Science is awesome. But I expect you already knew that, dear readers o’mine. In science laboratories across the world, every day dedicated researchers are testing ideas, generating and evaluating hypotheses, critically analyzing observations, and thereby, making significant contribution to the humanity’s attempts to understand in greater depth and detail the wonderful natural world that surrounds us, of which we, along with other living beings and non-living objects, form a part.
A UK case report on Occupational Health and Safety, published in August, came to my attention today. Two NHS Occupational Health investigators from UK, Charles Poole of the Northern General Hospital, Sheffield, and M Wong of the Dudley & Walsall NHS Trust Health Center, presented two clinical cases associated with a relatively new occupational industry in that nation: “The separation of garden waste from domestic waste, its collection and processing in industrial composting sites, so as to reduce biodegradable waste going to landfill“.
It is well known that any kind of disturbance created in a given environment, for any reason, can often potentially release harmful substances in air in form of aerosols, or minute particles capable of floating in air. We have seen that with the yeast-like fungal pathogen, Cryptococcus gattii, which was found, via environmental studies, to be present in high concentrations in the soil of Vancouver Island (British Columbia, Canada), and to spread during dry summer weather likely as airborne particles (a.k.a. “propagules”). Release and dispersal of spores of various molds during large-scale air-disturbing activities such as construction, renovation and/or demolition of buildings is a well-studied phenomenon in the fields of Infection Control and Epidemiology; for example, see Krasinski et al., 1985; Streifel et al., 1983. The waste separation, collection and processing appear to be no different. The investigators write:
The process of composting organic matter encourages the production of bacteria, fungi, spores and endotoxins, which may be released to air in bioaerosols. Levels of bacteria and fungi up to 106 colony forming units/m3 in ambient air have been reported in relation to composting…
The problem has not been studied well at all in the population of waste-composting workers, because – as the investigators indicate – reports of illness in these workers are relatively rare. As a result, no safe levels of exposure to such potentially hazardous aerosols have been defined in this context, nor have been the exact conditions conducive to exposure; we don’t know if, and/or how much of, the exposure depends on variables such as composition of the compost, weather conditions, steps and systemic controls engaged during the separation and collection process.
In the existing clinical literature, one of the major culprits implicated in these environment-related diseases is the ubiquitous, spore-producing mold, Aspergillus, in form of its various species, mostly commonly Aspergillus fumigatus which is the etiological agent behind various diseases involving the upper (nose and upper part of the air-tube) and lower (lower part of the air-tube and the lungs) respiratory tract. Untreated or incompletely treated, these diseases can be severe and chronic. One particularly important manifestation is the Allergic Broncho-Pulmonary Aspergillosis (ABPA, in short), which is a complex or multi-component, immunologic, inflammatory response similar to allergies or hypersensitivities – which if not detected and treated early (with antifungals and steroid immune-suppressants) can lead to serious lung damage. ABPA is generally observed in people with certain debilitating conditions, such as cystic fibrosis, or immunosuppression, but rarely in otherwise healthy individuals. In ABPA, apart from classical respiratory symptoms, reduction in lung functions, and lung abnormalities observed under X-ray, certain allergy-related responses are noted in blood (more precisely, serum) – such as:
- Type I hypersensitivity to bits and pieces of Aspergillus (all recognized as antigens by the immune system), leading to the excessive generation of allergy-associated antibody, called Immunoglobulin E (IgE). By its action, IgE causes release of highly inflammatory mediators, such as histamine, leukotriene, and prostaglandin, from immune cells, which have both immediate and long term deleterious effects.
- Type III hypersensitivity to Aspergillus antigens, in which small complexes of these antigens with antibody run amok through the body, depositing in blood vessels, kidneys and joints – eventually leading to immune-mediated destruction of tissues at those sites.
- Eosinophilia, in which eosinophils, a type of white blood cells, markedly increase in number in blood and/or tissues, a common occurrence in allergy and asthma, and in parasitic (worm) infections. Activated eosinophils, a member of immune defence, are capable of causing tissue damage by various mechanisms.
The UK case report describes two late-thirties, early-forties patients, both garden waste collectors by profession, and both diagnosed with ABPA at occupational health clinics; both responded to treatment and were released with the advice not to work with waste and compost. Another member of their team, who though not ill had symptoms of asthma and tested positive for high serum IgE to Aspergillus antigens (indicating exposure) was given the same advice.
The investigators go on to make some recommendations at the end of the report. They write:
Until the results of large epidemiological studies of garden waste collectors and industrial compost workers are known, the few case reports of ABPA […] would indicate that workers with asthma who are sensitized to A. fumigatus or who have cystic fibrosis, bronchiectasis or are immunosuppressed should not work with garden waste or compost, unless their exposure to airborne fungi can be controlled. Whether asthmatics who are SPT positive or specific IgE positive to A. fumigatus will go on to develop ABPA is unknown, but they should be made aware of the theoretical risk.
Annual health surveillance by way of a respiratory questionnaire and skin prick testing is also recommended for these workers. Other cases of ABPA or EAA in garden waste and compost workers should be sought and reported, until such time that the results of a national study of UK compost workers are known.
The recommendations gave rise to some germane questions in my mind. These are, of course, valid from a clinical standpoint, and made keeping the health and welfare of the patients in mind. But given that these are related to occupational health, how do these situations play out from the perspective of the employer? How are these situations different in the UK as opposed to in the United States? For example:
- Can/should the employers (say, a waste management firm) mandate pre-employment testing for Aspergillus-specific IgE and skin prick hypersensitivity testing?
- Can/should the employers refuse employment to a person who tests positive for IgE and hypersensitivity because of a theoretical risk? Relatedly, can/should such an employee be made aware of this theoretical risk?
- Should such an employee choose to ignore this theoretical risk and accept the job (or continue on the job after a diagnosis) and become inflicted with ABPA, can/should the employee be able to claim occupational exposure and Worker’s Compensation?
- Specifically in the US context, can a Health Insurance company demand the results of these surveillance tests for a person engaged in the waste management profession, and if positive, treat this as a pre-existing condition and refuse payment in the event the employee becomes ill and needs treatment?
I don’t have the answers to any of these questions. Perhaps someone conversant with labor and/or occupational health-related laws would care to illuminate me in the comments?
Commenting on a recent study from her institution (McMaster University, Ontario, Canada), published in the journal Mucosal Immunology, Dr. Stephanie Swift, my blog colleague at Scilogs and a postdoctoral scientist working in host-pathogen interaction, wrote the other day about how vaccines can be used to train the innate immune system to recognize and repel dreaded pathogens, such as Mycobacterium tuberculosis (“M.tb”), the bacterium responsible for tuberculosis (‘TB’). [I shall highlight a few related points here, but do go read her post: it is informative and interesting.]
Immunologically speaking, the body’s defence mechanisms are dual layered. The first line of defence, called “innate” immune response, is a non-specific, general system; it is comprised of barrier mechanisms that hinder the entry of micro-organisms, as well as an immune cell-based (‘cellular’) and a non-cellular, protein-based (‘humoral’) compartment. The second line of defence – a more robust, specific and sustained response – is provided by another system, called “adaptive” immunity, which also employs many cellular and humoral mechanisms. As the names suggest, innate immunity is ‘always on’; when microbes try to invade, innate immune mechanisms stave off the first attack, and help recruit the components of adaptive immunity. Adaptive immunity serves as immunological memory that primes and programs the defence mechanisms, so that in the event of a second attack, the immune system responds in a focused manner, targeting the specific invading pathogen.
