Bioterrorism: The Reality of Risk

Speaker:
Gregory A. Poland, Mayo Clinic Director, Mayo Vaccine Research Group and the Program on Translational Immunovirology and Biodefense; and Group Leader, Region V Regional Center of Excellence

Highlights

• Biologic terrorism is a real and present threat.
• Complete prevention or countering of bioterrorism is not currently possible.
• Full detection and interdiction of those intending to use biologic weapons is not currently possible.
• Provision of protective vaccines to the civilian population (including first responders) is not currently possible.
• Regional Centers of Excellence play a critical role in addressing the above issues.

"Multidimensional" risk

“ People have very different opinions about the risk of bioterrorism in the United States, the risk in the rest of the world, and their personal risk,” said Gregory Poland, MD, Director of the Mayo Clinic’s Vaccine Research Group and Program on Translational Immunovirology and Biodefense, in the opening teleconference. “The fact is, risk is a multidimensional thing. You might be attacked directly; you could be at risk because of collateral or bystander exposure; you could be at risk—depending on the organism—hours, days, or even decades later from environmental contamination; and, of course, health care workers who provide direct patient care can be at risk from that exposure.”

Despite these real dangers, the threat of bioterrorism often is dismissed. “Up until recently, precedent had suggested that bioweapons wouldn’t be used; that they were morally repugnant—at least, in our society’s concept of war; that the science of production and dispersal was too difficult; and that, like nuclear winter, the destructiveness of bioweapons is essentially unthinkable.” More recently, people have also dismissed the danger by noting that no weapons of mass destruction were found in Iraq.

However, Poland emphasized, “the fact that weapons of mass destruction have not, to date, been found in Iraq does not negate the threat that biologic weapons pose. The technology exists. The intent and will exists. Money is not an obstacle. Expertise is not an obstacle. And multiple countries have, in fact, successfully developed, or attempted to develop, such weapons.”

Adding to the risk is the fact that America is facing an enemy that is not readily identifiable. “They are both everywhere and nowhere. There is no really ‘safe’ place and, more importantly, there is no single commander to negotiate with or to surrender,” he said. The “enemy” represents splintered, widely dispersed groups with different agendas and with different aims and outcomes.

Biological weapons remain a threat, even if none have been found in Iraq.

Perpetrators of bioterrorism may be state-sponsored terrorists, lone terrorists, single-issue groups, nationalists, separatist groups, or apocalyptic cults. Common characteristics of such terrorists are diffuse objectives, making it hard to understand their ideology and infiltrate their groups; a sense of grandiosity; and a paranoid conspiratorial or apocalyptic world view that leads to defensive aggression.

Their motivations, he noted, include getting attention, economic terrorism, millennialism (the idea of the new dawn after a cleansing apocalypse), revenge and the creation of chaos, the feeling of mimicking God, giving ascendancy to a religious jihad, creating an aura of science and technology (i.e., look at the weapon we have; you’d better negotiate with us as a serious threat), and copycatting (imitating previous bioterrorist activities).

Formula for bioterrorism

Bioterrorism is, essentially, “the use of a biologic agent in order to induce a state of intense fear or terror, as a means of intimidation or coercion,” said Poland.

“If your goal is to harm, kill, terrify, or demoralize your opponent,” he noted, “what would prevent your use of any weapon that you thought was effective? More importantly, how might that play out if you believe that you have a philosophical or religious imperative to eliminate your opponent?”

For terrorism to occur, three elements must be in place, Poland observed: 1) a vulnerable target; 2) technical and organizational capability on the part of the perpetrators to carry out an attack; and 3) the intent to attack.

A biologic weapon, like conventional munitions, has four components, he explained: a payload—that is, a biologic agent; a munition—a container keeping the payload intact and virulent up till the point of delivery; a delivery system—typically a missile or shell; and a dispersal mechanism—an explosive force or spray device to dispense agent.

“Sometimes, the munition or container is as simple as a 3-cent envelope [as in the anthrax envelope attack], and sometimes the delivery system is our own technology used against us,” he noted.

Bioweapons have also been called “the poor man’s nuclear bomb.” For a large-scale operation against a civilian population, the cost of producing casualties, per square kilometer, is roughly $2000 for conventional weapons, $800 for nuclear weapons, $600 for nerve gas weapons, and $1 for biologic weapons, Poland said.

Anthrax and smallpox as bioweapons

Poland went on to briefly summarize the potential of anthrax and smallpox as bioweapons. The two organisms are the “poster children,” so to speak, of bioterrorism, he said, because they are highly lethal, stable for transmission in aerosol, capable of large-scale production, odorless, and tasteless, and they have a delayed onset of symptoms, allowing the perpetrators time to escape. In addition, “they induce panic and devastating psychological effects, as we saw after the anthrax attack on this country. There’s limited, if any, vaccine availability. And there’s the capability for large outbreaks over large geographic areas.”

