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An Ebola Question

Olytico-Question-Mark-1024x576I’ve had a question about Ebola posted on the StackExchange Biology page for some time without getting any answers. Basically, I was wondering about how antibody responses to Ebola can drive either sterilizing immunity (the goal) or actually improve the virus’s entry into host cells (a big problem). The idea that Ebola antibodies may be detrimental to the host was first raised by Baize et al, and my question is how this has impacted efforts to develop an effective vaccine. For background, I’ve written about this topic previously.

If anyone knows what the current thinking is in this area, please point them my way.

 
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Posted by on September 4, 2015 in Uncategorized

 

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Congressional hearing on Vaccination

schuchatIn the wake of several months of nation wide fear of ebola,  a new outbreak strikes home. One can only wonder whether the timing of these events will have a lasting effect on the country.

as a side note, it’s interesting to hear Dr Schuchat, the Director of the National Center for Immunization and Respiratory Diseases at the CDC, bring up the number of annual deaths attributed to influenza in the US as between 5-30,000. Because of the way deaths are reported, it is actually tricky to get a very accurate number for this, but a CDC study attempting to do so sets the range as falling between two recent extremes of  “3,349 in 1986–87 to 48,614 in 2003–04.”  For perspective, the 2014 / 15 outbreak of ebola in Africa has claimed just over 9,000 lives.

Despite the high annual mortality of flu, only about 40% of American adults get vaccinated each year.

Screen Shot 2015-02-11 at 1.10.13 PM

Measles has a track record of much higher mortality rates than flu, and is much more contagious. The WHO warns, “[t]he highly contagious virus is spread by coughing and sneezing, close personal contact or direct contact with infected nasal or throat secretions.

The virus remains active and contagious in the air or on infected surfaces for up to 2 hours.”

In 2004, Perry and Halsey summarized what we, as a population have forgotten, “Before the introduction of measles vaccines, measles virus infected 95%–98% of children by age 18 years, and measles was considered an inevitable rite of passage.”

Measles is associated with a number of complications, including pneumonia (either directly as a result of measles or from another agent) which is associated with the majority of deaths attributed to the disease. Over the years, as medical interventions have improved, the number of measles-associated fatalities has dropped from “One hundred years ago in Scotland, the measles case-fatality rate was 30–40 deaths per 1000 cases. In the United States, mortality from measles decreased from 25 per 1000 reported cases in 1912 to 1 per 1000 reported cases in 1962.”(see figure below) Nevertheless, it remains a dangerous disease capable of causing a number of complications and death. (all those deaths from flu mentioned above, flu kills only about 1.4 to 16.7 deaths per 100,000 persons.)

Screen Shot 2015-02-11 at 1.30.17 PM

Perry and Halsey conclude:

Measles vaccination is one of the most cost-effective health interventions ever developed. Without the vaccine, 5 million children would die each year from measles-assuming an estimated case-fatality rate of 2%–3%. Without measles vaccination, the costs of caring for those with measles in the United States would be ~$2.2 billion annually, and the indirect costs would be an additional $1.6 billion. Each dollar spent on measles vaccine saves $12–$ 17 in direct and indirect costs.

With this in mind, here is the full video broadcast of CSPAN’s coverage of the the hearing on childhood vaccination:

http://www.c-span.org/video/?324253-1/hearing-childhood-vaccination

 
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Posted by on February 11, 2015 in Uncategorized

 

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Virus, Vaccine and Passive Antibody Therapy

The immune system is a many-layered construction that protects the body through barrier defences, additional non-specific responses including phagocytosis and chemokines, an antibody-mediated humoral response capable of neutralizing viral particles, and a cellular response for eliminating infected cells.

Ebola: Disease and Response

mapEbola is a viral disease first identified during a first appeared in 1976 in two simultaneous outbreaks, one in Nzara, Sudan, and the other in Yambuku, Democratic Republic of Congo.  It is reasonable to suspect that Ebola has infected humans prior to this time without being identified specifically. This is a reasonable assertion because, like the first, all subsequent outbreaks have occurred in remote areas of Western African countries that are largely isolated. Although infamous for its lethality, this remoteness has proved self-limiting in terms spread.