Tuberculosis remains a significant threat to public health globally; co-infection with HIV has driven its resurgence despite efforts to reduce its impact. An estimated 1.4 million people succumb to TB each year (WHO, 2011), with the largest burden observed in Africa and Southeast Asia. Although anti-tubercular drug discovery has revived itself lately with renewed vigor, focusing on adaptive immunity has been the mainstay of immune-based TB therapies – as Dr. Swift pointed out in her essay, too.
And is there a good reason for this focus? Yes, it appears, there is, but we need to remember some fundamental facts about this pervasive pathogen. M.tb, which likely evolved from soil-dwelling ancestors to become a human pathogen around 10000 years ago, is endowed with unique abilities geared towards survival and persistence:
- M.tb is considered aerobic-to-facultative-anaerobe, which means it normally grows in presence of oxygen, but if oxygen is deficient, it can shift to other mechanisms of respiration that doesn’t require oxygen.
- When oxygen and nutrients are plentiful, and temperature is 37˚C (as, say, in the lungs), M.tb makes more copies of itself in 18-24h (3-4 weeks on artificial medium in vitro). For a pathogen, this is an extremely slow rate of division.
- When conditions are not propitious, M.tb has the unique ability to enter a dormant, non-replicative state with low respiratory rate or metabolic activities. When in this zen-like state, it is not affected by either host immune mechanisms or anti-tubercular drugs (which otherwise kill the growing bug). It also modifies its metabolism, via genetic changes, to adapt to the nutrient limitations.
- M.tb possesses an unusual cell wall, thick and impermeable, composed of complex sugars (polysaccharides), amino acid-containing sugars (peptidoglycans), sugar-containing fatty acids (glycolipids) and long-chain fatty acids (e.g. mycolic acid). It is these latter lipid-components that are considered to protect the bacteria against weak disinfectants and desiccation.
M.tb revealed with a special stain specific for Mycobacteria, acid-fast Ziehl-Neelsen stain; Magnified 1000X. Image courtesy: CDC/Dr. George P. Kubica (1979).
Macrophages, members of first-line defence, are immune cells that engulf microbes via a process called phagocytosis, and kill them internally by:
- Creating an acidic (low pH) environment inside the bubble-like enclosure, called a phagosome;
- Producing hyper-reactive (and corrosive) chemical derivatives of oxygen and nitrogen, which causes damage to microbial DNA, lipids, proteins and other structural components;
- Flooding the microbes with enzymes which, under the acidic condition, can break down (‘hydrolyze’) lipids and proteins in the outer layers of the microbes;
- Releasing peptides (small proteins) with potent antimicrobial properties (such as cathelicidin, hepcidin, etc.), which pokes holes into the outer layers of the microbe, and
- Undergoing a regulated suicide process known as apoptosis, in case the microbe is able to escape its enclosure and wade into the cytoplasm, the fluid-filled intracellular space, of the macrophage itself.
Immune messenger proteins – called cytokines, notably Interferon-γ – secreted by certain immune cells influence these defensive process by acting on and activating the same and other immune cells. Macrophages and another immune cell, known as dendritic cells, can pick up pieces of the destroyed microbes and their products, and display it to yet another immune cellular component, the T-lymphocytes, at which point the immune response traverses to the adaptive side; T-lymphocytes instruct B-lymphocytes to produce specific antibodies against the microbe, and create memory B-lymphocytes and memory T-lymphocytes in preparation of subsequent attacks; other immune cells are also appropriately instructed to recognize the pathogenic components for unleashing their destructive power in a focused manner on the microbe in the event of the next attack.
But… M.tb’s unique abilities include not only escaping elimination, but also surviving inside macrophages. It is thought that their unusual cell-wall composition allows them to invade resting macrophages silently. Once inside the resting or active macrophages, M.tb can shut down the internal processes by which macrophages kill and digest microbes. In addition, virulent M.tb can prevent the macrophage from undergoing apoptosis by blocking the self-destruct signal and making quick repairs to the already-destroyed cellular structures. (Note: If you are familiar with Stargate SG-1, this is an easy parallel with Replicators!)
In addition, inside activated macrophages, M.tb can tolerate the low pH environment by reducing the macrophage protein pumps responsible for acidification, up-scaling the production of its own urease enzyme, which produces ammonia to neutralize the excess acid, and programming a set of its own genes called aprABC, whose functions allow it to adapt to the acidic micro-environment inside the host cells. Therefore, M.tb essentially reprograms the host cells after entry to prevent its own destruction and ensure its persistence.
And that is not all. Once inside macrophages, M.tb has the ability to use a host-derived carbon source (most likely, cholesterol and glycerol) for its metabolic needs; it modifies both its lipid metabolism and its toxic waste disposal mechanism to suit its environment. To prevent damage from corrosive ionic derivatives of oxygen and nitrogen, M.tb enhances the production of several key enzymes (such as, superoxide dismutases, peroxidases, and reductases) that can neutralize these ions, as well as repair the damages caused. There is some evidence that macrophages may use excess zinc ions inside the phagosome to kill M.tb, but recently it was discovered that the bacteria can safely pump out the zinc ions. It even counteracts the effect of the antimicrobial peptides by neutralizing their negative charge via a positively-charged lipid molecule on its membrane.
But wait, there is more! Ordinarily, macrophages with engulfed microbes migrate to tissue sites, where other cell types, such as monocytes, lymphocytes, and neutrophils are signaled and recruited to create a confined environment, called a granuloma, where a delicate balance is established between the host immune cells and the pathogen. Formation of well-organized granulomas, comprising different immune cells, is a characteristic feature of several microbes, mainly some respiratory fungal pathogens (such as Cryptococcus, Histoplasma etc.), and – of course – M.tb. In TB, a primary lesion or point of damage becomes a solid granuloma involving macrophages, monocytes, dendritic cells, T- and B-lymphocytes; solid granulomata in lungs may represent foci in which M.tb remain in a latent, quiescent condition – but not dead. Under certain circumstances, especially if the host immunity falters for some reason, they can be reactivated, and progress to active disease. The granuloma in this case becomes necrotic or caseous (filled with dead tissue and dissolved material), which allows for the bacteria to escape its immune-prison chamber, and spread elsewhere via blood, and to other persons via breathed/sneezed out air from the lungs.
Image: Necrotizing granulomas (G) localized around an airway (A). (B) is a blood vessel. ©Dr. Yale Rosen; you can view more of the pulmonary pathology photos by fabulous Dr. Yale Rosen on Flickr.
It is clear that while the host innate immune system, led by the macrophages, can boast of a remarkable arsenal of microbicidal mechanisms in general, when it comes to M.tb, their efficacy is not guaranteed; during co-evolution with mammalian hosts for thousands of years, M.tb has been conditioned to defeat the hostile intracellular environment and persist in the host. Therefore, it is hardly surprising that most of the current vaccine candidates are not aimed at preventing or eliminating primary TB infection, but focus on priming the players of adaptive immunity to stave off the emergence of active disease by targeting the metabolically active, replicating pathogen.
