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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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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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Using Antibodies as vaccine delivery vehicles

Antibodies are glycoprotein molecules synthesized by plasma cells (mature, activated B cells) with the capacity of binding to any potential antigen epitope. For a review of lymphocytes and how they are activated, see this link where you will find more information about antibody production in response to ’challenge’.

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An (IgG) antibody with basic structural features labeled

Antibodies are the natural products of these plasma cells and function in a variety of ways to effect immunity. Most basically, they bind and may interrupt the function of the target molecules or trigger a response disadvantageous to the pathogen. In addition, a number of other functions are mediated by these molecules, including recruitment of complement and of phagocytic cells that will digest and inactivate the cell / antigen.

Therapies, such as vaccines, are designed to separate and eliminate the disease-causing elements of a pathogen from those that generate an immune response, thereby initiating a normal immune response to antigens without the dangerous exposure to live pathogens. Most often, these are prophylactic vaccines that initiate the development of immune ’memory’ prior to any disease exposure.

In some cases, therapeutic vaccines do much the same job, but are used to ’jump-start’ an immune response that has failed to initiate naturally for some reason (this may be because the target of the therapy is very similar to ’self’ as is the case with cancer), or because a long-term, chronic disease has fooled the body into tolerating an unwanted condition.

Additionally, some molecular therapies provide passive immunity by administering exogenous antibody that fulfills these functions. A weakness of these therapies is that, by providing pre-made antibody, potential antigens are blocked and no endogenous antibody response will be elicited.

A final use of antibodies, to be elaborated further here, is to provide targeted delivery of toxins to pathogens or infected cells or to deliver antigens to the immune system.

Purpose: to trigger / amplify immunity to an ongoing infection or disease

Considerations:

1. Target protein or cell – what cell and what protein on that cell should be targeted to elicit the desired immune response?

2. How to get antibody to the site where target cells are present?

3. What is the desired response / activity of the target cell?

4. What, if any, molecule is being delivered to these cells?

5. Lastly, how can efficacy be measured and what are the objective endpoints that will be used to determine whether therapy is effective?

Although this antibody is not currently in use therapeutically, I will use, as an example, one that I made while working for a biotech company some years ago.

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An antibody with an antigen conjugated to the Fc portion

The antibody we used specifically bound to the macrophage mannose receptor (MMR) expressed by macrophages and the similar phagocyte cells, dendritic cells. Natively, this protein binds to a sugar, mannose, that is commonly charged to protein molecules. Once bound, the MMR will direct receptor-mediated endocytosis of the bound protein and deliver it to endolysosomes for processing and presentation upon MHC class II molecules (see animation below). As explained in the link, processing and presentation lead to the activation of T Cells and the resulting immune response.

Using an antibody that targets this molecule (MMR), a target compound can be fused to the antibody (chemically or genetically) leading to the precise delivery of this compound into the cell and the generation of a response. The antibody will guide the (tumor) antigen to the phagocytic cell. In this way, the antibody serves only as a vehicle. This vehicle takes its passenger, the antigen that we would like to generate an immune response against, and inserts this antigen into the processing and presenting apparatus of these ‘professional’ antigen presenting cells.

Animation of Antibody delivering a Target Antigen to an APC:

 
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Posted by on December 26, 2013 in Uncategorized

 

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Antigen Presentation #2: B Cells

Antigen Presentation

Presentation by B Cells

Before thinking about B Cells presenting antigen, first recall that B Cells are lymphocytes bearing antigen receptors on their surface called B Cell Receptors (BCRs). These BCRs have been randomized during development such that every B Cell can theoretically bind a unique antigen. See Lymphocyte Development for a refresher on this if you need it.  The major function of B Cells is to make antibody that is nearly identical to its receptor protein, which will be secreted and can then bind to antigens of the same shape.

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B Cell with specific BCR engages an antigen on a bacterium (Left). After activation this B Cell will become a Plasma Cell secreting antibody with identical specificity as the original BCR (Right).

A major distinction between B Cell phagocytosis and that by Macrophages is that B Cells only take up materials they have bound with their BCRs, while macrophages take up material indiscriminately. The reason for this, of course, is that B Cells are gearing up to produce antibody, and the best way to ensure this antibody will bind anything of use is if only B Cells bearing specific BCRs known to bind antigen are activated. Macrophages have no antigen-specific receptors, so this specificity is not required by those cells. The membrane bound BCR is exactly the same molecule as secreted antibody – except for the small portion that anchors the BCR to the membrane.

Like macrophages, B cells are ‘professional’ antigen presenting cells (APCs) that take up exogenous antigen, break it down within lysosomes and present the resulting peptide fragments within MHC Class II Molecules. As with other professional APCs, this is intended to pick up foreign, invasive particles for present them to T cells to elicit a specific immune response.

ImageJust by binding to antigen with their BCRs, the B Cell will become (at least partially) activated, stimulating proliferation of this cell and processing/presentation of antigen as indicated above. In order to complete its activation, this B Cell must receive ‘help’ from T Cells capable of binding the presented antigen in the context of MHC II. Because T Cells have also been selected for ‘Non-Self’ exclusivity, this provides additional insurance that this B Cell was truly activated by a ‘Non-Self’ antigen.  The MHC II :: TCR + CD4 interaction between the antigen-presenting B Cell and the helper T Cell results in activation of the T Cell, that immediately gives activation signals (cytokines) back to the B Cell.

 

Keep in Mind the Big Picture!