The current epidemic has defied these rules resulting in escape from the remote areas of West African villages to larger population centers, and for the first time ever, even resulting in at least one case presenting in the United States. (citation)

In general, although viral infections are not treatable by classical antibiotics, vaccines against these types of organisms have been largely successful. Although it is impossible to know exactly why a specific vaccine works, it is reasonable to assume that a humoral response (i.e. mediated by antibodies) is involved in most cases as antibody titer correlates well with protection.

I the case of Ebola, there is data regarding the type of immune responses mounted by patients who have survived the disease compared to those who have not. Baize et al report that “early and increasing levels of IgG, directed mainly against the nucleoprotein and the 40-kDa viral protein, were followed by clearance of circulating viral antigen and activation of cytotoxic T cells” in survivors of disease. While “fatal infection was characterized by impaired humoral responses, with absent specific IgG and barely detectable IgM.” Again, this supports the idea that an effective humoral response is key to protection.

More evidence of the centrality of the humoral response comes from data published by Villinger, et al (citation) showing that “IL-6 levels are unusually low among fatal cases.” They suggest that this points to a deficiency of the endothelial cells that produce this cytokine leading to failure to protect. An alternative explanation may be that macrophages, which are key targets of ebola infection – and are producers of IL-6, are also failing to respond appropriately due to their involvement as targets. This leads to an obvious defect in immune response as IL-6 supports the growth of B cells and is antagonistic to regulatory responses (i.e. regulatory T cells).

If antibodies are so important to response, what are the targets of these antibodies and what issues are there related to this response?

Ebola Virus:

Eboal5Ebola has only one known surface protein found on virions and infected cells. It is presumed that this protein, a ‘sugar-coated’ glycoprotein (GP), is what enables virions to adhere to target cells, a vital first step in the infection of host cells by animal viruses. As neutralizing immunity against viruses is presumed to be a result of the opsinization of viral particles by antibody, the Ebola GP is the obvious target of these antibodies. However, there are still a number of epitopes (regions of the protein to which immune reactions develop) on the GP protein to which antibodies bind. And, furthermore, two versions of GP are made, one in the viral envelope (membrane) and one that is secreted from infected cells. Together, this means that there are a lot of different spots for antibodies to bind, and some spots may be better for protective immunity, while others have no protective effect at all.

Vaccines against ebola are currently being developed with the hope of bringing these to affected areas to either prevent – or at least control- outbreaks at their source. The benefits of developing an effective vaccine include actively inducing life-long immunity.

A second method of fighting disease is to treat with previously generated antibodies in a way that the virus is neutralized, but life-long protection is not induced. One way of accomplishing this treatment is by harvesting serum from patients who were infected, but survived the disease. This has obvious limitations logistically and there is insufficient data on these treatments to know whether they were actually helpful in treating patients. Another way to transfer this sort of ‘passive’ immunity is by making large amounts of a single antibody in cell culture. These ‘monoclonal’ antibodies are highly standardized and can be produced in very large quantities.

A number of monoclonal antibodies targeting different epitopes on the Ebola GP have been developed and show protective effects when administered after viral exposure (i.e. therapeutically). One example of this kind of therapy is ZMapp  from Mapp biopharmaceutical. In studies with animals, they found that “a combination of monoclonal antibodies (ZMapp), optimized from two previous antibody cocktails, is able to rescue 100% of rhesus macaques when treatment is initiated up to 5 days post-challenge.”

Treatment of Ebola patients with Convalescent Serum

Treatment of Ebola patients with Convalescent Serum

I’ve written before in this space about one of the challenges that antibody treatment against ebola. Because ebola infects macrophages as one of its targets, and because one of the jobs of macrophages is to clear opsonized (antibody-coated) particles, ebola appears to have co-opted this function as a mechanism for penetrating and infecting cells. This characteristic is termed Antibody-Dependent Enhancement (ADE) of infection and has been shown to increase the infectivity of the embryonic kidney cell line, HEK-293, in vitro (Takeda et al 2003). Reportedly, the mechanism for this enhancement is via the complement protein, C1q, and receptors on the host cells.

Together, these data beg the question of whether antibody treatments, such as ZMapp, or vaccines leading to humoral responses will be helpful or harmful in the treatment and protection of patients.

“On 11 August, a group of experts convened by WHO reached consensus that the use of experimental medicines and vaccines under the exceptional circumstances of the Ebola epidemic is ethically acceptable.” So, we may find out the answers to these questions much sooner than we would otherwise expect.