Vaccine antigens are antigens which, when used to immunize, prime the immune system to recognize the same or similar structures in the invading pathogens and mount an immune response. A booster is one or more subsequent doses of the same or similar vaccine antigens, designed to further hone the ability of the immune system to focus on the target. The desired effect of this strategy is the establishment of a state of immunological memory, which allows the immune system to respond more rapidly and effectively to previously-encountered pathogens. The memory responses – which are often the principal antimicrobial response in an immunized host – are dependent on antigen-specific B- and T-lymphocyte populations made up of genetically identical (‘clonal’) cells, and are known to also differ qualitatively from primary responses, in terms of both antibody production and T-cell memory.
For the longest time, we have had the BCG vaccine, derived from a cousin of M.tb, the Mycobacterium bovis strain Bacillus Calmette-Guérin. Following WHO recommendations, BCG is routinely administered to neonates or infants in parts of the world endemic for TB; BCG is at least partially effective in preventing development of serious TB manifestations in children, such as TB-meningitis and disseminated (“miliary”) TB. However, it appears that for some reason, BCG vaccination is unable to sustain immunological memory, the sine qua non for adaptive immunity; this leads to highly variable protection from pulmonary TB as far as adults are concerned. To counter this, various investigators have started considering combining BCG vaccine with another vaccine antigen in a prime-boost strategy designed to maximize the benefits of both. Recombinant BCG vaccines, with improved immunogenicity profile, are in clinical trials now.
In addition, several new candidate vaccine antigens are being studied. Incidentally, a team of researchers from my institution (Johns Hopkins University, Baltimore) also published this month an interesting study in PLoS One, in which they identified a potential vaccine antigen that can protect against TB meningitis. This antigen is an Mtb protein, known as PknD, whose function involves helping the bacteria cross the barrier – known as the blood-brain barrier – that ordinarily keeps microbes from entering the brain. They had earlier discovered that an antibody that specifically recognizes this protein is able to neutralize its action. Therein lies the rationale of this vaccine program, which can potentially produce such an antibody in an immunized person, as well as produce T- and B-lymphocyte memory of this protein. This would likely protect specifically against TB meningitis, a high mortality outcome of disseminated TB.
However, as I indicated before, during the period of latency/dormancy, M.tb modifies its genetic program; as a consequence, the character of the antigens usually associated with the bacterium changes, and separate antigens expressed by the dormant pathogen predominate. Therefore, many investigators now consider that a sustained and effective control of dormant M.tb would require the vaccine-induced immune response to recognize target antigens on the latent bacterium. Mouse studies are ongoing, with encouraging results, using a latency-associated M.tb antigen, a protein called Rv2660c, which is selectively expressed during nutrient starvation.
The prime-boost immunization strategies, focused towards developing immunological memory, have shown promise in battling M.tb, but the mechanism of their actions, particularly how they impact innate and adaptive immunity is yet unclear.
In the McMaster study that Dr. Swift commented on, the researchers used two engineered (recombinant) viral vaccines; the gene for an immunodominant early secreted M.tb protein Ag85A, was inserted into either of two viral vectors, derived from the genomes of adenovirus (Ad) and vesicular stomatitis virus (VSV). Once these vectors are taken up into host cells, this protein would be continuously produced within the host cells, and serve as the vaccine. Using the prime-boost strategy in a mouse model, they found both elicited similar M.tb-specific T-lymphocyte responses (that is, adaptive immunity) in the lung and spleen.
However, the picture changed drastically when they challenged the immunized mice with the bacterium, and studied the signatures (a.k.a. markers) of protection. The Ad-based boosting regimen was able to greatly clear M.tb from the lungs and spleen of the mice, and elicit high levels of a macrophage-stimulating cytokine called IL-12 in the lungs. In contrast, the VSV-based boosting showed no enhancement of protection against TB, and even elicited high levels of another cytokine called IL-10, which profoundly shuts down the anti-M.tb activity of macrophages. These cytokines were released from phagocytic cells that represent innate immunity. Therefore, clearly, Ad-based boost strategy enhanced the activation of innate immune system in the lung, which presumably resulted in the greater protection to TB.
The researchers figured out that the main difference between the two vector vaccines was in how they interacted with – and primed – the innate immune cells at the site of immunization. IL-12 is known to enhance the production of interferon-γ, as well as of the reactive Nitric Oxides, from these immune cells. In short, the immune cells exposed to the Ad-based boost became more efficient killers of M.tb.
In contrast, the researchers found that exposure to VSV-based boost led to increased release of another cytokine, called interferon-β, which has grossly different actions compared to interferon-γ. Increased interferon-β severely blunted the beneficial IL-12 responses in the M.tb-infected phagocytic innate immune cells; in addition, exposure to interferon-β made the immune cells inefficient in controlling the numbers of engulfed (phagocytosed) M.tb.
Whereas T-lymphocyte mediated adaptive immunity is essential and beneficial for many microbial pathogens, effective elimination of pathogens that hide in plain sight within immune cells itself, such as M.tb and HIV, requires the engagement of innate immunity which encounters them first, independent of the T-cells. To succeed against M.tb, a vaccine would need to engage both wings of the defence. As Dr. Swift pointed out, each of the viruses from which the respective vectors were derived:
“… enters lung cells in a certain way, attaching at different sites, activating different intracellular pathways, expressing different viral products and consequently engaging different parts of the immune system.”
Viral vectors offer an attractive choice for engaging adaptive immunity against different pathogens. This study shows how they can be used to strengthen innate immunity, too. These are important considerations for a rational vaccine design.
Two major studies discussed:
- Jeyanathan M, Damjanovic D, Shaler CR, Lai R, Wortzman M, Yin C, Zganiacz A, Lichty BD, & Xing Z (2013). Differentially imprinted innate immunity by mucosal boost vaccination determines antituberculosis immune protective outcomes, independent of T-cell immunity. Mucosal immunology, 6 (3), 612-25 PMID: 23131783
- Skerry, C., Pokkali, S., Pinn, M., Be, N., Harper, J., Karakousis, P., & Jain, S. (2013). Vaccination with Recombinant Mycobacterium tuberculosis PknD Attenuates Bacterial Dissemination to the Brain in Guinea Pigs PLoS ONE, 8 (6) DOI: 10.1371/journal.pone.0066310
Much have been made in the media recently, of a February 2013 paper, published by a German group in the Annals of Internal Medicine, claiming that acupuncture may help relieve seasonal allergies. Always interested in examining the bold claims of efficacy by various forms of pseudoscientific, wannabe-medicine modalities (such as homeopathy, naturopathy, and so forth), I elected to go to the source; the paper was behind an annoying paywall, but thankfully, I had institutional access, and dove in.
