“In each year’s flu season, most deaths are in infants and the aged, but none of the first ones in Mexico were in people over 60 or under 3 years old, a W.H.O. spokeswoman said. When a new virus emerges, deaths may occur in healthy adults who mount the strongest immune reactions. Their own defenses — inflammation and leaking fluid in lung cells — can essentially drown them from inside.” The New York Times

Pathogenicity is a complex question and a delicate balance of host response and virus load.

Cytokines play an extremely important role in the immune system’s defense against infection. We don’t fully understand the effects of intervening with the cytokine response, since this can alter the balance between the protective and damaging effects of these signaling molecules during an infection. We need to learn much more about the role and effects of cytokines during an infection. Much more work remains to be done to understand the interplay between the highly pathogenic virus and the host.

When the immune system is fighting pathogens, cytokines signal immune cells such as T-cells and macrophages to travel to the site of infection. In addition, cytokines activate those cells, stimulating them to produce more cytokines. Normally, this feedback loop is kept in check by the body. However, in some instances, the reaction becomes uncontrolled, and too many immune cells are activated in a single place. The precise reason for this is not entirely understood but may be caused by an exaggerated response when the immune system encounters a new and highly pathogenic invader. The body must limit the multiplication of the virus itself as well, as not create a cytokine storm, by an over reactive immune system.

A cytokine storm is a blizzard of signaling proteins called cytokines that is generated by various immune system cells as they coordinate an attack on an invading microorganism. But if this response runs out of control, it can cause potentially fatal inflammation and damage to the lungs. And that is what many researchers have thought kills people who are infected with flu virus. Perhaps, this is why many Mexicans have died from the swine flu, whereas people from other countries have not.

We think of external microbes as our worst enemy during an outbreak of influenza or bronchitis but our own immune system can be potentially lethal, as well. When our body detects pathogens, indicating an infection, our body might respond by over-protecting the site of infection.

The body may race so many antibodies to the infection site that they collect in a cytokine storm. When the infection is in the lungs, for example, a cytokine storm can potentially block airways and result in suffocation. Medical researchers have identified the causes and stages of the cytokine storm and are working on treatments to weaken an overactive immune response.

20090429-1

At all times, white blood cells circulate in the bloodstream and are the first to sense if a virus or bacteria has infiltrated the body. Immediately, our body sends immune cells, including T-cells and macrophages, to attack the infection. During this stage, our immunity functions properly, and immune cells attack the microbes so they do not get too strong a foothold in our lungs.

For reasons not completely known, too many immune cells can be sent to the infection site. This happens when a particular type of molecule in the body, known as cytokines, activate the immune cells at the infection site and cause more immune cells to flood the site of infection. This propagates what is referred to as a cytokine storm where far too many immune cells are caught in an endless loop of calling more and more immune cells to fight the infection. The cytokine storm ends up inflaming the tissue surrounding the infection.

When the infection is in the lungs, severe inflammation caused by a cytokine storm can cause permanent lung damage. A prolonged cytokine storm will eventually shut down breathing altogether. Airducts get clogged and cells no longer properly absorb oxygen. This is what makes the cytokine storm so deadly in certain epidemic strains, such as bird flu.

Even bronchitis, other varieties of influenza, pneumonia, sepsis and possibly rheumatoid arthritis are susceptible to triggering a cytokine storm.
Of course, flu vaccines are usually effective at preventing the flu during its peak season. But they are no guarantee, especially when flu strains mutate after the vaccine has been manufactured. Therefore, researchers are pursuing other methods of preventing the cytokine storm by bioengineering a drug that could slow the snowball effect of antibodies. They hope to force the cytokines to recirculate in the bloodstream, rather than pool in the lungs. Experts predict that a major influenza pandemic could kill millions of people worldwide as it has done in centuries past.

20090429-2
Technician Working on Egg-Based Production of Inflluenza Vaccine

Aventis Pasteur MSD/Getty Images.

20090429-3
A train in Mexico City, ground zero of the epidemic;

Ganesh Suntharalingam, F.R.C.A., Meghan R. Perry, M.R.C.P., Stephen Ward, F.R.C.A., Stephen J. Brett, M.D., Andrew Castello-Cortes, F.R.C.A., Michael D. Brunner, F.R.C.A., and Nicki Panoskaltsis, M.D., Ph.D.