Anthrax and smallpox are the “poster children of bioterrorism.”

Bacillus anthracis is a gram-positive bacterium that forms long-living, durable spores .“In Minnesota, we continue to have outbreaks of anthrax in cattle related to the inhalation or ingestion of anthrax spores found along the cattle trails of a century ago,” Poland noted. Anthrax causes three distinct syndromes: cutaneous (affecting the skin), gastrointestinal, and inhalational. The organism is nearly 100% lethal to unvaccinated or untreated persons. After an incubation period of one to six days, it causes abrupt respiratory distress and a flu-like syndrome; this is followed by transitory improvement two to four days later, and then by shock, massive edema, hemorrhage, and death.

“Of course, it didn’t happen that way in the 2001 anthrax attacks, and I think that served as a warning to all of us, in terms of our scientific knowledge,” said Poland. “Large-dose exposure of an agent in the context of bioterrorism might very well present and act differently from what we expect in natural transmission.”

We learned the additional lessons from the 2001 attack, he noted: the technology exists for producing inhalational anthrax; weapons-grade anthrax is available; low-tech delivery mechanisms, such as envelopes, are useful and can work; and pass-through cross-contamination can occur, as happened with the 92-year-old woman who died after handling a contaminated envelope.

“We also learned that with known exposure and proper treatment, anthrax is treatable and survivable,” as was the case with the 7-month-old infant who somehow came in contact with cutaneous anthrax.

“Finally, and perhaps most importantly, the lesson learned by the terrorists was that infection can be produced, along with panic and disruption of the entire government and economy.”

Smallpox, unlike anthrax, is transmissible from person to person, and about 25 to 30 percent of unvaccinated people who are exposed and infected will die. The United States stopped routinely immunizing against smallpox in 1972, and there is no licensed treatment available. Moreover, there are published articles and statements suggesting that smallpox is obtainable on the international black market trade in weapons of mass destruction.

“I’m sometimes asked,” Poland noted, “‘Is this viral infection really any big deal?’” In response, he quoted Dr. Steven Block at Stanford University, who calculated that “by the end of the second millennium, smallpox had killed, crippled, blinded, or disfigured one-tenth of all humankind that had ever lived.”

There is an estimated total of 60 million doses of smallpox vaccine available worldwide: 15 million in the USA, 15 million in Canada, 5 million in France, and the rest scattered among a variety of countries. Recently, it was revealed that Aventis Pasteur had an additional 90 million doses available.

“Smallpox is obtainable on the international black market.”

“In 1991, the World Health Organization destroyed 200 million doses of smallpox vaccine because it was going to cost $25,000 to store it,” Poland said. “The United States has already committed well over a billion dollars to regain the capacity to manufacture the vaccine.”

Risk is real. . . and increasing

Poland underscored the increasing risk of bioterrorism in today’s world with a quote from former CIA director George Tenet, who noted, “People that say the terrorist risk is exaggerated aren’t looking at the same world I’m looking at.” Conventional terrorism, explained Poland, involves political acts with “calculated levels of violence. . . but not so severe that it alienates supporters, or triggers overwhelming responses from the authorities.”

That’s different from the current era of “postmodern terrorism, or superterrorism.” The goal of terrorist activities today could include maximizing casualties, maximizing fear and panic, or maximizing damage to the target as an end in itself. “This sort of terrorism is unconventional, unsuspected, and asymmetric, and frequently done to please God, or a religious figure, or a holy calling.”

The World Terrorism Index 2003/4 (World Markets Research Centre)—which ranks risk based on such factors as the motivation of terrorists, the presence of terrorist groups in countries, the skill and frequency of past attacks, the efficacy of the groups in those past attacks, and the number of attacks—shows the United States at fourth-highest risk, behind Colombia, Israel, and Pakistan.

Poland went on to describe the history of bioweapons involvement by Iraq and the former Soviet Union as examples of “how hostile countries can engage in this [bioterrorist] work pretty much unfettered and, oftentimes, without our knowledge.”

Iraqi bioweapons program

Highlights of the history of the Iraqi bioweapons program include the following, according to Poland.