The clinical entity of Chronic Fatigue Syndrome1 (CFS) has so long eluded explanation. Patients of CFS complain of extreme and prolonged fatigue that is disproportionate to their physical and mental activity, and is not alleviated by any amount of rest. The condition may well last for more than 6 months at a time, and may be accompanied by a variety of other symptoms, such as pain in the muscles and/or joints without swelling, memory impairment, significant lapse of concentration, headaches, painful lymph nodes in the neck or armpit, and so forth. Physicians currently employ the 1994 case definition in which persistent (>6 months) fatigue is to be present along with at least 4 of 8 known associated symptoms, for the condition to qualify as CFS; if these criteria aren’t fully met, the condition is referred to as ‘idiopathic’ (without known cause) fatigue. Management of both conditions are practically identical.
For patients of CFS, the bouts can be debilitating, perhaps made worse by the fact that scientific research has not yet identified the root cause of the condition, and can, therefore, offer no solution beyond symptomatic relief.
Many theories as to the cause of CFS abound, such as:
- Direct effect of viral infections;
- Specific induction of host immunity as a result of invasion by some pathogenic microbe, et cetera.
- Non-specific activation of the patients’ immune systems, a subset of which may be expressed as allergies;
- Direct involvement of the central nervous system, resulting in abnormal, neurally-mediated lowering of blood pressure, which may cause light-headedness and compensatory tachycardia (i.e. increase in heart rate);
- Indirect action of the brain, via the HPA (‘Hypothalamic-Pituitary-Adrenal’) axis, which may disturb the release of various stress-associated hormones.
In addition, symptoms in CFS may resemble those seen in many physiological, neurological, as well as psychological illnesses.
This, understandably, poses a diagnostic challenge; the problem is that all these phenomena in the human body are processed through physiological pathways that are highly inter-related, a fact which underscores the difficulty in arriving at a single factor responsible for CFS. Current thinking is, therefore, that CFS may be multi-factorial, i.e. triggered by a combination of an unknown number of factors.
In part, this is also the reason why there is no diagnostic test or ‘biomarker’ (an observable phenomenon that can be specifically attributed to the condition) for CFS, and why the diagnosis must be exclusionary, via a process of elimination of other possible conditions that may explain the symptoms. What makes diagnosis even more difficult – not to mention, controversial – is that the number, types and even severity of these symptoms are highly variable amongst patients, and the condition periodically goes into remission and relapses.
When symptoms arise, management – in absence of a cure – focuses on treating primarily those symptoms that disrupt life and activities most, such as pain, lack of sleep, memory problems, depression, anxiety, et cetera. Long term care involves specially-developed activity programs, behavioral therapy and other interventions that aim to mitigate the physiological and psychological effects of this chronic illness.
Given that many CFS symptoms mimic those of certain immune dysfunctions involving unregulated inflammation, a lot of research has focused on understanding the inflammatory pathophysiology of CFS patients. One recent study from the Stanford University medical school, published in the Journal of Translational Medicine2, investigated the role of cytokines in this condition. Cytokines are a group of small protein molecules produced and released by various types of cells in the body, including cells which comprise the immune system. Cytokines take part in cell-to-cell signaling; released by one type of cells, cytokines affect other cells, either in their immediate environment or elsewhere in the body, by binding to receptor molecules present on the surface of these cells. These receptors recognize specific cytokines, and the binding at the cell surface initiates cascades of sub-cellular (inside the cell) biochemical reactions which lead to a specific effect. For example, some cytokines are active in regulation of developmental processes3 during the implantation of the embryo and maintenance of pregnancy. Again, ‘pro-inflammatory’ cytokines, released by certain leukocytes of the innate immune system, can recruit other leukocytes and bring them to the site of infection or injury, in order to mediate various effects4.
The Stanford group, led by Elizabeth Stringer, hypothesized that the daily variability of the levels of various cytokines in the serum may correlate with the observed variations in the severity of CFS symptoms. In a pilot study they had monitored the daily levels of 51 different cytokines in 3 women with fibromyalgia (another chronic painful condition) and CFS, and discovered that one adipokine (cytokine released by fat cells, ‘adipocytes’), called Leptin, stood out. Leptin, which regulates appetite, metabolism and behavior5, and has profound inflammatory effects, as well as a protective role in mucosal immunity6, was found to correlate significantly with the self-reported fatigue severity.
In the current study, participants (CFS patients and healthy controls, all female) were chosen carefully to account for or exclude other existing conditions that may confound (i.e. not allow proper interpretation of) the observations. Twice a day, 20 participants answered questions about the severity of fatigue, muscle/joint pain and sleep quality that they experienced during the study period, which included blood draws for 25 consecutive days. In the CFS patients, serum Leptin levels correlated strongly with daily levels of fatigue; although Leptin levels were associated with a plethora of pro-inflammatory cytokines, no other direct correlation was found, indicating that Leptin may be the central player in the CFS-associated inflammatory process mediated by a network of cytokines. None of these associations were observed in healthy controls. Leptin levels were predictive of daily fatigue levels in women with CFS, and using cytokine predictors, the authors were able to distinguish between high fatigue and low fatigue days with 78% accuracy.
Illustrative image composite made from parts of Fig. 2 & 3 of Stringer et al. (Ref. 2)
Interestingly, absolute Leptin levels, as well as the range of daily fluctuations, were not abnormal in CFS patients, which suggests that Leptin alone may not be responsible for causing the inflammation in CFS. As the authors indicate, larger and more detailed studies are necessary to explore a causal role of Leptin and/or its cytokine network in driving CFS severity, and uncover hitherto elusive diagnostic biomarker(s) and therapeutic targets.
- CDC information website on Chronic Fatigue Syndrome.
- Stringer, EA, et al. Journal of Translational Medicine 2013, 11:93; doi:10.1186/1479-5876-11-93.
- Saito, S. Journal of Reproductive Immunology 2001, 52:15-33; PMID: 11600175
- Whitney, NP, et al. Journal of Neurochemistry 2009, 108:1343-59; PMCID: 2707502.
- Gautron L, Elmquist JK. Journal of Clinical Investigation 2011, 121:2087-93; PMCID: 3104762.
- Mackey-Lawrence NM, Petri WA Jr. Mucosal Immunology 2012, 5:472-9; PMCID: 3425733.
As I asked in the previous part: Why is the capsule a virulence factor? What special properties make it a key player in the process by which Cryptococcus neoformans (CN) causes disease?
Well, while acapsular mutants (lack external capsule, but contain GalXM on the cell wall), avirulent in normal hosts, may still cause disease in severely immunocompromised hosts, the burden of virulence associated with CN has traditionally fallen on the capsule, because of the extra-ordinary effects that it has on the immune system (quick reminder: these studies were largely done with what we now know as the ‘exopolysaccharide’, i.e. the shed capsular material).
Interestingly, CN appears to have the ability to modify the size and structure of the capsule in response to various stimuli, including the micro-environment of the target organ, stage of infection, stage of fungal cellular growth, and so forth. In mouse infection models, the capsule size increases rapidly (within hours) following infection; strains isolated from severely immunocompromised individuals (such as HIV/AIDS patients) often show large capsular diameter. The capsule composition (including ratio of various sugars, and arrangement of repeating units) may change at various stages during the infection. Since the host immune system presumably encounters the capsule first, these changes likely modulate how the host immune components react against CN.
