in the gut very long because sandflies don't have a very long lifespan (~25 days) and only take bloodmeals 3-4 times during their lifetime. They must get in and get out.

Leishmania promastigotes in the skin after sandfly bite

- mouth cuts through epidermis, looking for blood and delivering promastigotes (metacyclics)
- neutrophils and macrophages are predominantly infected
- *replication only occurs in macrophages*
- Depending on leishmania species, the amastigotes within infected macrophages (or dendritic cells) can either leave the skin and enter the macrophages of visceral organs, bone marrow, liver, and spleen (in the case of visceral leishmaniasis, i.e., L. donovani and L. infantum) or remain in the skin (in the case of cutaneous leishmaniasis, i.e., L. major and L. tropica).
- The heavily infected macrophage remains in the skin. The immune system recognizes this as an invasion by an infectious disease, driving a massive immune response and targeting the parasite. B cells produce antibodies, T cells produce a cellular immune response, cytokines are released. The strong immune response induces localized destruction of the skin at the location of the sandfly bite, resulting in a lesion.
- antibodies have no effect on leishmania, *only cellular immunity plays a role in clearance*

Leishmania treatment with pentavalent antimony

- first treatment developed against VL
- still used in many parts of the world for CL and VL, most common treatment in 100 years
- need to inject up to 20 mL at a time (large quantity)
- sometimes needs to be injected into the muscle which is very painful
- antimony is heavy metal just below arsenic with similar toxicity
- produces a dark blue colour during oxidation and was first used as a cosmetic in Egypt
- used to treat syphilis initially
- requires 20 injections of a large volume and done at a hospital

Treatment of leishmaniasis with Ambisome

- treats fungal infections but also works against VL
- Gilead provides the treatment for free specifically for visceral leishmaniasis in developing nations because they cannot afford it. - In the West, it is expensive and only used for fungal infections.
- Preferential pricing by Gilead allows developing nations to treat leishmaniasis.
- *Doesn't work as well in Africa, where a combination of antimony and paromomycin is used instead*
- Administered by IV.
- Comes in a powder, dissolved in dextrose solution, takes about 2 hours to provide treatment.
- Needs to be done in a hospital setting.
- Only one treatment is necessary to cure an infected individual.

Post kala azar dermal leishmaniasis

- Occurs in ~20% of individuals that have been treated for visceral leishmaniasis (L. donovani only).
- More common in Africa than in India.
- No transmission occurs between family members.
- The parasite leaves the visceral organs and goes to the skin to avoid destruction by the treatment agents.
- Certain areas of the skin have less pigmentation (the parasite is found in these areas). No other symptoms.
- In some individuals PKDL is self-healing, but in others it remains for a number of years or the whole lifetime.
- Doesn't really cause problems for the patient.
- Very difficult to treat.
- Requires multiple doses of AmBisome or a long-term treatment with antimony.
- PKDL is more common in patients treated with antimony, but it can occur with any treatment. It is unknown whether people with PKDL are a reservoir for transmission.

Leishmania - Mammalian host macrophage

- The promastigote from the sandfly enters the host to replicate in the human macrophage. It can also infect other cells, but replication only occurs in macrophages.
- The *phagolysosome is the site in the macrophage where the promastigote replicates and converts into the amastigote*.
- The phagolysosome is a part of the macrophage that normally takes in infectious agents (bacteria, viruses, parasites) or other debris to destroy them.
- Leishmania has developed ways of protecting itself and surviving within the phagolysosomes, where it replicates in high levels.

Clinical vs. asymptomatic cases

- The majority of people who get infected with Leishmania do not develop clinical disease (asymptomatic infection).
- Only 1 clinical disease case for 5 to 20 asymptomatic healthy cases (probably closer to 1 in 20).
- Asymptomatic infections occur with many infectious diseases, such as COVID-19.
- The immune response against Leishmania is well understood, probably more than the vast majority of other diseases. In fact, Leishmania is used as a model to study the immune response.

Explain the immune response to Leishmania infection

- Promastigotes entering the dermis can also infect dendritic cells but there is not a lot of Leishmania replication. Dendritic cells take up the pathogens and present them to the immune system.
- Dendritic cells leave the site of infection and go to lymph nodes, where they present the parasite to naïve CD4+ T cells.
- The activated T cells interact with the naïve T cell. Depending on this interaction, these naïve T cells will differentiate in one of two directions: the Th1 or Th2 immune response. Sometimes, you can have both types, but a primarily Th1-based immune response is needed for successful parasite clearance.