• The US Defense Intelligence Agency (DIA) found that 8 of 71 (about 10%) Iraqi Gulf War prisoners of war had smallpox antibodies.
• United Nations Special Commission inspectors noted that Iraq was immunizing troops against smallpox as late as 1990, well after the disease was declared to have been eradicated.
• In 1990, senior Iraqi virologist Hazem Ali defected to the United States and revealed a bioweapons program using camelpox as an ethnic bioweapon.
• In 1991, the British intelligence service reported the presence of Vektor scientists [from the former Soviet Union] in Baghdad.
• In 1994, the DIA learned that Soviet scientists had transferred smallpox cultures to Iraq in the early 1990s.
• In 1995, Saddam Hussein’s son-in-law defected to the United States, bringing with him documentation acknowledging production of more than 20,000 liters of botulinum toxin, 8000 liters of anthrax spore suspension, SCUD missiles carrying 400-pound aerial bombs fitted with anthrax warheads, and drone aircraft that had been outfitted and tested in the United States with aerosol disposal systems.

Russian bioweapons program

Poland then reviewed highlights of the Russian bioweapons program, which began in 1947, shortly after World War II.

• Their doctrine for strategic biowarfare called for massive quantities of contagious agents that would be delivered at urban targets; cause panic, social disruption, and civil unrest; overwhelm the enemy’s medical system and ability to respond; spawn widespread epidemics that would be impossible to control; and be used in a war of mutual destruction that few would survive.
• By the 1970s, smallpox weapons had been deployed on intercontinental ballistic missiles in silos near the Arctic Circle, in a launch-ready status and aimed at the United States and the People Republic of China.
• In 1973, the Soviet Politburo formed the Biopreparat, an agency designed to carry out offensive biologic weapons production concealed behind civil biotechnology research. It had 52 sites with a capacity to produce hundreds of tons of biologic agents.
• In 1985, KGB headquarters developed Vektor, “the most ambitious program for biologic weapons development ever devised,” with a $1 billion budget.
• In 1988, the Soviet Ministry of Defense authorized the use of SS-18 intercontinental ballistic missiles that could deliver ten independently targetable warheads over a range of 6000 miles; each warhead contained 150 bomblets capable of delivering 375 kilograms of smallpox suspension over a geographic area of 150 square kilometers.
• In 1997, Vektor scientists successfully inserted an Ebola virus gene into the smallpox genome, with the goal of creating a hybrid smallpox-Ebola weapon.

Aum Shinrikyo: worldwide cult

Religious cults such as Aum Shinrikyo also pose bioterrorist threats. This worldwide cult with an estimated 20,000 to 40,000 members has yearly net revenues of more than $30 million and an estimated net worth of $1.5 billion, Poland said.

• From 1990 to 1994, the group made three attempts to disperse botulinum toxin and one attempt to disperse anthrax spores.
• In 1993, the organizers led a “missionary group” to obtain Ebola virus from Zaire.
• In 1994, they were responsible for the computer-controlled release of the neurotoxin sarin in Matsumoto, which left people dead and 200 hospitalized.
• In 1995, they released sarin in the Tokyo subway system, leaving 12 dead and 1,000 hospitalized.
• In 1998, the cult made eight attempts to aerosolize anthrax and botulinum toxin.

“Other nightmare scenarios exist,” Poland emphasized. “One example would be hostile states supplying biologic weapons to terrorist cells such as Hamas, Hizbollah, Abu Nidal, or al Qaeda, and officially denying any responsibility. Other scenarios are hostile states or terrorist groups on the brink of destruction deploying biologic weapons, in a stance of defensive aggression.”

Conclusion

“My view is that biologic terrorism remains a valid threat; that completely preventing or countering bioterrorism is not currently possible; that full detection and interdiction of those intending to use biologic weapons is currently impossible; and that it is currently not possible to provide prophylactic protective vaccines to the civilian population, including first responders,” concluded Poland.

“To me, the role of the Regional Centers of Excellence is crucial in addressing these issues.”

Smallpox: a research perspective

Speaker:
R. Mark Buller, Saint Louis University School of Medicine

Highlights

• The upper respiratory tract is the most frequent site of smallpox infection.
• It is likely that a low dose, perhaps as little as one virion, is needed to cause infection.
• More work is needed to clarify smallpox pathogenesis, including how the virus is transmitted from person to person and spreads from the upper respiratory tract to the rest of the body.
• A number of vaccines and antivirals are in development to combat smallpox.
• The virus’ potential use as bioweapon has revitalized interest in smallpox research.

In a comprehensive and insightful presentation on smallpox research, R. Mark Buller of the Saint Louis University School of Medicine in St. Louis provided a succinct look at our current knowledge of how smallpox is spread, the course of the disease, and vaccines and treatments in development.

Smallpox is caused by variola virus, which is a poxvirus that is in many ways similar to poxviruses that infect animals. Therefore, most of what is known about smallpox comes from various animal models. These include ectromelia virus (mousepox), cowpox virus, vaccinia virus, rabbitpox virus, and monkeypox virus. Studies of these poxviruses focus both on poxvirus biology and on testing of vaccines and antivirals to treat potential smallpox outbreaks.