 
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Posted by on November 5, 2014 in Uncategorized

 

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Back to School: Zombies, Ebola and some cool tunes

Ebola is in the news a lot right now.

Could this be The Coming Plague that Laurie Garret warned us about in 1994?

By the late 1980s, with the world shaken by the strangest and deadliest arrival of all – HIV and AIDS – Garrett traveled widely in search of understanding: Why did new viruses and bacteria appear, seemingly out of nowhere? Why couldn’t modern medicine vanquish HIV and other newly emerging microbes? How were scientists battling these diseases? Had hubris put the arrogant biomedical world of the late 20th Century at peril?

– from her website

A recent depiction (below) of the rise of Ebola cases and deaths (cumulative numbers) appears on the wikipedia site.

Diseased_Ebola_2014

The CDC is probably the most reliable source of information on the virus today. They provide a wealth of information about the virus, including that infection does not spread through the air, water or food (with the possible exception of some bushmeat – likely bats acting as a reservoir for the virus). And further, although Ebola does have a frighteningly long incubation period (of about 21 days), there is no evidence that asymptomatic persons can spread the disease.

When an infection does occur in humans, the virus can be spread in several ways to others. The virus is spread through direct contact (through broken skin or mucous membranes) with

  • a sick person’s blood or body fluids (urine, saliva, feces, vomit, and semen)

  • objects (such as needles) that have been contaminated with infected body fluids

  • infected animals

In recent news, two items sound eerily similar to those scrolling across the newswire in the game Pandemic 2:

August 8, 2014 – Experts at the World Health Organization declare the Ebola epidemic ravaging West Africa an international health emergency that requires a coordinated global approach, describing it as the worst outbreak in the four-decade history of tracking the disease.

August 19, 2014 – Liberia’s President Ellen Johnson Sirleaf declares a nationwide curfew beginning August 20 and orders two communities to be completely quarantined, with no movement in or out of the areas.

With all this in mind, maybe it’s a good time to pack up your emergency preparedness kit. And, while you’re at it, check out this comic from the CDC to help determine what you need to include:

Zombie_GN_Final

Imagine a ven diagram illustrating preparedness. How prepared should you be for flooding? fire? tornado? active shooter? zombies?? If you’re prepared for the apocalypse, surely you can handle a flood.

prepared

While you’re huddled in the basement waiting for the threat to pass, enjoy some music to keep your spirits up.

 
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Posted by on August 20, 2014 in Education, Uncategorized

 

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Working Overtime to Prevent Sudden Death

Do you have Ebola? If you're reading this, then probably not.

Do you have Ebola?
If you’re reading this, then probably not.

My cousin, in Philadelphia, tipped me off that I should write a blog article about the current Ebola outbreak that has occurred this year in Western Africa. One of the reasons that this interested him was that the story of the outbreak was being shadowed by another story of a ‘secret’ serum that was being used to treat some of the American victims of the disease. I think ‘Secret’ was the operative word. I had definitely heard about the outbreak, but this was actually the first time that I heard about this serum – and it immediately tipped of my BS / Conspiracy theory detector because of the suggestion that America actually had a secret ‘cure’ for Ebola. It almost begs for allegations by people wearing tinfoil anti-alien hats that America was engineering some Apocalypse Bringing Disease a la I am Legend or Dawn of the Planet of the Apes.

So, I was interested to hear that there actually is a serum to treat Ebola – it’s just not secret, and it’s not an approved treatment, but an experimental one. Ebola VirusFirst, something about the virus…

There is currently no vaccine available for Ebola virus infection and the standard of care remains supportive therapy aimed at maintaining the body’s electrolytes, blood pressure and to prevent / treat additional infections that may otherwise complicate care(1). Coupled with an extraordinarily high fatality rate (up to 90%) and horrifying symptoms including internal and external bleeding, fever and