Explain the Th1 immune response

- In the majority of the cases, CD4+ T cells differentiate into Th1 cells, which are characterized by their release of a specific set of cytokines including IL-2, IFN-Υ, TNF-α, IL-12. When the Th1 cells come into contact with the macrophages after releasing IFN-Υ, the macrophages turn into killer cells releasing toxic molecules such as nitric oxide, inducible nitric oxide synthase (iNOS), free radicals, and proteases. The activated macrophages kill the parasites inside them mainly with iNOS, which is toxic to intracellular pathogens, which results in an asymptomatic infection.

Explain the Th2 immune response

- It is very good at producing antibodies, but not so good at producing IFN-Υ to activate macrophages. Also, antibodies are not protective against Leishmania. The macrophages do not become fully activated to kill intracellular parasites so they are permissive to the proliferation of the parasite, and you can have the development of the disease.

Are there responses that are mixed

- There is some crosstalk involved between the two pathways, particularly with the cytokine IL-10 produced by Th2 cells. IL-10 can suppress the Th1 immune response, which can lead to the survival of the parasite inside macrophages.
- The two cytokines that really control the whole response is *IFN-Υ*, which *activates macrophages*, or *IL-10*, which *suppresses the activation of macrophages*. We want more IFN-Υ for an asymptomatic infection.

Toll-like receptors (TLRs)

- In the last 20 years, research has focused on molecules that can activate toll-like receptors (TLRs) early on, to help direct the response towards a Th1 immune response.
- There are different types of TLRs on the surface of cells, and they can recognize common molecules.
- TLR1 and TLR4 recognize bacterial LPS, TLR2
recognizes other bacterial molecules also present on fungi, TLR5 recognizes bacterial flagellin.
- The is a number of TLRs on phagolysosome, which mostly recognize nucleic acids (double and single strand RNA, and DNA)
- When the pathogens are destroyed in the
phagolysosome, their free-flowing DNA or RNA activates the TLRs, which can stimulate the Th1 immune response.

TLR stimulating drugs

- Imiquimod (IMQ) is a drug developed 20 years ago by 3M' pharmaceuticals. It was used to stimulate TLRs to activate the immune system against Papillomavirus infections.

How does Imiquimod work?

- Imiquimod stimulates TLRs in the phagolysosome (*TLR7/TLR8*), it can also be used against leishmaniasis by stimulating a Th1 immune response. It can also directly interact with macrophage cells, to secrete iNOS, which can kill Leishmania.
- In experiments done over 20 years ago,
macrophage cells were infected with L. donovani. If you add Imiquimod, the parasite cannot replicate because the macrophage is now non-permissible for replication, which was shown with different strains of L. donovani.
- Imiquimod was an approved molecule because it was already being used to treat Human Papillomavirus. If you have an approved molecule for one disease, it is possible to try to see if you can use it to treat another disease, called off-target use of a drug.
- Comes as a topical cream (*Aldara*) treatment
- There is healing but only some have complete healing, some scarring and reduction of inflammation
- Aldara is given topically because if it was given systemically it would activate all your TLRs, causing a cytokine storm and severe illness.

Combination (Aldara + Antimony)

- the standard treatment
(Antimony injected intravenously 20 days in a row) in Peru was given in one arm, while the other arm was supplemented with Imiquimod (topical treatment of Aldara).
- The cure rate increased when Imiquimod was used in combination. By adding topical Imiquimod where you are activating the macrophages, you could increase the cure rate, starting at one month.
- This is a WHO approved treatment for Cutaneous Leishmaniasis, that is now commonly used in Peru when someone does not respond to antimony alone.