Cryptococcal cells of various capsule sizes (all images under same magnification)
Let me try to summarize some of the major deleterious effects which the capsular polysaccharides (including the exopolysaccharide) have on the host immune system. Needless to say, these effects are crucial in helping CN establish the disease in the host. [Note: The host immunity plays an unwitting role in the establishment of the cryptococcal disease, but we shall discuss those factors later.]
- Capsule interferes with normal functioning and immune functions of epithelial cells: Two types of epithelial cells (lining the surface of cavities) encounter inhaled CN first, the bronchial epithelium (inner lining of the branches of trachea, the air-tube) and the alveolar epithelium (inner lining of the air sacs – pockets of air – where exchange of gases takes place). Both epithelial cell types are capable of responding to the capsule, initiate a signal via release of messenger proteins called cytokines, and call for backup in form of an immune cell called neutrophil, which can kill microbes. However, the capsule suppresses cytokine release from bronchial epithelial cells; in alveolar epithelium, however, the capsule causes CN to be internalized in the epithelial cell, which kills the host cells. The resulting host cell damage likely allows CN to cross the epithelial barrier to reach inside the lung tissue.
- Capsule helps CN evade capture by largely inhibiting phagocytosis: Phagocytic cells (cells which engulf and kill foreign substances, including invading microbes), such as the Macrophages in the alveolar pockets of the lung tissue, are among the first line of defence against invading microbial pathogens. The engulfment is initiated either by direct interaction between macrophages and the microbe, or via components of the humoral (i.e. non-cellular) immune system (antibodies and/or complements) of the host. Apart from killing the microbe, this process also presents bits and pieces of microbial material (‘antigen presentation’) to another immune cell, the helper T-lymphocytes (‘T-cells’), which ultimately allow the antibody-producing B-lymphocytes (‘B-cells’) to produce specific antibodies recognizing the microbe, as well as create immunologic memory. The cryptococcal capsule helps the microbe evade capture (thereby, also interfering with antigen presentation) by largely inhibiting the phagocytic process
- Capsule also interferes with additional immune mechanisms that enable phagocytosis: Even in presence of capsule, antibodies and/or complements (mentioned above) can bind to the capsule and/or cell wall, and enable phagocytosis. However, a large capsule may effectively mask the binding sites for these immune proteins.
- Capsule allows intracellular parasitism of phagocytosed CN: Even when the first-line immune mechanisms can successfully help Macrophages engulf the cryptococcal cell, the capsule actively interferes with their antimicrobial properties, so that internalized CN is not killed, but thrives, with the ability to spread to other cells, tissues, and organs.
- Capsule aids dissemination of CN: Along with other mechanisms (including various cryptococcal enzymes), the capsule allows the spread of CN from lungs to the rest of the body. The microbe is able to change the composition of the capsule, presumably in order to adapt to different micro-environments. The structure and composition of the capsule are also important for the ability of CN to cross various physiological barriers to reach the target tissue.
- Capsule is able to induce immunological unresponsiveness to CN (a.k.a. immune paralysis): The polysaccharide is known to be able to inhibit antibody production by B-cells, and growth and proliferation of T-cells; this is achieved via inhibition of antigen presentation by phagocytes (mentioned above), modulation of the production and release of certain cytokines, as well as induction of a certain subset of T-cells whose secreted products make other T-cells unresponsive. In addition, the direct binding of the polysaccharide to certain receptors (known as FCRγII) in various phagocytic cells leads to a profound immune suppression. The capsule also interferes with the functions of various other immune effector cells, such as neutrophils. Most of these effects have been described for the GXM component (see previous part) of the capsule, but GalXM and the mannoprotein components have also been implicated in similar effects. GXM and GalXM both can induce antigen-presenting phagocytic cells and T-cells to commit suicide (via a process called ‘apoptosis’).
Click to embiggen: Various types of cryptococcal colony morphologies on artificial medium; it has been hypothesized that changes in colony morphology, controlled genetically, may be associated with changes in structure and composition of the capsule.
CN is abundantly present in the environment (especially in endemic areas, in association with certain trees and birds); therefore, one enduring mystery is the reason why it would need to evolve to make a capsule with such high virulence potential in mammalian hosts (primates, quadrupeds, as well as aquatic mammals). Extensive work done in the laboratory of Arturo Casadevall at the Albert Einstein College of Medicine in New York, as well as elsewhere, have shown that, in the environment, CN has to interact with various hosts, including certain amoebas, slime mould, nematode worms and insects – many of which can kill the microbe; the capsule affords protection against those marauders in a way that is remarkably similar to its interaction with the mammalian immune cells. Pathogenicity and virulence of CN in mammalian hosts may, therefore, be a consequence of that process.
In the next installment, I shall describe some different types of the cryptococcal organism, including their clinical significance.
Review of the Cryptococcal Capsule, by Oscar Zaragoza et al. Advances in Applied Microbiology, 2009, 68:133-216.
In the recent issue of the Morbidity Mortality weekly report (dated March 22, 2013), the CDC has published detailed recommendations for prevention and control of meningococcal disease. Meningococcal disease refers to diseases caused by the Gram-negative (stains pink by the Gram Stain), diplococcus (round-shaped cells usually occurring in clusters of two) bacterium known as Neisseria meningitidis (a.k.a. the meningococcus)—that is responsible for a variety of serious, often life-threatening, diseases, including bacterial meningitis (inflammation of the meninges, or covering of the brain and the central nervous system), bacteremia (more precisely, meningococcemia; meningococcal invasion of blood) often leading to septic shock, as well as a type of pneumonia.
It is well-said that a picture is worth a thousand words. I have always found that a good, illustrative graphic can make a great impact upon the understanding of complex cellular pathways. And when one is visualizing dynamic processes, such as the processes occurring within the physiological system, newer technologies such as animations can be of a tremendous help. Of course, in order to be useful, it must be well-researched (so as to be scientifically accurate) as well as well-executed. This is why I was so excited about an animation depicting an immune process in the mammalian intestines presented by Nature Immunology.
As research into safer and more efficacious options for the therapeutic use of monoclonal antibodies (MAbs) goes on, interesting and innovative technologies have been discovered and refined by scientists, which represent the next level of accomplishment in the overall effort to develop “fully human” MAbs (which I have touched upon in the THIRD PART).
The Fully Human MAbs are generally made in one of two ways; either, naïve human B-cells are processed (via phage-display; gene-transfer to transgenic mice; or fusion of human B-cells) in order to, say, generate libraries of Immunoglobulin (Ig) V-regions. Screening techniques are utilized, at various levels thereafter, to select clones bearing desirable antibodies that are specific to the target antigen. In other words, the MAbs are made antigen-specific ex post facto.
Alternatively, memory B-cells are generated by training the immune system via immunization. For example, humanized transgenic mice are immunized with the target antigen, allowing them to generate specific, antigen-recognizing human antibodies. These B-cells are isolated and processed. However, in this process, there are several important caveats: (a) the antigen is still recognized in vivo in a murine context, (b) the human V-region incorporation into mouse genome may be restricted, and therefore, have limited diversity, and (c) there is the possibility that V(D)J rearrangements inside the murine B-cells may not be adequate. These difficulties – which may lead to the generation of low affinity, low frequency human antibodies, requiring extra steps of in vitro affinity maturation – are known issues in the development of human MAbs using transgenic mice.