SUMMARY

Six healthy young male volunteers at a contract research organization were enrolled in the first phase 1 clinical trial of TGN1412, a novel superagonist anti-CD28 monoclonal antibody that directly stimulates T cells. Within 90 minutes after receiving a single intravenous dose of the drug, all six volunteers had a systemic inflammatory response characterized by a rapid induction of proinflammatory cytokines and accompanied by headache, myalgias, nausea, diarrhea, erythema, vasodilatation, and hypotension. Within 12 to 16 hours after infusion, they became critically ill, with pulmonary infiltrates and lung injury, renal failure, and disseminated intravascular coagulation. Severe and unexpected depletion of lymphocytes and monocytes occurred within 24 hours after infusion. All six patients were transferred to the care of the authors at an intensive care unit at a public hospital, where they received intensive cardiopulmonary support (including dialysis), high-dose methylprednisolone, and an anti–interleukin-2 receptor antagonist antibody. Prolonged cardiovascular shock and acute respiratory distress syndrome developed in two patients, who required intensive organ support for 8 and 16 days. Despite evidence of the multiple cytokine-release syndrome, all six patients survived. Documentation of the clinical course occurring over the 30 days after infusion offers insight into the systemic inflammatory response syndrome in the absence of contaminating pathogens, endotoxin, or underlying disease.

On March 13, 2006, eight healthy male volunteers participated in a double-blind, randomized, placebo-controlled phase 1 study of the safety of TGN1412 (TeGenero), a novel monoclonal antibody. The study drug is a recombinantly expressed, humanized superagonist anti-CD28 monoclonal antibody of the IgG4 subclass that stimulates and expands T cells independently of the ligation of the T-cell receptor.1 In contrast to other antibodies in clinical use or in clinical trials, TGN1412 directly stimulates the immune response in vivo. In preclinical models, the stimulation of CD28 with TGN1412 (or with murine-antibody counterparts) preferentially activated and expanded type 2 helper T cells2 and, in particular, CD4+CD25+ regulatory T cells, resulting in transient lymphocytosis with no detectable toxic or proinflammatory effects.1,2,3,4

On the day of the trial, six of the eight volunteers received TGN1412 and two received placebo. Subsequently, the six volunteers in the treatment group, who had multiorgan failure with an unknown mechanism and an unpredictable severity, were all admitted to the on-site critical care unit at Northwick Park and St. Mark’s Hospital, a National Health Service (NHS) hospital in London. We detail the clinical and pathological findings during the first 30 days after the infusion.

Methods

Trial Conduct

TeGenero sponsored the trial of the monoclonal antibody TGN1412, which was manufactured by Boehringer Ingelheim. The trial was conducted by Parexel International, a contract research organization that operates an independent clinical trials unit in leased space on the premises of Northwick Park and St. Mark’s Hospital.

The authors of this report are a group of NHS clinicians who assumed clinical responsibility for the secondary care of these patients after they were transferred to the NHS (between 12 hours [one patient] and 16 hours [five patients] after infusion). The authors have no contractual or operational relationship with either Parexel International or TeGenero.

Patients and Sources of Data

All six patients provided written informed consent to the NHS for the publication of data obtained during clinical case management. Clinical data obtained before admission to the NHS, and selected laboratory data obtained before the complications were observed, are reproduced here with permission from TeGenero. The trial was suspended owing to the serious adverse events, and no further tests were performed for research purposes. There was full disclosure of drug information, scientific data, and trial documentation by TeGenero and Parexel International, in order to assist in clinical management decisions at the time of the incident.

Cytokine and Cell Subgroup Determinations

Data on subgroups of cytokines and lymphocytes were subsequently collected for clinical purposes during the course of the illnesses. For details on the cytokine assays and the cell subgroups, see the Supplementary Appendix (available with the full text of this article at www.nejm.org).

Results

All six patients who received the trial drug were male, with a median age of 29.5 years (range, 19 to 34) (Table 1). None had a notable medical history, and all were clinically well during the 2 weeks before the study; baseline laboratory values were normal (Table 2). Beginning at 8 a.m. on day 1, each volunteer received an intravenous infusion, 10 minutes apart, of either the study drug or placebo. Each infusion lasted 3 to 6 minutes. The six volunteers in the treatment group each received 0.1 mg of TGN1412 per kilogram of body weight, infused at a rate of 2 mg per minute; the remaining two volunteers received a similar volume of saline.