Imiquimod for reducing vector-borne viral infections

- If a mosquito injects a virus, such as Dengue virus, it will infect the epithelial cells in the skin and then it starts to replicate. In Figure 6, the blue particles are the virus replicating in epithelial cells within the skin. Once the infection is established or once the virus infects the skin, it moves to other parts of the body, and you get the disease.
- This applies to other mosquito-borne disease such as Chikungunya virus and Yellow Fever virus.
- In the study, they added topical Aldara cream on mosquito bites. This activates the local inflammatory cells, such as macrophage cells in the skin to kill the virus. The viruses don't replicate inside macrophage cells, but in epithelial cells. Nevertheless, the activated macrophages release a number of cytokines that stimulate a local inflammatory response that brings in other immune cells such as neutrophils.
- This local inflammatory immune response is enough to kill the virus before it causes disease.
- The caveat here is that you *have to add the Imiquimod no longer than 24 hours after the bite*.

Malaria and discovery

- Charles Laveran, a French military doctor in the 1880s, would take some blood to look at under the microscope before treating his patients. He wanted to determine if he could see why these people were developing malaria.
- He would draw on paper the structures he would see under the microscope, such as Plasmodium falciparum gametocytes, which causes malaria.
- Other people looked under the microscope, but they could not see them because their eyes were not trained and the microscope was primitive, so they did not believe him and he had a hard time publishing.
- In 1884, a new microscope which used oil immersion was developed, a big advance in microscopy that gave the ability to look under the microscope and see things much more clearly.
- Other people could then see the same structures Laveran had drawn and he was able to publish his work in the 1880s.
- In 1907, Charles Laveran won the Nobel Prize because he was the first one to discover that the parasite caused malaria. Still, they did not know how people were getting the disease.
- Malaria in Latin means bad air, because people thought they were getting the disease by breathing bad air since they would get it near swamps, where the air was bad but also where there were a lot of mosquitoes.

Malaria and Ronald Ross

- A scientist from the British army who was working in Calcutta (India) was convinced that malaria was transmitted by mosquitoes.
- He would take mosquitoes and feed them on people who had malaria, and then he would dissect the mosquitoes and look if he could see the same organisms that Charles Laveran had seen.
- He spent 2 years drawing what he could see but there was never the development of a mature parasite inside the mosquito because he was working with Culex mosquitoes, similar to what is found in North America.
- Once be was brought a new batch of mosquitoes, Anopheles, the right species that transmit malaria, he could observe the development of the parasite inside the mosquito.
- In 1902, he won a Nobel Prize because he proved that mosquitoes were transmitting malaria.

Plasmodium life cycle

- Anopheles mosquito bite *injects sporozoites* into the individual. Sporozoites enter bloodstream, travel through the body.
- *Target of sporozoites: liver*
- Once liver is reached, sporozoites *infect hepatocytes*. After about a week, they *convert into merozoites*.
- Once merozoites mature (in about a week), they return to the blood and invade the red blood cells.
- *Merozoites replicate in the red blood cells*. This causes the pathology of the infection.
- Most severe pathology of merozoite replication is death
- Some of the *merozoites eventually convert into gametocytes*.
- Gametocytes are taken up by the mosquito during their bloodmeal. The female and male *gametocytes form a zygote* in the gut of the mosquito, *transforming into an oocyst and then into sporozoites*, which then re-enter the salivary gland and can be passed to the next human.

What are the malaria endemic regions

- Sub-Saharan Africa.
- Have been endemic in this part of the world for a very long time, slowing the countries' development. It continues to be a devastating disease in this side of the world.
- Other locations of malaria cases: Southern and South-East Asia, South America.

Malaria and human genetic adaptation

- Genetic polymorphisms in some human genes were selected because they protect against malaria in Sub- Saharan Africa.
- Sickle cell anemia: oval-shaped red blood cells caused by a mutation in the hemoglobin gene prevent merozoites from replicating effectively in the red blood cells.
- You can survive the disease, but circulation is quite poor. Patients also have low energy.
Interestingly, sickle cell anemia is the first disease to be cured using CRISPR gene editing since it is produced due to a single-gene polymorphism.
- Victoria Gray in the patient that was cured in 2020.
- Normally, hemoglobin is a tetramer. A single nucleotide mutation changes the a.a. sequence (from *Pro-Glu-Glu to Pro-Val-Glu*) and prevents Hb from forming a normal tetramer, resulting in a formation of long inflexible hemoglobin chains, which "stretch" the red blood cell and give it the sickle cell phenotype.