“Currently, there are only two official repositories for the smallpox virus, at the Centers for Disease Control and Prevention (CDC) in Atlanta, Georgia, USA, and the VECTOR Institute in Kotsovo of the former Soviet Union,” Buller noted. Although work on variola virus at these sites has been restricted, two recent, albeit, preliminary, decisions by the World Health Organization (WHO) could open up new avenues of research, he said. One would allow the introduction into variola virus of a foreign gene—a reporter gene—to aid in the development of antivirals; the other would allow the recombination of variola virus genes into other orthopoxvirus species to evaluate their function. If approved, such work would help research “tremendously,” he observed. In the meantime, he focused in his talk on what is currently known about the virus.

How smallpox spreads

Smallpox is less contagious than such viral illnesses as influenza or measles, but it is still considered to be highly contagious. Person-to-person transmission most often occurs through exposure to large droplets of aerosol from an infected individual. The smallpox virus can also be spread through contaminated bedding and clothing. According to Buller, the upper respiratory tract is likely to be the most frequent site of infection.

Five factors contribute to transmission, Buller observed. Most important is the amount of virus produced in the upper respiratory tract of infected individuals. Other factors include whether or not the infected person is coughing and sneezing; the proximity of susceptible people to the infected person during the transmission period (two to five days after a rash appears see Henderson); how long the virion remains stable in the environment (the smallpox virus tends to be very stable regardless of temperature and humidity); and the form of the virus (virus particles in scabs and skin debris are less infectious than those in the air).

Smallpox is less contagious than influenza or measles, but is still highly contagious.

How much virus is needed to cause disease? Very little, Buller said. Studies in animal models suggest that perhaps as little as a single virion is sufficient. Two epidemiological studies seem to support this conclusion: one concerning the 1970 outbreak in Meschede Hospital, which involved the importation of smallpox from an endemic country into smallpox-free Germany (see Poland and Henderson); and another a 1971 outbreak in the Aral’sk region of Kazakhstan.

In 1971, in Aral'sk, a Russian biologist working on a trawler called the Lev Berg in the Aral Sea contracted smallpox. At the time, she was in an area that was not friendly to the Soviets, and was not allowed to leave the ship. Thus, said Buller, she must have been infected on board the ship—probably during periods on deck, where she spent considerable amounts of time. No other shipmates were infected. The source of the virus was likely the island of Vozrozhdeniye, where the Soviets maintained a biological weapons field testing facility that was 15 kilometers north of the Lev Berg. Therefore, the biologist's infection was deemed to be an example of long-distance transmission of smallpox, from the island to the trawler.

"Both epidemiological studies suggest that the infectious dose of variola is probably very, very low," said Buller, and if it were given in the form of an aerosol that was intentionally released, the efficacy of the aerosol could be "very, very high in the presence of a non-vaccinated population."

Under the microscope

As noted earlier, smallpox is thought to be transmitted primarily by large droplets of aerosol, and most frequently to the upper respiratory tract. After reviewing the complex processes involved in viral replication, Buller went on to give his vision of how human smallpox infection occurs, noting that there is still much work to be done in elucidating both the cellular pathogenesis of smallpox and host cell-mediated immunity to smallpox infection.

Virus infection of the upper respiratory epithelium is mediated by extracellular enveloped virus (EEV) and/or intracellular mature virus (IMV), he explained. The EEV is an IMV virion wrapped in a second membrane [SLIDES 7-9 provide a high-resolution electron micrograph of the binding of EEV to the cell], which disrupts the outer membrane of the EEV; this allows the attachment of the IMV particle and the fusion of its membrane with the cellular plasma membrane, which releases the core into the cytoplasm. The transcription machinery in the virion is then activated, and the virus replication cycle commences.

The life cycle of the virus occurs in entirely in cytoplasm, although it requires certain nuclear functions. During replication, the virus produces an array of molecules that modulate the internal and external host responses to infection, contributing to virus persistence at the site of infection and spread in the animal host. The virus has a very complex morphogenesis pathway [SLIDE 11 partially describes the morphogenesis pathway of smallpox virus].

Using the example of an ectromelia virus infection in the epidermis, Buller explained that infection induces a host response that leads to the production of a wide spectrum of cytokines, chemokines, and interferon. These proinflammatory substances activate the proximal epithelial cells as well as macrophages and NK cells in the dermis.

Next, the cytokines and chemokines form a gradient and upregulate the adhesion molecules ICAM-1, E-selectin, and VCAM, which normally would facilitate adhesion and movement of inflammatory cells into the tissue surrounding the site of infection; however, virus-encoded binding proteins for key cytokines and chemokines are thought to prevent this process.