Initial Response after Infusion of TGN1412

A series of adverse effects began in the treatment group after infusion, starting with the onset of severe headache in five patients after a median of 60 minutes (range, 50 to 90), accompanied by lumbar myalgia in all six patients after a median of 77 minutes (range, 57 to 95) (Figure 1). Subsequently, during this early phase, the patients were restless and had varying degrees of nausea, vomiting, bowel urgency, or diarrhea (Table 1). Five subjects had short amnestic episodes associated with severe pyrexia, restlessness, or both. All patients had a systemic inflammatory response that included erythema and peripheral vasodilatation (the timing of which was undocumented), with recorded rigors in four patients at a median of 59 minutes (range, 58 to 120) after infusion. Hypotension (defined by a decline in systolic blood pressure of 20 mm Hg or more) developed in all patients a median of 240 minutes (range, 210 to 280) after infusion, accompanied by tachycardia, with maximal heart rates of 110 to 145 beats per minute. All patients received intravenous lactated Ringer’s solution during this time. Body temperatures of 39.5 to 40.0°C were recorded a median of 280 minutes (range, 240 to 390) after infusion. At 300 minutes after infusion, Patient 1 had signs of respiratory failure, with tachypnea and a partial pressure of arterial oxygen (PaO2) of 52 mm Hg while breathing ambient air; the PaO2 increased with the addition of supplemental oxygen. Chest radiography revealed pulmonary infiltrates; these findings were not consistent with the expected response of a fit young man to the infusion of less than 4 liters of fluid at this stage. There was no clinical evidence of bronchospasm or laryngeal edema.

All patients were initially empirically treated in the independent clinical trials unit. A dose of 200 mg of hydrocortisone was administered intravenously in divided doses (with the initial 100-mg bolus a median of 331 minutes [range, 315 to 346] after infusion), in addition to 10 mg of chlorpheniramine intravenously, 1 g of acetaminophen intravenously, 4 to 8 mg of ondansetron intravenously, and 0.5 to 3.0 mg of metaraminol intravenously (in divided doses, titrated to effect). Blood samples were analyzed 8 hours after infusion at an off-site private laboratory (according to the study protocol) and therefore were not available as the situation evolved; the results were abnormal (Table 2).

Subsequent Events

After an initial recovery, Patient 6 became hypotensive (blood pressure, 65/40 mm Hg), and 12 hours after infusion, he had metabolic acidosis and marked respiratory distress with hypoxemia that was refractory to treatment with supplemental oxygen. He underwent intubation and mechanical ventilation, after which he was admitted to the intensive care unit (ICU) at Northwick Park and St. Mark’s Hospital. He had severely abnormal hemodynamics, coagulation, and pulmonary function, with a PaO2 of 84 mm Hg while breathing 100% oxygen (ratio of PaO2 to the fraction of inspired oxygen, 84) (Table 1).5 Because there was concern that all patients would follow a similar course of rapid deterioration, all remaining patients were transferred to NHS ICU facilities 16 hours after infusion.

Further Treatment

Between 16 and 20 hours after infusion of TGN1412, the patients had further signs of respiratory deterioration: all six had signs of tachypnea, use of accessory muscles, inability to complete spoken sentences, and bilateral pulmonary infiltrates on chest radiography (Figure 2A and 2B), and two had symptoms of dyspnea. There was also evidence of substantial renal impairment and disseminated intravascular coagulation, as indicated by an elevated prothrombin time, low fibrinogen level, high level of D-dimers, and decreased platelet counts in all six patients (Table 2). All patients had severe lymphopenia and monocytopenia, with sparing of neutrophils. Blood smears showed toxic granulation with Döhle’s bodies and a dysplastic appearance of the neutrophils, with pseudo–Pelger–Huët anomaly (Figure 2C and 2D).

There was no clinical evidence of primary cardiogenic shock, nor was there bronchospasm, laryngeal edema, or cutaneous signs indicating anaphylaxis. There were no overt or focal neurologic symptoms or signs that suggested neurogenic vasodilatory shock. All electrocardiograms and echocardiograms were normal (Table 1), and there was no clinical indication for lumbar puncture or electroencephalography.