Malaria etiology

4 species where humans are the only reservoir (stage 5, anthroponotic).
- Plasmodium falciparum.
- Plasmodium vivax.
- Plasmodium ovale.
- Plasmodium malariae.
1 species has monkey as the reservoir but can also infect humans (stage 4, zoonotic).
- Plasmodium knowlesi.
- Most cases are caused by P. falciparum and P. vivax, and most deaths are caused by P. falciparum.
- P. vivax is more commonly found in Asia. Vivax causes a really high fever but does not typically lead to death.
- There are hundreds of species of Plasmodium, but most do not infect humans.

Malaria intervention and prevention

1) Vaccination.
- The most advanced through clinical trials is the RTS,S vaccine made of the sporozoite surface protein, circum sporozoite protein (CSP). Poor efficacy.
- Infants 5-17 months exhibit 40% protection, less against severe malaria.
- Requires 4 doses.
2) Chemotherapy.
- A number of effective drugs are available including Quinine, Chloroquine, Mefloquine, Artemisinin, and other. Now ACT (artemisinin combination therapy) is always recommended to reduce development of drug resistance.
- Needs to be given before the patient is too ill. If someone develops cerebral malaria, it is probably too late to treat them with these drugs.
3) Chemoprevention.
- Antimalaria drugs are recommended for all travelers to endemic areas.
- Recommended for pregnant women living in Africa because they have a slightly suppressed immune system, leaving them exposed to malaria.
- Now recommended that infants (3-60 months) are treated with 4 doses per year to remove any
plasmodium that they can become exposed to. Prophylactic treatment. Children suffer most from infection.
- This may be the best option for kids until a more effective vaccine becomes available.
4) Vector control: Pyrethroid insecticide-treated mosquito nets reduce mortality by 20% in children younger than 5 years. - Bed nets treated with insecticide stop the mosquito from biting, but also kill the mosquito the
insecticide that's present on the net. Biting mostly occurs at night.

Who is dying from malaria

- mostly children under the age of 5 since they have not had the time to develop immunity
- Targets set by the WHO.
- Intended to distribute 247 million drugs, only distributed 100 million.
- Many great drugs are available, but they aren't being distributed to the right people at the right time, resulting in a high mortality due to poor health infrastructure and poor health institutions found in affected countries.
- Drug targets not being met because of lack of funding to provide the correct infrastructure in order to distribute drugs, bed nets, and diagnostic tests. There is simply not enough monetary support to provide all the necessities.
- Similar with COVID vaccines, which aren't reaching countries such as Sub-Saharan Africa due to a lack of infrastructure needed to deliver vaccines.

Malaria drug dev

- Quinine, first seen in Peru
- Chloroquine, dev around the turn of the 20th century but wasn't used to treat malaria until the Second WW
- Artemisinin, Vietnam war -\> resistance to chloroquine became prevalent so soliders used arteminisin instead, became backbone
- now artemisinin combo therapy w derivatives of previous drugs. ACT used to suppress the dev of drug resistance

How do malaria drugs work

- *Quinine and mefloquine* inhibit merozoite protein translation.
- *Chloroquine and artemisinin* inhibit hemozoin formation
- Plasmodium uses amino acids from hemoglobin found in the red blood cells to build merozoites. - However, heme, which is released when hemoglobin is broken down, is toxic for the parasite; in order to avoid toxicity, *parasites crystallize heme into hemozoin*.
- Chloroquine and artemisinin inhibit the crystallization process. Without hemozoin formation, the parasite is exposed to heme, which results in parasite killing.

Plasmodium in human lifecycle

- mosquito bites individual and injects several hundred sporozoites into dermis, migrate into tissue until they reach capillary but can also enter lymph
- travel via blood vessels
- 2 signals within the blood vessels allow the sporozoites to know that they have reached the liver:
- heparin sulphate proteoglycans (HSPG) are derived from hepatocytes. *Sporozoites bind HSPG with circumsporozoite proteins and move towards hepatocytes*
- Kupffer cells found within endothelial cells: macrophages that are only present in the liver, recognized by sporozoites and they can enter here

Plasmodium in human lifecycle (2)

- Once sporozoites are in the liver, they move from one hepatocyte to the next, using lipases and proteases found on their surface. Eventually stops at one of the cells and differentiates into a schizont.
- The *schizont* is like a sac that eventually ruptures, *releasing merozoites*. Merozoites are released back into the bloodstream. This starts the erythrocytic cycle.
- Red blood cells are a great choice of target. Once the merozoites reach red blood cells, they are not exposed to the immune system because red blood cells don't have an MHC. Additionally, red blood cells contain hemoglobin, which contains a lot of amino acids that are useful for merozoite multiplication.
- *Within the red blood cells, they convert back into a schizont*, which then ruptures, once again releasing merozoites that continue to infect other red blood cells.
- The infection of red blood cells causes the pathology of disease and the symptoms of malaria.