In addition to encoding homologs of host cytokines and chemokines receptors, the virus also encodes other host-response modifiers that contribute to the inflammatory process: serpins (protease inhibitors), which can affect a wide range of biological processes; an initiation factor 2α homolog, which is thought to be important in allowing the virus to escape the antiviral effect of interferon; the proinflammatory cytokine interleukin-1β-like antagonist; the inhibitor of complement enzyme, which may be an important virulence factor in variola; inhibitors of apoptosis to damp down apoptosis signals; CD47-like homolog, whose function is not yet known; inhibitors of the antiviral enzymes PKR and RNase L; and superoxide dismutase-like protein.

Host-response modifiers coded by the virus contribute to blocking the inflammatory process.

But although the factors that allow viral replication have been identified, how the virus spreads from the site of primary infection in the upper respiratory tract to the rest of the body is not clear, Buller emphasized. “The conventional wisdom is that the virus spreads through the body as cell-associated viremia, but there’s never been a very thorough study to support this. The pathogenesis scheme for variola is based on that of ectromelia [mouse poxvirus]. However, the two viruses are very different, and so this scheme really represents only our best guess.”

Combating smallpox

Although the correlates for immunity in smallpox are not yet known, it is likely that protective approaches will need to focus on both cell-mediated and humoral (antibody-mediated) responses, Buller noted. Among traditional vaccines, Dryvax (dried, calf lymph type) is the “gold standard.” Also in development are derivatives of Dryvax, which include Acambis 2000, a Dryvax clone; MVA, which comes from a different strain of vaccinia virus and has not yet been tested against smallpox; and LC16m8, a live virus derived from the Lister vaccine strain that also has not been tested against smallpox.

Novel vaccine candidates include a conditionally lethal vaccinia virus with an inactivated E3L gene, which has been shown to be effective in a mouse model; a live recombinant Streptococcus gordonii vaccine that encodes certain protective antigens; and DNA and subunit vaccines that use antigens from the EEV or IMV forms.

Among the candidate antivirals, only cidofovir, which is licensed for treating human cytomegalovirus infections, has been tested for safety in humans and shown in vitro and in animal models to be active against variola virus. An orally available derivative of cidofovir, hexadecyloxypropyl-cidofovir (HDP-cidofovir), has not yet been safety-tested in humans, but is active against variola virus in vitro.

A third compound, ST-246, works by a different mechanism. It seems to block EEV formation, thereby blocking viral spread. It, too, has yet to be safety-tested in humans, although it has been shown in vitro to be active against variola virus. A fourth compound, TTP-6171, targets a protease, 17L, that is encoded by the vaccinia virus and conserved in the variola virus, so it has a high degree of specificity. “It hasn’t been tested for safety or for efficacy against variola virus, but it’s a very promising idea,” Buller concluded.

Anthrax: a research perspective

Speaker:
Theresa M. Koehler, University of Texas-Houston Health Science Center

Highlights

• The main virulence factors for B. anthracis are the anthrax toxin protein and the capsule.
• The atxA gene is the "elusive master regulator" of toxin and capsule gene expression.
• Genetically complete strains, containing both toxin and capsule, should be used to study virulence gene expression and function.
• The B. anthracis capsule is a potential target for detection, treatment, or prevention of anthrax disease.

A virulent disease

While it has been estimated that 25% of patients with untreated cutaneous anthrax will die, more than 85% of those who go untreated for the inhalational form of the disease will meet this fate. (Click here to see report.) Why is inhalational anthrax so lethal? Can researchers pinpoint the causes and pave the way to new treatment targets?

On January 10, 2005, Theresa M. Koehler, associate professor in the department of microbiology and molecular genetics at the University of Texas-Houston Health Science Center, addressed these and other questions related to anthrax pathogenesis in her thought-provoking presentation on virulence gene expression in anthrax.

Koehler's talk focused on three main elements of current research: the anthrax toxin protein and B. anthracis capsule, which are the main virulence factors for the disease; the atxA gene, dubbed the "elusive master regulator" of both toxin and capsule gene expression; and the new models that are being used in vitro and in vivo to study the basic mechanisms involved in anthrax virulence and to identify potential targets for treatment and prevention. She also described experiments conducted by her group and others as they fill in the knowledge gaps in these complex processes.

The anthrax toxin protein and the B. anthracis capsule are the main virulence factors for disease.

Koehler noted that the anthrax toxin is actually comprised of three different proteins: protective antigen (PA), edema factor (EF), and lethal factor (LF). "B. anthracis secretes these three proteins, and entry of the toxin into a susceptible host cell begins when PA binds a receptor on the host cell. Following cleavage of PA by a cell-associated protease, processed PA forms a heptameric ring with high-affinity binding sites for EF and LF," she explained. The toxin complex is then taken up by endocytosis. Acidification of the endocytic compartment results in translocation of EF and LF to the cytoplasm, ultimately resulting in cell death.