To bypass this problem, a technology that incorporates immunological and genetic principles has been described  for rapid generation of MAbs specific to a vaccinating antigen, where immunized and/or infected-recovered donors – whose serum, therefore, contains the specific antibody – provide B-cells; these B-cells are sorted into separate enclosures, cultured and screened. From those B-cells that are secreting the desirable specific antibodies, the heavy and light chain genes are isolated and cloned into a human cell line, Human Embryonic Kidney (HEK)-293, which produces the human antibodies in culture. Using this technique, diagnostic and immunotherapeutic MAbs that can recognize and neutralize Anthrax have been generated .
Exton, PA-based company, Morphotek, using a similar technology , isolated B-cells from healthy individuals with serum antibodies to Staphylococcal entertoxin B (SEB), cultured them in vitro and fused with a mouse/human heteromyeloma cell line. The clones that produced the desired MAbs were identified, and their light and heavy Ig chain genes were sequenced and cloned into a mammalian cell line (Chinese Hamster Ovary, CHO, cells) for expression; CHO cells are known to be able  to produce the recombinant MAb protein molecule with the appropriate glycosylation (a process by which carbohydrate moieties are attached to the antibody molecule, which is crucial for the antibody function ). Using this technique, Morphotek has successfully generated human MAbs that are capable of neutralizing high doses of SEB both in vitro and in vivo.
However, some of the technologies used for the in vitro manipulation of the primary structure of the antibodies possibly introduces a degree of immunogenicity (as evidenced from human anti-human antibody or “HAHA” reactions), and may change the safety, tolerability, and in vivo performance characteristics of these therapeutic MAbs. For example, chimeric MAb Infliximab and transgenic-mouse-derived Fully Human MAb Adalimumab, both recognizing TNFα and used to treat Rheumatoid Arthritis, have been reported  to give rise to antibody response that neutralizes them and reduces their efficacy greatly. And this undesirable immunogenicity of the therapeutic MAbs themselves – according to a 2010 study  – resides, in part, in the CDR or V-region, the domain of the antibody molecule that is directly responsible for recognizing its cognate antigen! The good news is that, as the study contends, it is possible, via molecular genetics tools, to modify these CDRs such that immunogenicity of the MAb is reduced while retaining its biological activity. Neat, huh!
A combination of these approaches has been taken by an Austin, Texas-based biopharmaceutical company, XBiotech, which considers its human MAbs to be a generation ahead of the existing ‘Fully Human’ MAbs, and therefore, has designated its technology to be True Human™. But before I describe this technology, here is a little primer in Immunogenetics.
Remember the antibody structure I had talked about in the FIRST POST of this series, how heavy and light chains had antigen-recognizing Variable (V) regions and Constant (C) regions?
The V-regions of the Heavy (VH) and Light (VL) chains, that form the CDR or antigen-binding site, are produced in the B-cell by an interesting process, called VDJ recombination (a.k.a. somatic recombination). In the naïve B-cell, from the moment of its formation, there are 3 distinct type of gene segments (Variable, Diverse, and Joining segments) in the chromosome, present in what is known as a ‘germline’ configuration; for example, the area on chromosome 14, responsible for human Ig Heavy Chain (a.k.a. Heavy Chain locus), contains a total of 44 V-genes  (classifiable into 7 different families, VH1-7), 27 D-genes (of which 25 are involved in antibody generation), and 6 J-genes . Prior to the generation of the antibody protein molecules, there occurs a VDJ recombination event, a nearly random rearrangement of the germline gene segments, eventually bringing together one of each, from which the final protein molecule is made. The Light chains (of which there are two types, λ and κ) similarly have V and J recombination (lacking the D segment). The randomness of the segment selection process is responsible for the generation of massive diversity in the antigen-recognition capability. To illustrate:
In addition to this recombinatorial process that occurs in the germline gene, another process introduces further diversity to the antigen-recognition capabilities of the antibody, once the B-cell has encountered an antigen. This secondary process is known as Somatic Hypermutation; antigen-recognition by a naïve B-cell stimulates its proliferation, during which the chromosomal locus responsible for antibody generation mutates (mostly point mutations, in form of single base substitutions) extremely rapidly (100,000-1 million fold higher rate) to create a high degree of variation in primary structure. These mutations occur mostly at DNA “hotspots” known as hypervariable regions that correspond to CDRs, and result in enhanced affinity for the target, cognate antigen.
Somatic Hypermutation is followed by another process known as Clonal Selection; hypermutated B-cells are presented with the cognate antigens by a type of cells known as the follicular dendritic cells (FDCs), present in the germinal centers of lymphoid tissue. The B-cell clones/progeny with the highest affinities for the presented antigen are selected to survive, whereas other clones bind antigen with lower affinity are destroyed. The Clonal Selection process thus effectively increases the affinities of secreted antibodies for the target antigen. Taken together, Somatic Hypermutation and Clonal Selection are known as in vivo Affinity Maturation of the antibody.
The antigen that stimulates this process may be extrinsic, such as a vaccine antigen, or antigen from an infecting microbe, or intrinsic/autologous, such as a component of the host’s own body. While the former leads to a beneficial immune response, it is not hard to imagine that the latter may cause debilitating ‘autoimmune’ disorders if host’s own antibodies recognize and attack internal physiological components. The immune system has a checkpoint to prevent this, a process called Central Tolerance, that – for B-cells – occurs in the bone marrow and regulates autoreactive B-cells in several ways, including receptor editing (gene rearrangements to change reactive Ig molecules on the B-cell surface to non-reactive ones), clonal deletion (antigen-induced loss of cells from the B-cell repertoire via programmed cell death/apoptosis), and the induction of anergy (a.k.a. non-responsiveness). An additional checkpoint for those mature self-reacting B-cells that may survive this process and reach the peripheral lymphoid tissues is the requirement of T-lymphocyte signals. B-cells need co-stimulatory signals from T-cells in presence of the cognate antigen in order to proliferate and produce antibodies; therefore, mature B-cells recognizing self-antigens are denied T-cell help in the periphery and eliminated by apoptosis or anergy – the process being known as Peripheral Tolerance.
One important fact to remember is that the Central Tolerance requires B-cells to be exposed to self-antigens during maturation, i.e. antigens present in primary lymphoid organs. In the bone marrow, this exposure is naturally limited to bone-marrow specific antigens either present in the bone-marrow, or imported by circulation. Therefore, in a relatively healthy host, there may be a small proportion of mature B-cells that escaped these checkpoints and continue to secrete low-affinity antibodies to endogenous substances. For example, a 1996 paper  reported that naturally-occurring IgG and IgA autoantibodies specific for the pro-inflammatory cytokine Interleukin (IL)-1α occur in the sera of 5-25% healthy individuals. The other important concept to remember in the context of this discussion is that therapeutic MAbs produced in vitro do not undergo these in vivo checkpoints to eliminate self-reactive antibodies.