Merozoite

- The merozoite contains a nucleus with genes that express proteins required for production of more merozoites.
- The merozoite is capable of producing its own proteins. Doesn't need the help of the red blood cell, which lacks a nucleus.
- *Vesicles, dense granules, and microneme contain proteins that are secreted into the red blood cells to replicate the merozoites*.
- The merozoite contains an opening (apical prominence) that allows them to secrete the proteins.

Entry of merozoite into red blood cells

- Merozoite binds a red blood cell, orienting itself so that its secretion port can begin to inject proteins into the red blood cell.
- Needs an interaction of *CD147 (red blood cell) with RH5 and RIPR (merozoite)*, among others.
- Proteins are injected into the red blood cells.
- *RON2 protein* enters the red blood cell and subsequently goes to the surface of the red blood cell to tightly interact with *AMA1* found on the surface of the merozoite. This stimulates the movement of the *actomyosin motor* (muscle) present in the merozoite to drive the merozoite into the red blood cell by an active process.
- Once the merozoite is found in the red blood cell, it continues injecting proteins.
- The red blood cell is overtaken and used for replication of more and more merozoites.

Receptor interaction: Evolution from a gorilla parasite (P. adleri) to a human parasite (P. falciparum)

- For any pathogen to enter into a human population, there has to be an initial receptor interaction.
- Interaction between CD147 and RH5 + RIPR is critical. Without it, the merozoite cannot bind the red blood cell.
- It's believed that *gorilla plasmodium RH5 mutated to bind to human CD147*.
- Humans became a better reservoir than gorilla.
- Mutations are key to evolution of pathogens to infect humans, notably receptors.

Erythrocyte cycle

1) The merozoite binds to the red blood cells and gets into the cell through anchoring and actinomycin activation *within 5 minutes*.
- Normally, there is *only one merozoite infecting each red blood cell*
2) The protein expression starts 12 hours later (*ring stage*) to form the foundation for making more merozoites.
3) The merozoite enters the *trophozoite stage in about 24 hours*. In this stage, a lot of the proteins are starting to be produced and the *parasite precipitates heme into hemozoin*, which forms a reddish or brownish color.
- Heme is a breakdown product of hemoglobin and is toxic to the parasite.
4) By *36 hours* you can start to see more formation of new merozoites and production of hemozoin. There are *proteins deposited on the surface of the red blood cells called the Plasmodium falciparum erythrocyte membrane protein (PfEMP1 or PfP)*.
5) After about *2 days, it forms the schizont* (~40 hours). Within 48 hours, the red blood cell converts into a factory producing either *16 or 32 merozoites per round of replication*, which are released to infect more red blood cells.

Role of the PfEMP1 proteins

- These proteins on the surface of the red blood cells are like knobs that stick out of the cells. They bind to receptors on endothelial cells.
- Endothelial cells are the cells that make up blood vessels, which have many receptors on them that differ in different organs
- The P. falciparum-infected RBCs bind to the endothelial cell receptors. It causes the infected RBCs to stick to blood vessels.
- It wants to stick to the endothelial cells to avoid going to the spleen; if the infected RBC ends up in the spleen, it will get destroyed and removed by splenic macrophages that recognize it as damaged

Plasmodium Infected red blood cells avoid spleen

- When blood cells go from the afferent to the efferent arteries of the highly vascularized spleen, they interact with the immune system. It activates the immune response if there are any infected cells, which can be destroyed by splenic macrophages.
- The more the infected red blood cells stay out of the spleen, the better the infection for the plasmodium.