All patients received empirical treatment with 1 g of methylprednisolone sodium succinate intravenously a median of 16 hours (range, 15.5 to 17) after infusion with TGN1412, with subsequent doses 40 hours and 64 hours after. Because of the expected effects of TGN1412 on T cells, all patients were empirically treated daily for 3 days with an anti–interleukin-2 receptor antagonist antibody, daclizumab (Roche), beginning a median of 25.5 hours (range, 23.5 to 28.0) after infusion. This treatment was stopped after 3 days in the absence of TGN1412-induced lymphocytosis. In addition, potential activation of a histaminergic response was treated with 50 mg of intravenous ranitidine every 8 hours and 10 mg of intravenous chlorpheniramine maleate every 8 hours (continued from earlier doses).

Supportive Management

Patients 1 through 4 received continuous positive airway pressure of 10 cm H2O by means of a tight-fitting face mask. Patients 5 and 6 underwent mechanical ventilation, with tidal volumes limited to 6 to 8 ml per kilogram of dry body weight and positive end-expiratory pressure maintained at 15 to 20 cm H2O. All six patients had oliguria, metabolic acidosis, and increasing creatinine levels; they therefore received renal support by means of continuous venovenous hemodiafiltration with the use of a standard polyacrylonitrile membrane (Gambro Hospal U.K.) within 36 hours after their exposure to TGN1412. Dialysate rates were set to 1 liter per hour and were subsequently increased to 4 liters per hour.

All patients required the replacement of blood components by means of the infusion of fresh-frozen plasma and cryoprecipitate to correct coagulopathy. Owing to their severe lymphopenia, the patients were treated according to a protocol of infusions of irradiated red cells and platelets, as required, to prevent possible graft-versus-host disease.

Clinical Progression

Patients 1, 2, 3, and 4 continued to have intermittent fever, myalgia, and diffuse erythematous flushing for 48 hours, at which point their clinical symptoms and signs diminished markedly. Immunomodulatory treatment in these four patients was reduced to a tapering dose of intravenous hydrocortisone followed by oral prednisolone (total duration of corticosteroid treatment in each case, 21 days). Continuous venovenous hemodiafiltration was stopped after a median of 28 hours (range, 22 to 35), and continuous positive airway pressure was stopped after 4 hours in Patient 1 and after a median of 77 hours (range, 57 to 82) in Patients 2, 3, and 4 (Figure 4A and 4B through 7A and 7B in the Supplementary Appendix). Patient 2 was also successfully treated for presumed nosocomial Klebsiella pneumoniae bacteremia, isolated on day 6 after TGN1412 infusion.

Patients 5 and 6 had a more complex course, as detailed in the Supplementary Appendix. Although both patients initially had diminished erythema and fever 48 hours after infusion, they subsequently had recurrent fever, increased peripheral vascular permeability, and episodes of diffuse erythematous flushing lasting several days. Both patients required intubation and mechanical ventilation. Peripheral ischemia was observed in a glove-and-stocking distribution in Patient 6. It fluctuated over time, independently of the changing vasopressor dose. Most of the peripheral ischemia slowly resolved, except in patches of necrosis on the fingers of both hands and all the toes.

Over the next 30 days, all patients had generalized desquamation (most marked in Patients 5 and 6) and muscle weakness on discharge from the ICU. Five patients had late myalgia, headache after the discontinuation of corticosteroids, difficulties with concentration, and short-term difficulties in finding words (particularly names). Three patients had delayed hyperalgesia, and two had peripheral numbness. None had documented lymphadenopathy or splenomegaly while in the ICU or after discharge.

Discussion

The intravenous infusion of TGN1412 in healthy persons produced a sudden and rapid release of proinflammatory cytokines. These unexpected clinical data provide insight into the natural course of the cytokine storm and the systemic inflammatory response syndrome (SIRS) in the absence of contaminating organic factors. Regulatory authorities, who tested TGN1412 from the same batch as the infused drug, found no errors in its manufacture, formulation, or administration and found no contamination with endotoxin, pyrogen, or microbiologic or other agents. This type of cytokine release had not been observed in the preclinical studies of TGN1412, and it is currently unclear whether the severe effects of this type of cytokine release in vivo in humans is caused by the direct ligation of CD28 on T cells or by the ligation and activation of other cell types, leading to the release of preformed TNF- , which then triggers the remainder of the cascade. The Secretary of State for Health has convened an expert scientific group to study the events of the clinical trial in greater detail.