This also leads to the point where I found XBiotech’s True Human™ antibody technology and innovation to be interesting. Let me explain. As I have been informed, this company has access to a library of blood samples derived from normal individuals with functioning immune systems and no evidence of disease. They use a proprietary screening methodology to identify in those blood samples native human antibodies that recognize the target antigen they desire. Consider what that means; it means that the original B-cells that secreted these antibodies were already primed by antigen-exposure, and consequently, had already undergone somatic hypermutation of its genes, followed by clonal selection. Via XBiotech’s proprietary technology, they can thus find even low titer antibodies that recognize some endogenous substance as antigen with high affinity. They isolate the corresponding mature B-cells (with their full complement of ready-to-secrete antibodies) and use proprietary cloning strategies to clone the already-rearranged, ready-to-be-used Ig genes into CHO cells, and generate MAbs.
The beauty of this system? The in vivo B-cell selection and maturation process undergoes in the endogenous human microenvironments (germinal centers in bone marrow, lymph node or spleen); in addition, since the variable regions, including the CDRs (which are responsible for undesirable immunogenicity, as I mentioned above), are derived from native antibodies, True Human™ MAbs show no significant immunogenicity in the human host. Indeed, with their current True Human™ lead antibody, MAb-p1 (which recognizes human IL-1α), has been administered in Phase 1/2 studies to over 145 patients, in 457 doses, and the patient with the longest treatment has been over 785 days. They have observed no dose limiting toxicities and no evidence of any deleterious HAHA response (Data source: John Simard, Founder, President and CEO, XBiotech; see disclaimer below). It should be very interesting to follow the progress of the applications of this therapeutic technology.
DISCLAIMER: This post describes proprietary technology in a purely informative manner, partly based on information provided by the inventor of this technology, a company called XBiotech. This does not constitute a comparative analysis of competing technologies, in whichever form they may exist, nor an endorsement of this technology I am describing. CONFLICT OF INTEREST: I declare that I have no financial interest whatsoever in writing about this technology. My interest in XBiotech and its technological achievements is purely academic. However, in the interest of transparency, I declare that this company, via its representative, made some of their data available to me at a time when it was embargoed.
- Smith et al. (2009) Rapid generation of fully human monoclonal antibodies specific to a vaccinating antigen. Nature Protocols, 4(3): 372–384; doi: 10.1038/nprot.2009.3; BACK TO TOP
- Smith et al. (2012) Human monoclonal antibodies generated following vaccination with AVA provide neutralization by blocking furin cleavage but not by preventing oligomerization. Vaccine, 30(28):4276-83. BACK TO TOP
- Drozdowski et al. (2010) Generation and characterization of high affinity human monoclonal antibodies that neutralize staphylococcal enterotoxin B. Journal of Immune Based Therapies and Vaccines, 8:9; doi: 10.1186/1476-8518-8-9 BACK TO TOP
- Sheeley et al. (1997) Characterization of monoclonal antibody glycosylation: comparison of expression systems and identification of terminal alpha-linked galactose. Analytical Biochemistry, 247(1):102-10. BACK TO TOP
- Zheng et al. (2011) The impact of glycosylation on monoclonal antibody conformation and stability. MAbs, 3(6):568-76; doi: 10.4161/mabs.3.6.17922. BACK TO TOP
- Radstake et al. (2009) Formation of antibodies against infliximab and adalimumab strongly correlates with functional drug levels and clinical responses in rheumatoid arthritis. Annals of Rheumatic Disease, 68(11):1739-45. BACK TO TOP
- Harding et al. (2010) The immunogenicity of humanized and fully human antibodies: residual immunogenicity resides in the CDR regions. MAbs, 2(3):256-65. BACK TO TOP
- Matsuda et al. (1998) The Complete Nucleotide Sequence of the Human Immunoglobulin Heavy Chain Variable Region Locus. Journal of Experimental Medicine, 188(11): 2151–2162. BACK TO TOP
- Li et al. (2004) Utilization of Ig heavy chain variable, diversity, and joining gene segments in children with B-lineage acute lymphoblastic leukemia: implications for the mechanisms of VDJ recombination and for pathogenesis. Blood, 103(12):4602-9. BACK TO TOP
- Garrone et al. (1996) Generation and characterization of a human monoclonal autoantibody that acts as a high affinity interleukin-1 alpha specific inhibitor. Molecular Immunology, 33(7-8):649-58. BACK TO TOP
Let me start with a quick re-cap of how Monoclonal Antibodies (MAbs) are made in the laboratory.
The journey of MAb therapeutics to its current state has been interesting, with several important iterations.
Murine: These were the first generation MAbs, in which immunized mouse splenocytes (spleen cells, including B-lymphocytes) were fused with a myeloma partner to form a hybridoma. The successful hybridomas would be screened for clones that would recognize the specific antigen. The resultant monoclonal antibody would have both heavy and light chains of mouse origin.
The first ever therapeutic MAb, described in 1986 and licensed by the US FDA in 1992, was a murine monoclonal against CD3 surface protein of T-lymphocytes; named ‘Muromonab-CD3’, this MAb of the class IgG2a was indicated for use to prevent heart, liver or kidney transplant rejection. In the World Health Organization’s International Nonproprietary Names (INN) system, a murine antibody is identified by the pre-stem -o- in its INN; muromonab, around since before INN existed, is the only exception. One example is edrecolomab, approved in Germany since 1995), a mouse IgG2a against a protein antigen called EpCAM (Epithelial Cell Adhesion Molecule), carried on the surface of many tumor cells.
Theoretically, a related species of MAbs may come from rats, one in which both chain types are of rat origin, and a rat antibody would be identified by the pre-stem -a- in its INN. However, currently there are no purely rat therapeutic MAbs in the market. There is a rat-mouse hybrid MAb, engineered with binding sites for two different antigens (class known as ‘trifunctional antibodies’) with the substem -axo- in its INN, called catumaxomab, but more about that later; by design, this MAb qualifies as the next-generation of therapeutic MAbs, the ‘Chimeric’ antibodies.
Murine MAbs, although in use, are considered to have limited therapeutic value. Recognized by the human body as foreign substances, the murine MAbs often elicit a human immune response (known as HAMA or “human anti-mouse antibody” response) which neutralizes the foreign antibodies, prevents their ability to engage immune cells as intended, and accelerates their removal. This, obviously, limits the effectiveness of murine MAbs, and may even result in deleterious side effects reminiscent of the ‘serum sickness‘ observed during the days of serum therapy, such as fever, chills, arthralgia and life-threatening anaphylaxis. The Chimeric MAbs came off the efforts to solve this problem.
Chimeric: These were the second-generation MAbs; a chimeric antibody is encoded by genes from more than one species, technically a fusion molecule, produced by genetic engineering methods. Remember the basic antibody structure from the FIRST PART of this series? For the heavy and the light chains, a chimeric chain contains a foreign Variable (VH and VL) domain (originating from one species other than human, or synthetic) which is connected to a Constant region (CH and CL) of human origin. The heavy chain constant region comprising the Fc portion is recognized by the immune effector cells for binding and bringing about many of the antibody’s cell-mediated effects, such as opsonophagocytosis, ADCC and so forth. Therefore, in a chimeric MAb, while the foreign variable regions confers epitope specificity for the intended therapeutic target, the human constant domain – introduced by genetic engineering to replace the analogous foreign constant domain – eliminates most of the potential immunogenicity of (i.e. the possibility of HAMA response against) the MAb without altering its specificity. Therapeutic chimeric antibodies are identified by the pre-stem -xi- in its INN; for example, abciximab, infliximab and the widely successful rituximab. There are other examples in the SECOND PART of this series.