What are the different PfEMP1 protein receptors

- express a lot of different domains because there are so many different types of receptors on the endothelial cells, changing depending on the organ
- *ICAM-1* receptors on capillaries within the *brain*
- *CSA* receptors in the *placenta*
- *CD36* receptors on *normal tissues*
- *CR1* receptors on *other RBCs* (can be uninfected).
- Binding to different parts of the body causes different pathologies.
- If many infected red blood cells bind the capillaries of the brain (via ICAM-1), it can cause cerebral malaria.
- Binding CSA in the placenta can be bad for the fetus because the placenta cannot properly function. This could lead to low birth weight, prematurity, and death of fetus.

Why does malaria cause death

- Malaria causes death because the brain is starved for oxygen because of the blocked blood vessels (binding of RBCs to one another causes rosetting and vascular obstruction)
- Malaria does not kill people so fast that it blocks transmission, and when mosquitoes bite, there are many merozoites in the blood that can be transmitted.
- There must be a balance because there is no advantage for the pathogen to kill the host before transmission.

Explain humoral response to malaria

- Our body recognizes that we are assaulted by this pathogen, and we produce antibodies against foreign proteins on the red blood cells (PfEMP1) to protect ourselves.
- If you bind the PfEMP1 surface proteins with antibodies, then you do not have effective binding to the endothelial cells and the infected cells can start to be removed in the spleen.
- Plasmodium responds to this by having 60 different PfEMP1 proteins (variants) so that the antibodies are not able to neutralize all PfEMP1 surface proteins.

Var genes

- The PfEMP1 proteins are expressed by the Var genes and there are at least 60 of these genes within the plasmodium, leading *up to 60 variants*
- parasites do not need to wait for evolution to develop new variants since they already have these genes in their genome. Only one gene is expressed at a time, *switching occurs to provide antigenic variation*
- When one antibody is effective at blocking the infection, the plasmodium switches to another var gene, and there is enough antigenic difference between var genes to keep the parasite infecting the host.

Malaria susceptibility and age

- Young children below age 5 are highly susceptible to malaria because they did not have the chance yet to develop good antibodies against the PfEMP1 proteins.
- From 5 to 10 years old, children are also susceptible, but they get mild disease since they have some antibodies and some immunity, which is enough to reduce the parasite level.
- From 15 to 25 years old, people can have parasitaemia (green zone), which is similar to an asymptomatic infection.
- The level of parasite is probably high enough to get passed on to the next mosquito but maybe not too effectively.

Premunition

- In these endemic regions, adults have high levels of antibodies against different PfEMP1 proteins and are probably getting boosted every year.
- The antibodies protect them, and they get mild to no disease (asymptomatic infection), called premunition, which is naturally acquired immunity maintained by repeated antigen exposure from infective bites.
- If those people were to leave a region where it is endemic (Africa, Asia, South/Central America) to North America for 5-6 years, their antibody levels would go down and since the memory response is not very good, they would be more susceptible to the infection when they go back to the endemic region.
- There is no vaccine for malaria and PfEMP1 proteins are not a good target because we cannot inject all the different variants. We would need a vaccine that enables the production of an antibody against all these proteins. Efforts have been made but they were quite unsuccessful.

RIFIN and STEVOR

- merozoite-derived surface proteins (like PfEMP1)
- also have lots of variability: *\>130 members in RIFIN family*, *\>30 members in STEVOR family*
- located near Var genes within the Plasmodium genome
- bind surface proteins on RBCs

*RIFIN*
- binds group A glycoporin
- Type O blood \= less severe malaria (no RIFIN binding site)

*STEVOR*
- binds glycoporin C

Parasite and Virus comparison

- The advantage of parasites is that they have a large genome to express all kinds of genes to deal with the host. In contrast, viruses are more limited in their genome and only have 10-12 genes, so they have to hope that variants will happen on their own.
- That is one reason why parasites tend to survive longer and cause more chronic diseases than viruses, which tend to cause more acute infections.
- Exception: HIV is a long-term virus due to its integration into the genome.

African Trypanosomiasis

- When EU was trying to colonize parts of Africa, they were bringing their animals and trying to reproduce the normal farming practice they had in their country. The animals developed Nagana disease, cows would lose weight
- Brits send vet David bruce who took blood samples from animals dying from the disease and found organisms called Trypanosoma brucei
- Trypano: meaning it is a screw type organism.
- Soma: meaning body.
- Brucei: from the name of the doctor (David Bruce).
- He observed that the wild animals also had the disease, but they had developed some resistance to prevent them from dying like the European animals.
- His solution was to kill all the wild animals to remove all the reservoirs.
- He determined that the disease was being transmitted by the painful bites of tsetse flies, which replicate near rivers.
- Local African people realized that living near rivers was not a good idea, but Europeans liked to live near rivers.