The second virulence factor, the capsule, is unique in that, unlike other bacterial capsules, it is made entirely of protein—specifically, poly-D-glutamic acid. But although its composition is unique, it behaves like other bacterial capsules in that it prevents the host's phagocytes from destroying the vegetative bacterium.

Discussing the structure of the bacterium further, Koehler explained that B. anthracis harbors two virulent plasmids, pXO1 and pXO2. The structural genes for the anthrax proteins PA, EF, and LF are located on pXO1. An operon of four genes, called capBCAD, is located on pXO2. The operon encodes proteins that are involved in the biosynthesis of the capsule.

New models

Most studies of virulence gene expression in anthrax use established, attenuated strains of B. anthracis: the Pasteur strain, which is missing the pXO1 plasmid, and thus the anthrax toxin; and the Sterne strain, which is missing the pXO2 plasmid, and thereby, the capsule.

By contrast, Koehler is working with a genetically complete strain of B. anthracis that harbors both virulence plasmids, thereby providing a more complete window into virulence gene expression. For in vivo studies of the function of virulence genes and their regulators, she is collaborating with Rick Lyons at the University of New Mexico Health Science Center, who has developed a new mouse model of inhalation anthrax.

A genetically complete strain of B. anthracis harbors both virulence plasmids.

"The animal studies are also unique," she said. "Rather than using an injection model for mice, which is commonly seen in the literature, we are using the inhalational model to address how the capsule genes affect pathogenesis, and whether the function of regulators in vitro match what we're seeing in vivo." Koehler's work thus has the potential to expose the mechanisms behind the most virulent form of the disease.

"Elusive" master regulator

Previous studies suggest that expression of the three toxin genes is controlled by the atxA gene, which also plays a role, along with acpA, in regulating the capsule biosynthetic operon, capBCAD. But just how atxA accomplishes this "master regulator" function is not known. "We've been trying to associate some molecular function to the AtxA protein for a long time, and simply have not been able to do so," Koehler said.

Nonetheless, her group and others have pinpointed certain characteristics of the gene, which she summarized as follows:

• atxA encodes a 56-kD soluble basic protein.
• AtxA has amino acid sequence similarity to AcpA.
• AtxA has no demonstrated, specific nucleic acid-binding activity and atxA-regulated promoters have no obvious similarities.
• atxA-regulated genes respond to CO2/bicarbonate.

These findings were a starting point for additional studies. The first question Koehler's group asked was whether AtxA had other targets in addition to the toxin proteins. A series of experiments using the genetically complete anthrax strain revealed that atxA not only controlled the EF, PA, and LF genes, but also a number of other genes on both plasmids—including acpA

Koehler's next question was to inquire why acpA exists. "If atxA can do acpA's job," she asked, "isn't it redundant to have this gene in B. anthracis?" It turned out that although acpA does not seem to affect expression of genes other than the capsule biosynthetic operon and one other pXO2-encoded gene, the group was excited to find synergy between atxA and acpA.

Equally exciting was the discovery of a gene (formerly called pXO2-63; now called acpB) on pXO2 that is downstream of the capsule biosynthetic operon. "We're interested in the product of this gene because its predicted amino acid sequence is 62% similar to that of AcpA," she explained, "which suggests that it, too, may affect capsule synthesis."

Subsequent experiments with various mutant strains revealed that, indeed, acpB does have an effect on the capsule biosynthetic operon; that atxA's effect on capsule gene expression is mediated by acpA or acpB; and that there is some functional similarity between these two regulators, although acpA seems to have a greater effect on capsule gene expression than does acpB.

Although knowledge of the complex regulation of virulence gene expression is increasing, the atxA gene remains in charge, controlling all known virulence factors and potentially some new factors that are yet to be determined. In terms of bacterial targets for specifically shutting down virulence, atxA appears to represent a viable candidate.

Virulence in vivo

The second part of Koehler's presentation summarized studies using the mouse inhalational anthrax model. These showed that atxA controls virulence in mice, just as it does in vitro. Surprisingly, though, acpB has a greater role in virulence in the animal model than predicted from the in vitro studies. In fact, knocking out AcpB from mice increased mean time to death 100-fold compared with the parent (non-mutant) strain, whereas knocking out AcpA had no effect on time to death. Additional studies showed that the capsule is required for dissemination of B. anthracis from the lung to the spleen.

"We're actively engaged now in determining the function of these virulence genes," Koehler noted, "and in exploring another pathway, sigH, that seems to influence the transcription and expression of the three toxin genes. And we're trying hard to find some link between this pathway and atxA."