Western Africa

Western Africa

intense weakness, it remains one of the most feared diseases in the world (2). Ebola is so debilitating and deadly, in fact, that its severity has actually functioned to keep it contained within a relatively small area of western Africa. Most cases tend to occur in and around poor, unsanitary hospitals where virus spreads from a contaminated individual or cadaver to a person (often serving as a healthcare worker). Often cases present with symptoms similar to more common, less lethal diseases and are not quarantined away from other patients leading to a rapid accumulation of nosocomial infections (3). One reason for the high mortality rate associated with Ebola infections may be due to a curious condition in which antibodies against the virus may, ironically, worsen the infection. The mechanism of this behavior appears to operate through the binding of antibody to viral glycoproteins, followed by antibody-mediated phagocytosis of virus by immune cells. This is confounding because it is this process that is utilized by immune cells to destroy viruses and may further impair the ability of researchers to develop an effective vaccine as most vaccines work by promoting antibody development (4). With Ebola, the interaction of a protein on the virus’ surface is bound by antibody, which is then bound by an immune cell that internalizes the virus, but instead of destroying the virus, it manages to escape destruction and infect the cell.

Rather than making you better, antibodies against Ebola may make you worse off.

Rather than making you better, antibodies against Ebola may make you worse off.

To make matters worse, this time around many more people are contracting the disease, so concern is elevated around the world, even some US Congressmen have been making hay about the possibility that undocumented immigrants from Central America may introduce Ebola into the US. Which brings me back to the conspiracy angle. What’s this about a secret serum again?


The serum is actually just an experimental treatment – one that is extremely early in the development process, called ZMapp. This is a product produced by Mapp Biopharmaceutical Inc. that is a combination of three monoclonal antibodies made in tobacco plants (this is a more common method than you might think). The idea being that these antibodies will provide passive protection against Ebola, much like the antibodies produced by a typical vaccine, but -hopefully – without the adverse effects associated with the antibodies that enhance infectivity. Reading the article describing the manufacture of these antibodies does not provide an explanation of how the antibody-mediated enhancement of infection will be evaded, but one may imagine the construction of neutralizing antibodies that lack the constant regions associated with FcR or C1q binding as the binding of these two proteins have been proposed as causing the adverse effect. As this drug lends passive immunity, it may (if effective) prevent infection of an exposed person – or at least lesson the severity of the infection, however it will NOT lead to the accumulation of antibodies by the patient as would a vaccine. Rather, this form of immunity is more akin to treatment with an anti-serum following a snake bite. With luck, a silver lining to this major outbreak may be the opportunity to test an early-stage treatment, possibly resulting in the first ray of hope in improving Ebola survival.

Can I catch this?

Can I catch this?

References:

  1. http://www.vox.com/2014/7/29/5945515/ebola-outbreak-virus-disease-symptoms-africa-facts-guinea-nigeria
  2. http://www.who.int/mediacentre/factsheets/fs103/en/
  3. https://microbewiki.kenyon.edu/index.php/Infection_Mechanism_of_Genus_Ebolavirus
  4. http://jid.oxfordjournals.org/content/196/Supplement_2/S347.full.pdf
 
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Posted by on August 7, 2014 in Uncategorized

 

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An Epidemiological Method: Using RFLP to Identify Strains of Pathogens

An excellent classroom resource for a case study in epidemiology is presented by the CDC. This study walks students through an outbreak of E. coli O157:H7 in Michigan.

The purpose of this study is to provide student investigators with the opportunity to walk through the procedures and rationale behind investigating the etiology and to develop experiments testing hypotheses generated by the students.

I am using this exercise as an end-of-semester project for my microbiology students to work through collaboratively now that we have completed our discussion of Paul Offit’s Vaccinated.

The study begins:

PART I – OUTBREAK DETECTION

 

Escherichia coli O157:H7 was first identified as a human pathogen in 1982 in the United States of America, following an outbreak of bloody diarrhea associated with contaminated hamburger meat. Sporadic infections and outbreaks have since been reported from many parts of the world, including North America, Western Europe, Australia, Asia, and Africa. Although other animals are capable of carrying and transmitting the infection, cattle are the primary reservoir for E. coli O157:H7. Implicated foods are typically those derived from cattle (e.g., beef, hamburger, raw milk); however, the infection has also been transmitted through contact with infected persons, contaminated water, and other contaminated food products.

Infection with E. coli O157:H7 is diagnosed by detecting the bacterium in the stool. Most laboratories that culture stool do not routinely test for E. coli O157:H7, but require a special request from the health care provider. Only recently has E. coli O157:H7 infection become nationally notifiable in the U.S. Outside the U.S., reporting is limited to a few but increasing number of countries.