Sleeping sickness

- Hundreds of years before David Bruce discovered what was happening in cattle, missionaries would go to Africa, and they would see that people had a lethargic disease that they would call sleeping disease, because all people would do was lie down.
- In stage 1 of the disease, Trypanosoma brucei is present in the blood and people have winterbottom sign (swelling of the cervical lymph node).
- In stage 2 of the disease, Trypanosoma brucei is present in the central nervous system and people have sleeping sickness, lethargy, confusion, and it can be fatal.

Sleeping sickness 2

- Slave traders (1500-1600s) would not take people with swelling of the cervical node on their neck.
- The lymph node was swollen due to the immune response and people would not live very long.
- It was around 1903 when Castellani took blood from these people that he also saw this trypanosome in the blood of humans.
- Brucei argued that Castellani stole his idea even though he had not published at the time.
- This disease is specific to Sub-Saharan Africa.

African Trypanosomiasis (T. brucei)

- nagana disease in domestic animals
- winterbottom sign: swelling of cervical lymph node, *stage 1* where T. brucei is present in *blood*
- sleeping sickness: lethargy, confusion, fatal if untreated, *stage 2* is when T. brucei is present in the *CNS*.

What are some treatment options for african trypanosomiasis

- *Stage 1* (blood, recurrent fever): *pentamidine*.
- Developed ~70-80 years ago, administered via IV.
- *Stage 2* (brain, neurological disorder, confusion, sleepiness): *melarsoprol* (IV administration).
- Stage 1 and 2 can now be treated with a newer drug: *fexinidazole*, administered *orally*.
- Developed by Drugs for Neglected Diseases initiative (DNDi) in 2019. This is the leading organization in developing drugs for neglected diseases.

Explain the human african trypanosomiasis distribution

T. brucei *rhodesiense* (infects cattle and humans - Stage 4) *East Africa*.
- causes *5% of trypanosomiasis*, zoonotic and has animal reservoir
T. brucei *gambiense* (infects only humans): *West Africa*
- causes *95% of trypanosomiasis*
- *stage 5* disease, anthroponotic

T. brucei physiology

- Typical eukaryotic cells.
▪ Nucleus with nuclear DNA.
▪ Endoplasmic reticulum.
▪ Ribosomes.
▪ Golgi apparatus.
▪ Large mitochondria.
- Has its own kinetoplast (genome involved in the production of energy - ATP).
- Variant surface glycoprotein (VSG).
- The whole parasite, flagella and body, is covered by a single glycoprotein.

Kinetoplastida class

- Kingdom: Protista
- Phylum: Euglenozoa
- Class: Kinetoplastida. All of these parasites have kinetoplast DNA (maxi and minicircle)
- Order: Trypanosomatida, splits into two genera:
- Trypanosoma: many more species than leishmania
- Leishmania: ~20 known species
- Many different species of the Trypanosoma parasites are found in Africa

Different species of Trypanosome parasites

- Different animal reservoirs.
- Only T. b. gambiense is a stage 5 anthroponotic disease, all others have animal resevoirs.
- Different mode of transmission.
- A number of Trypanosoma parasites are found in South America as well.
- New Trypanosoma species are constantly being discovered (mostly by a group of scientists from Czechoslovakia) by sampling animal blood in Africa and South America.

Trypanosoma brucei lifecycle

- Transmitted by the *tsetse fly* in Africa.
- Tsetse fly introduces the parasite into the blood. - The *trypomastigote* stays in the blood and *replicates in the blood* (doesn't need to enter any specific cells).
- Its presence in the blood is what causes the associated pathology.
- Tsetse fly feeds on an infected individual.
- *Trypomastigote gets converted into an epimastigote within the tsetse fly*.
- *Epimastigote converts back into trypomastigote* before the tsetse fly takes another bloodmeal to infect an individual.