Near the end of her talk, Koehler emphasized two take-home messages for researchers: First, it's important to use genetically complete strains for gene expression studies because of the synergy between two genes (atxA and acpA) encoded on two different plasmids and the presence of other plasmid-encoded regulators. Second, it's also crucial to remember that the Sterne strain, sometimes referred to as a "capsule" mutant, is missing more than the capsule, whereas the Pasteur strain, sometimes considered a "toxin" mutant, is missing more than the toxin. Both are missing whole plasmids "and a lot of genetic information that may be important."

Finally, Koehler explained that the new inhalational mouse model shows that the capsule is essential for dissemination. Looking ahead towards future research agendas, she concluded, "This really increases our interest in the capsule as a potential target for detection, treatment, or prevention of anthrax disease."

Dengue: a research perspective

Speaker:
Alan L. Rothman, Center for Infectious Disease and Vaccine Research University of Massachusetts Medical School

Highlights

• There are four dengue viruses, referred to as serotypes.
• Immunity to the same serotype is long lasting; immunity to other serotypes is partial and transient.
• More serious symptoms tend to be seen in secondary, rather than primary, infection.
• Plasma leakage distinguishes dengue fever from dengue hemorrhagic fever.
• Both viral and host factors influence the risk of developing dengue hemorrhagic fever.

Dengue virus infection: complex, multifaceted

Dengue is actually four viruses, not one. Because the viruses are defined based on serologic responses, they are referred to as dengue serotypes. "In understanding the implication of this, it has to be borne in mind that at least to the best of our knowledge, immunity to the same serotype-homotypic immunity—is long lasting, perhaps life long. But infection with one serotype confers, at best, only partial and transient immunity to other serotypes," explained Alan L. Rothman of the Center for Infectious Disease and Vaccine Research at the University of Massachusetts Medical School. "After this point, people who've been exposed to one virus are now susceptible again to infection with any of the other three serotypes of dengue virus. So we refer to the first time people are exposed to dengue as primary infection and subsequent exposure as secondary infection."

Dengue is transmitted to humans by mosquitoes, primarily Aedes aegypti. Most transmission occurs in urban areas, where infection can be endemic or epidemic. Humans and other primates seem to be the only natural vertebrate hosts for dengue virus infection; however, the viral strains that infect both groups are genetically distinct.

The risk of serious symptoms is greater in secondary infection.

Infection starts when the virus is injected, and viral replication begins at the inoculation site. Within about a day, however, virus can be found in regional lymph nodes. "Presumably, further replication goes on in the lymph nodes, and at some point the viral load increases to the point where it becomes spread throughout the body and disseminated. At this point, we detect viremia," Rothman continued. Within 12 to 24 hours, symptoms—for example, fever, malaise, headache, fatigue—appear. When the fever disappears, viremia ends and viral load declines rapidly.

The risk of more serious symptoms—bleeding diathesis and changes in vascular permeability, for example—seems to be greater in secondary, rather than primary, infection. This and other factors suggest an involvement of the immune response in the pathogenesis of dengue hemorrhagic fever, a life-threatening form of the disease.

From infection to disease

Many researchers are attempting to elucidate the connections between virus infection and disease, and to determine who is at greatest risk of developing dengue hemorrhagic fever. However, the models currently used to study the virus all have drawbacks, Rothman noted. For example, findings from cell culture systems for studying viral entry, replication, protein-protein interactions, and cellular responses to infection may be influenced by the type of viral strains used—that is, whether they are lab-adapted or natural.

Animal models also present problems. Primates, which are natural hosts for dengue virus, do not develop disease. So although primate studies can be used to study viral replication, they yield no information on what's happening in humans. Mice studies yield equivocal results, depending on the strain, and again, no disease that is comparable to human dengue fever.

Clinical studies also have drawbacks. Experimental challenges "pose risks [to participants] that are difficult to define," said Rothman. Studies of natural infections are subject to, among other factors, population differences (e.g., genetics, viral strains, environmental factors), lack of knowledge of previous exposures and immunologic history, a low virus isolation rate in the late phases of illness, and imprecise definitions of disease.

Despite the lack of a good disease model, researchers are making significant headway in understanding dengue virology immunology, and pathogenesis, said Rothman, who presented a detailed overview of current knowledge in these areas. Some key findings of dengue virology include: elucidation of the structure of the E glycoprotein, the major surface glycoprotein of dengue fever; the recent observation that dendritic cells are highly susceptible to dengue in vitro and in vivo, and that DC-SIGN is a key viral receptor; and the notion that dengue virus inhibits interferon signaling, and that this is a key component of the interaction of dengue with the host cell.

Recent immunologic studies have shown that the major targets of the antibody response to dengue are the E glycoprotein and a non-structural protein, NS1. Smaller proteins such as preM protein and the non-structural protein NS3 are also involved, but in ways that are not yet understood.