The above mentioned Catumaxomab, which I considered ‘chimeric’, is a different type of hybrid; one half of the antibody (one heavy IgG2a chain and one kappa light chain) is of mouse origin, recognizing tumor-associated EpCAM, whereas the other half (one heavy IgG2b chain and one lambda light chain) is of rat origin, recognizing CD3 on the surface of T-lymphocytes. The hybrid Fc segment of this engineered antibody is capable of binding to immune effector cells like any antibody. Therefore, this MAb binds to a tumor cell via one arm, to a T lymphocyte via the other arm, and to an Fc-receptor bearing immune effector cells, such as macrophage, a natural killer cell or a dendritic cell, via its Fc portion, triggering an immunological reaction against the cancer cell. It has been approved for malignant ascites in the European Union, and currently undergoing phase II/III clinical trials in the US for gastic and ovarian cancers.
Some chimeric MAb may still have issues. For instance, cetuximab, approved by the US FDA for metastatic colorectal cancer, is known to give rise to HAMA IgEs.
Although beyond the scope of this description, here is a quick reference to other non-antibody chimeric proteins in therapeutic use. Etanercept, for example, is a TNFα blocker biologic drug, to make which the gene for human tumor necrosis factor receptor (TNFR) was recombined via genetic engineering with the gene for human IgG1 Fc segment. In the resultant chimeric protein, the TNFR part provides specificity for the target, and the antibody Fc segment ensures stability and localized drug delivery. Etanercept has US FDA approval to treat rheumatoid arthritis, psoriatic arthritis, plaque psoriasis and ankylosing spondylitis.
Humanized: These were the third generation therapeutic MAbs, designed to be an improvement upon Chimeric MAbs. In the “humanization” process, the antibody protein sequence is genetically modified to increase its similarity with human antibodies, by interspersing human sequences within the murine complementarity determining region (CDR) segments in the epitope-specific Variable domain; the goal is to further reduce the change of an unwanted HAMA response. A humanized therapeutic MAb is identified by the pre-stem -zu- in its INN; for example, trastuzumab and alemtuzumab. Other examples are in the SECOND PART. Nevertheless, induced antibody responses against humanized therapeutic mAbs (termed ‘HAHA’, human anti-human antibody response) have also been observed, giving rise to the concern that other factors, such as the nature of the antigen, the disease process treated and the schedule of administration, may contribute to the unwanted immunogenicity of the MAb.
Human or ‘Fully Human’: This represents the fourth generation of therapeutic MAbs. A human antibody is one of which both chain types, and the J chain in the case of polymeric antibodies, are of human origin. A human antibody is identified by the pre-stem -u- in its INN; for example, adalimumab and ustekinumab. Other examples are in the SECOND PART. Different technologies were devised and studied to aid in production of human MAbs.
- Phage Display: ‘Phage’ or filamentous bacteriophage is a virus that can infect and replicate within bacteria. It offers an elegant and incredibly powerful technique to produce antibodies in vitro. In short, the genes for the Variable (V) regions of antibody heavy and light chains can be inserted separately into these phages, and they display the proteins on the surface. These genes can come from (a) naïve B-cells, or (b) immunized, antigen-specific memory B-cells. The proteins may be selected by screening against the antigen of choice, and the desirable proteins may be produced and secreted by bacteria infected by the specific phage. The secreted proteins can be reassembled in vitro to make functional antibodies. This technology is still developing and has great potential, but it is cumbersome, requiring multiple different steps. The antibodies made with this technology may also suffer from random heavy and light chain pairing, reducing the antibody efficacy. An example of fully human phage display derived therapeutic MAb is adalimumab.
- Transgenic Mice: In early 1990s, transgenic mouse strains, in which mouse Ig genes were replaced by human Ig genes, were developed. This mouse strain contains human genes and produces human antibodies, and murine-murine hybridomas – which immortalize antigen-activated B-cells – can produce large quantities of antigen-specific human MAbs; examples are golimumab and ustekinumab. However, the antibody diversity (in epitope recognition) is created in a mouse strain environment, where the antibodies undergo affinity maturation, as opposed to a in human environment. Although this gave rise to the concern of limited germline repertoire engagement, now there are other mouse strains with larger human Ig transgenes to address that issue. Moreover, the results of clinical trials have now started to come in and should permit better analysis of the efficacy of these transgenic MAbs.
- Primarily human system:This, of course, is the best-scenario for MAb therapeutics – the generation of human MAbs in a human system, thereby eliminating unwanted immunogenicity of the therapeutic agents. However, for human MAbs, the generation of human hybridoma cell lines of acceptable stability has posed a particularly galling challenge. For one, antigen-primed human B-lymphocytes needed for fusion are difficult to obtain on a routine basis; therefore, several alternative strategies have been devised for that purpose – which, nonetheless, has not been without problems.
- Mouse myeloma cell line transfected with human immunoglobulin genes – secrete low levels of human antibody.
- Fusion of human B cells with a murine myeloma partner – barring a few exceptions, mouse-human hybridomas cease human immunoglobulin production due to disturbances in gene expression; also, the peculiar phenomenon of selective loss of human chromosomes is well known; for some reason, mouse-human hybrids seem to preferentially retain human chromosomes 14 (H chain) and 22 (λ-L chain), and lose chromosome 2 (κ-L chain).
- Fusion of human B cells with a mouse-human hybrid ‘heteromyeloma’ – designed to solve the problem of chromosomal loss, but are unstable and secrete low levels of antibody.
- Transformation (and consequently, immortalization) of antigen-primed human B-cells with lymphotrophic (i.e. preferentially infecting lymphocytes) Epstein-Barr virus (EBV, a.k.a. human γ-herpesvirus 4) – moderately successful technique, but these transformed cells show unstable growth and secrete low levels of antibody, ceasing production altogether after a variable time period.
- Fusion of human B cells with a human lymphoblastoid cell line (LCL) – unfortunately, such hybridomas tend to secrete antibody molecules derived via permutation of the antibodies from both the fusion partners.
- Fusion of an EBV-transformed human B-cell line with a murine or human myeloma or a human LCL – secretes large amount of antibody. Because of the chromosomal loss in murine-human hybrids, human-human hybrids are considered more preferable. EBV transformation is beneficial because it causes polyclonal activation of B-cells, generating a greater V-region diversity, and makes the B-cell more competent for hybridization. These hybrids are stable and more efficient, and can be made to secrete specific antibodies.
In the next post, I shall focus on some very recent developments in antibody therapeutics and their applications. Stay tuned!
This is the THIRD PART of a multipart series on exciting new developments in the world of antibodies.
ACKNOWLEDGEMENTS with gratitude: The images of the mouse and rabbit have been modified from the product listing page of Charles River laboratories; the images of the myeloma cell and hybridoma cell have been modified and adapted from Figure 5 of Karpas et al. (PNAS USA 2001, 98:1799).