To summarize with an example:

  1. A bacteria gets into the host
  2. B Cells with BCRs capable of binding any part of that bacteria catch ahold of it
  3. These B Cells gobble up the bacteria (endocytosis)
  4. Inside the B Cell, the bacteria is killed and broken into a bunch of little pieces
  5. The little bacteria pieces are picked up by MHC II molecules
  6. MHC II molecules move to the cell surface and ‘present’ antigen
  7. T Cells with TCRs capable of binding this bacteria piece within MHC II, do so
  8. These T Cells become activated, proliferate and produce activation factors (cytokines)
  9. These activation factors trigger the B Cell to go on proliferating and changing into Plasma Cells.

10. Plasma Cells no longer make BCR on the surface, they make a soluble form of that BCR, called Antibody, and spew that forth in great amounts.

11. Antibody can coat, gum up, and signal the disposal of bacteria all over the body.

Resting and Activated T Cells from “Immune System History” by Dr. Harry Louis E. Trinidad 
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All that ER expansion is to accommodate the heavy load of secreted protein this cell will churn out.

 

 

 
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Posted by on December 8, 2013 in Uncategorized

 

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Immunology’s ‘dihybrid cross’ : Antibody response to different antigens

The progress of infection can be summarized as a pathogen going through a series of steps:

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Progress of Infection

The first three steps, ‘Portal of Entry’, through ‘Surviving Host Defences’ encapsulates all of the immune response. Some key events in the immunity are inflammation and the innate  response, antigen processing and presentation, adaptive immunity and memory.

Several of these topics I’ve described here before including an outline of the development of lymphocytes (B and T Cells – sorry NK Cells) in an article here. The activation of B cells here, immunological memory in several places including here.  Some of these topics I have yet to address (e.g. a good discussion of inflammation), and others (e.g. antigen processing and presentation) have been buried in other posts (see my lymphocyte development, B cell Activation or this post on Transmissible tumors). 

This time, I thought I’d prevent a sketch of the humoral immune response and how this illustrates, like Mendel’s traits in a dihybrid cross, that each immune reaction is ‘independent’. A typical immune response is outlined below showing the development of antibodies following a primary response and then a more rapid and robust secondary response. If we want to compare this response to Mendel’s monohybrid cross, we can see the same response for antigen after antigen just as Mendel saw the same pattern of inheritance for any single trait he observed.

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Response to a single antigen

 

Before we had the ability to ‘see’ this response on a molecular level, we could see its effects on people. Those who previously contracted a disease did not contract that same disease a second time. This immunological memory is the basis for vaccination, where we separate the disease-causing agent from the immunological memory-inducing agent for any given pathogen and then use only the later to vaccinate.

However, Mendel continued to examine traits and how they were inherited individually (i.e. the inheritance of one trait had no bearing on the inheritance of another). He called this independent assortment. Is there a similar experiment that can be done to show ‘independent immunity’?

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Response to two antigens independently

Borrowing a figure from Abul Abbas’ text on Cellular and Molecular Immunology, we see  that the response to one antigen has no bearing on the body’s response to a second, unique antigen. Like Mendel’s dihybrid crosses, the response to two antigens is, indeed, independent. (Note, the serum titer in this graph falls much lower than that in the first illustration – this second curve is more representative for real responses. Regardless, the antibody titer for a secondary response remains higher than that of the primary response.)

The primary response to antigen B is identical to the primary response to antigen A. The secondary response to antigen A results in a more rapid, robust response and eventually levels out to a higher steady-state of serum antibody.

To extend the analogy just a bit further, one might ask if there is such a thing in immunology that parallels the ‘linked genes’ of inheritance?

In fact, there is. The world’s first vaccine, developed by Edward Jenner in 1796, involved the use of cowpox pus to induce protective immunity to both cowpox and the related virus, smallpox. This seems to violate our rule of independent immunity, just as the Morgan lab found that genes for body color and wing formation were found to be inherited together in fruit flies, thus violating the Law of Independent Assortment (of alleles).

In the case of cowpox and smallpox, this comes from the similarity in antigens made by each of these viruses. That is, the cowpox antigens the body generates an immune response against are NOT (ENTIRELY) UNIQUE from antigens found in smallpox. When the vaccinated individual is challenged with smallpox, antibodies created to defend against a secondary challenge with cowpox react to the smallpox antigens as if this was a secondary response directed against smallpox.

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Primary reaction to Cowpox antigen (A) is used for vaccination. Secondary reaction to Smallpox antigen (A’) upon challenge.

 
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Posted by on November 25, 2013 in Uncategorized

 

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Complement – an antibody-guided innate immune response

That’s a complicated title. What it means is that complement is part of out innate immune response, i.e. it is pre-made, ready-to-go and does not adapt over time in response to immune challenges. However, it is guided by antibodies, which are part of the adaptive immune response.

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This bad guy, invading cell, has been opsonized by antibodies – they bind all over the cell 1)preventing it from binding other host cells and 2)recruiting complement and immune cells.

What complement is, is a number of proteins that come together and activate one another in a cascade that coordinates the formation of a hole- or pore- through the membrane of foreign cells. The cascade begins when the first of the complement proteins associates with antibodies that are opsonizing a foreign cell.

Once recruited, complement proteins will activate in a cascade (see movie) in which small parts of the proteins break off and act as anaphylatoxins recruiting and activating  immune cells to the region. Meanwhile, the larger protein elements will assemble into pore-forming complexes that will kill the invading cell.

With this in mind, watch the animation below looking for:

1) antibodies binding to foreign cells

2) early complement proteins being recruited

3) complement breaking into large and small proteins

4) the smaller ones floating away to recruit immune cells

5) the larger ones forming membrane pores and killing the invading cell.

(don’t worry about the sequence of events or the specific proteins involved)

 
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Posted by on April 11, 2013 in Uncategorized

 

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