Trypomastigotes in the blood

- unusual for a pathogenic organism to survive in the blood since it is normally sterile
- pathogens are quickly removed from the blood via antibodies, which bind directly to the pathogen
- antibodies can bind trypanosome cells directly, stimulating the complement system which would ultimately kill it

What does the Trypanosome parasite have that helps it survive in the blood

- The parasite has VSG on its surface. This is key for it to survive in the blood.
- VSG-coated membrane transport vesicles are ports which deposit the glycoprotein on the surface of the parasite. - Everything on the surface of the trypanosome parasite, including the flagella, is covered with VSG.
- *VSG anchors to the GPI anchor of the lipid membrane* of the trypanosome. The *conserved region is not exposed*, while the *variable region is exposed* to the blood.

Variant surface glycoprotein switching

- During infection, there is a wave in the parasites in the blood due to high replication. The immune system is activated, producing antibodies against the parasites. They clear the parasites and drive the level of parasites in the blood down.
- After a couple of weeks of infection, the parasite changes its surface glycoprotein variant (i.e., from VSG1 to VSG2) and the original antibodies no longer recognize it.
- Parasite replication increases again, and the immune system generates new antibodies to clear the infection. The new antibodies are effective at reducing the level of parasites (Fig. 5).
- The parasite changes the VSG again. The cycle repeats and continues over and over.
- VSG switching causes infected individuals to exhibit periodic waves of high and low parasite counts, corresponding with waves of new VSGs

VSG Genes

- The gene encoding the VSG has a region consists of multiple parts.
- Region that encodes the *GPI anchor* (~20 a.a., *C-terminus*).
- A *conserved region* (~100 a.a.).
- A *long variable region* (~360 a.a.).
- This is what changes from gene to gene.
- *Signal sequence* (~20 a.a., *N-terminus*)
- DNA sequencing of the genome has identified hundreds of VSG genes on numerous chromosomes.
- Each gene with *different variable region sequence* and identical signal, conserved, and GPI anchor regions.

VSG gene expression

- In the genome, certain VSG genes are not expressed; only one is expressed at a time.
- Only the *VSG gene closest to the telomere gets expressed*.
- After a few weeks of expression of one specific gene, switching begins.
- *Gene conversion event: the new gene to express is duplicated and introduced into the expression site (telomere). The original copy stays where it was. The gene that was expressed before gets deleted.*
- Now, the new gene is active.
- The expression of VSG genes occurs by *RNA polymerase I* (strongest promoter)
- RNA polymerase I usually expresses
ribosomal RNA but in trypanosome it is also used for VSG.

VSG switching induction

- When trypanosome is cultured in the lab, it also switches VSG genes, implying that the switch is pre-programmed and doesn't need the pressure of the host's immune system.
- This mechanism is specific to African trypanosomes.

Discontinuous transcription

- This mechanism is found in African and South American trypanosomiasis, but *also in leishmania*.
- Can happen with any gene.
- At the 5' end, there is a 39-nucleotide sequence that is present in all mRNA sequences (*spliced leader*). It is not present in the gene, but only in the mRNA.
- This happens through *discontinuous transcription*.
- A given gene is expressed in mRNA from chromosome B. However, this mRNA isn't complete.
- There's another chromosome (A) that expresses mini exon repeats. This is a small transcript of ~140 nucleotides.
- The two pieces of RNA - miniexon transcript and mRNA transcript - are added to one another through *trans-splicing* to produce the mature mRNA.

What's the point of discontinuous transcription to add the 39-nucleotide sequence at 5' end?

- It has a cap (modified guanine residue) which is needed for all mRNA.
- Not every Leishmania and Trypanosome gene has a separate promoter; they only have 1-2 promoters per chromosome, which express hundreds to thousands of genes (the whole chromosome). In order *to deal with this long polycistronic transcript* and mRNAs that are stuck to one another, *trans splicing* comes in and cuts, adding to the 5' end (Fig. 10).
- Essentially, this helps to separate the long RNA that contains genes from a whole chromosome into individual RNAs.
- Additionally, after the cut and the addition of the 39-nucleotide sequence to the 5' end takes place, the 3' end also gets poly-adenylated.
- Unlike human transcription, there is no regulation of the level of transcription of various genes. *mRNA levels are largely determined by RNA stability sequences in the 3' UTR*. This doesn't dictate the level of production of RNA, but its stability.