In the last week of June 1997, the Michigan Department of Community Health (MDCH) noticed an increase in laboratory reports of E. coli O157:H7 infection. Fifty-two infections had been reported that month, compared with 18 in June of 1996. In preliminary investigations, no obvious epidemiologic linkages between the patients were found.   The increase in cases continued into July.

Students are then asked a number of introductory questions and then presented with the following problem:

Compare the DNA fingerprints in Figure 2 from seven of the Michigan E. coli O157:H7 cases. Each isolate has its own vertical lane (i.e., column). Controls appear in lanes #1, 5, and 10. Which Michigan isolates appear similar?

This question requires some background in DNA Fingerprinting (aka Restriction Fragment Length Polymorphisms, or RFLPs), which I want to take some time to explain.

As the source material states, The purpose of this test is to identify common strains of organisms through their DNA banding pattern. “Different DNA composition will result in different PFGE banding patterns. Bacteria descended from the same original parent will have virtually identical DNA and their DNA fingerprints will be indistinguishable. Identification of a cluster of isolates with the same PFGE pattern suggests that they arose from the same parent and could be from the same source. “ (emphasis mine).

The method involves two core techniques. First, DNA from the target organism must be isolated and cut with one or more restriction enzyme(s). This will create a number of DNA fragments, where the precise number and size of fragments is determined by the sequence of that organism’s DNA.

As an example, let’s imagine a 10,000 base pair (bp) chromosome that we intend to cut with the restriction enzyme, EcoRI. EcoRI recognizes and cuts double stranded DNA at a specific sequence of 6 bases.

Image

Figure: DNA cut by the Restriction Enzyme, EcoRI. A. DNA sequence with EcoRI recognition site highlighted and cut pattern illustrated. B. Enzyme binds to DNA at the recognition site. C. DNA has been cleaved.

On average, this enzyme will cut a random sequence of DNA every 4096 bases (this can be estimated by 4 raised to the power of n, where n = the number of bases in the enzyme’s recognition sequence , or 46 = 4096 in this case.) In our example, this suggests that a 10,000 bp chromosome will have two EcoRI sites by random chance.

The circular chromosome should be cut twice by this enzyme, resulting in two fragments of DNA (see note #2, below). Let’s say the two bands are 4000 bp and 6000 bp.

We can see these two fragments by running them through agarose, which works as a molecular sieve, to separate the two fragments by size

How does this work?

DNA is a negatively charged molecule with that charge spread uniformly across the length of the fragment. Therefore, there is no difference in charge between our two fragments, except in proportion to their length. This means that as they run through the sieve, the only difference between the molecules comes from their lengths. As any sieve, smaller objects go through easier, while larger ones are held up.

ImageThe result is that the two fragments will appear as distinct bands on a gel, with the smaller fragment running farther through the agarose that the larger. (here, the smaller band at the bottom of the gel has migrated farther toward the positive electrode)

If someone new were to become infected with this bacteria, we could isolate it from them, digest the DNA and get the same banding pattern. A closely related bacteria may have one additional EcoRI site. This would result in one of the two bands being cut into two smaller fragments, meaning that the two strains could be easily distinguished.

Back to the question posed above…

Given this, examine the following compilation of samples. Controls appear in lanes #1, 5, and 10. Which of the remaining isolates appear similar?

Image

Definitions:

  1. Restriction Enzyme or Restriction Endonuclease– an enzyme that can recognize and cut DNA.
  2. Recognition Sequence – the sequence of bases that a restriction enzyme recognizes and binds to.

 

Notes:

  1. In my example, we are using the restriction enzyme, EcoRI, to cut DNA from E. coli. As the name suggests, EcoRI actually derives from E.coli, where it functions as a defence against invading DNA, i.e. a virus. In order to do this successfully, E. coli will either not have any EcoRI restriction sites in its own DNA, or it will protect them by methylation so that the enzyme does not destroy the host’s own DNA. I am ignoring the possibility that the DNA we are dealing with in our experiment may not be cleavable with this enzyme.
  2. Also note, that bacterial chromosomes are circular, rather than linear – interestingly, this means that they are not actually ‘chromosomes’ at all. Again, let’s ignore this.
 
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Posted by on April 18, 2014 in Uncategorized

 

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