Clinically, the most striking phenomenon in the cohort was the stereotypical response to the study drug in all six patients and in all organ systems affected (albeit to varying degrees) . All six patients initially had clinical signs that fit the criteria for SIRS. Subsequently, the most prominent clinical feature was the early appearance of respiratory distress and pulmonary infiltrates, accompanied by renal impairment and profound disseminated intravascular coagulation. This pattern of organ impairment may be consistent with a generalized multiorgan response to inflammation or critical illness. However, the rapid onset and concordance of the lung injury among patients seemed unusual, and in the presence of high cytokine (especially interferon- and TNF- ) levels, these features may be consistent with immune-mediated injury that is specific to the lung

Alveolar macrophages in humans are normally inefficient in the costimulation of T cells through the CD28 pathway; thus, our data suggest that anti-CD28 agonists in vivo may be able to potentiate immune activation and therefore lung injury. Neither cytokine storm nor lung injury was observed in the preclinical studies of TGN1412. This probably indicates that the presence of high levels of proinflammatory cytokines is a requirement for the pulmonary compromise, regardless of whether CD28 is ligated in the lung. In contrast to the pulmonary compromise that eventually ensues in SIRS, the more rapid onset of lung injury in our patients may have been due to the combination of the direct effects of the antibody and cytokines on lung tissue.

Equally striking was the consistent pattern of immunologic effects and recovery in all six patients. In particular, the severe lymphopenia observed in these patients was unexpected; a temporary lymphocytosis had been observed in preclinical studies of TGN1412 in animals. This unanticipated lymphopenia in humans may have reflected cell death or the migration of cells to other tissues such as lymph nodes, although lymphadenopathy was not detected. Lymphopenia has been observed as part of the cytokine storm induced by other monoclonal antibodies., However, the low cell numbers observed in these studies were anticipated, given the mechanism of action and the antilymphocyte specificity of the infused antibodies. Sepsis in humans may also induce lymphopenia that is selective for B cells and CD4+ T cells over the course of several days. In contrast, the onset of lymphopenia within 8 hours after infusion of TGN1412, and the involvement of all mononuclear cells (CD4+ and CD8+ T cells and monocytes), may suggest that the depletion of cells in our patients was a response to the infused T-cell agonist drug rather than to the cytokine storm alone.

The clinical progression after infusion of TGN1412 can be separated into four phases . Phase 1 began within an hour after infusion, continued through days 1 and 2 (and day 3, in Patients 5 and 6), and consisted of the cytokine storm, involving the rapid induction of type 1 and type 2 cytokines (to varying degrees) and severe lymphopenia and monocytopenia. Phase 2, the reactive phase, occurred from day 1 through day 3 (or days 1 through 8 in Patients 5 and 6, who were the most seriously ill); it consisted of renal failure, disseminated intravascular coagulation, pulmonary infiltrates, and respiratory failure. Phases 1 and 2 overlapped; phase 2 was not necessarily directly caused by the events in phase 1. The recovery phase, phase 3, occurred between day 3 and day 15 (or between day 5 and day 20, for the patients who were the sickest) and was characterized by the recovery of renal and pulmonary function. This recovery was reflected in thrombocytosis and increases in alanine aminotransferase and monocyte and lymphocyte levels (mostly in a 1:1 ratio of CD4+:CD8+ T cells). The last phase, phase 4, can be described as a plateau or steady-state phase. It began 15 days after infusion (or 20 days after in Patients 5 and 6) and consisted of normalization of the measured variables. As compared with reactions to the infusion of other immunomodulatory agents (such as anti-CD20, anti-CD3,and anti-CD52 monoclonal antibodies), the response to TGN1412 initially had similar kinetics, including the rapid increase in the levels of first TNF- and then interferon- and interleukin-6, followed by cardiovascular instability, and disseminated intravascular coagulation. However, from phase 2 onward, features unique to the response to TGN1412 were apparent — including early acute lung injury, diffuse erythema with late desquamation, neurologic sequelae, and post-illness myalgias .