Antibodies to the preM protein, E protein, and NS1 are cross-reactive across viral serotypes, suggesting that these proteins could be participating in secondary infection, Rothman noted. Antibodies to the E protein may also play a role in disease pathogenesis via enhancement of infection—the hypothesis that antibodies present from a previous dengue infection can bind to the virus but are not able to neutralize it, and may actually increase viral uptake into cells such as monocytes that have immunoglobulin receptors.

CD4 and CD8 responses are also cross-reactive with other serotypes. Thus, in a single individual, said Rothman, "you have some determinants in which the T-cell response links only to the same serotype, and other determinants that include T cells that react with either one or all of the other dengue serotypes." In the examples he showed, several individuals had robust responses to the virus with which they were infected, but also cross-reactive responses to at least one of the other serotypes.

Moreover, "cross-reactivity is not an all or none phenomenon," he explained. "We've observed what we call a partial agonist effect, in which T cells may recognize a particular peptide that differentiates, for example, dengue 2 from dengue 3, but the cells are not able to induce an interferon gamma response or proliferate fully. So again, this shows a great deal of complexity in the response."

Who's at risk for dengue hemorrhagic fever?

Rothman devoted the final section of his talk to reviewing the viral and host factors that seem to increase the risk of developing dengue hemorrhagic fever. He began by emphasizing that fever, headache, and myalgias are all common symptoms of both dengue fever and dengue hemorrhagic fever—they don't distinguish them.

Plasma leakage distinguishes dengue fever from dengue hemorrhagic fever.

"Rash can be present or absent in both dengue fever and dengue hemorrhagic fever," he noted. "Thrombocytopenia is used in the definition of hemorrhagic fever, and so it's almost always severe in these patients; however, some patients who have dengue fever also have severe thrombocytopenia, so this feature alone can't be used to define groups. Bleeding can be present or absent even in dengue hemorrhagic fever, and that's been very difficult for groups new to dengue to take into consideration. Plasma leakage, by contrast, is seen only in dengue hemorrhagic fever and so that is the criterion."

Many research groups have identified different viral and host factors that affect the risk of hemorrhagic fever. One potentially important viral factor is serotype. In a study of Thai children by Vaughn and colleagues, for example, about one-third of those symptomatic with dengue serotype 1 or 3 infection had primary infection whereas symptomatic primary infection with serotypes 2 or 4 was rarely seen.

The genotype of the virus also seems to play a role. Rothman reviewed molecular epidemiologic data on two distinct genotypes of dengue 2 virus: a Southeast Asian genotype and an American genotype. The Asian genotype that has been circulating in Asia since the 1950s was introduced into the Americas in the 1980s, and has been associated with epidemic dengue hemorrhagic fever in many regions. "And yet we now have convincing data that another group of viruses that had been circulating in the Americas in the 1960s through the 1980s, and in Peru in the 1990s, do not seem to be associated with hemorrhagic fever," he said. "They just don't seem to be capable, even when causing secondary infection, of causing the hemorrhagic fever syndrome." Differences in the non-coding regions of the gene and a single amino acid substitution in the E protein may relate to the ability of the Southeast Asian genotype, but not the American genotype, to replicate in dendritic cells, noted Rothman, "and so understanding this is going to be a key component of understanding disease pathogenesis."

Host factors that affect disease severity include genetic associations (for example, a study by Stephens and colleagues of HLA association showed that HLA-A*0207 was overrepresented in patients with severe disease, whereas -A*0203 was overrepresented in patients with mild disease); antibody and T-cell responses, particularly in the presence of previous infection; cytokine production; and levels of viremia.

The bottom line is that there is an interaction among both viral and host factors.

The bottom line is that there is an interaction among both viral and host factors, concluded Rothman. "When viremia is low, the antigen burden is low, and regardless of the T-cell background, cytokine production will be low and the risk of dengue hemorrhagic fever will be low." By contrast, he explained, "when you have high viremia and high antigen burden, we speculate that the outcome will relate to the T-cell profile, and it's only when you get a high TNF response that your risk of dengue hemorrhagic fever is elevated."

Plague: a clinical perspective

Speaker:
Paul Mead, Centers for Disease Control and Prevention, Fort Collins, Colorado

Highlights

• The three main clinical forms of plague are bubonic, septicemic, and pneumonic; the latter is the most lethal.
• Plague is mainly transmitted to humans by bites of infected rodent fleas.
• Inhaling respiratory droplets from infected animals is another source of human disease; the inhalational route can also be exploited for bioterrorist purposes.
• Plague is a serious, but treatable, illness; however, delay in seeking care or misdiagnosis with delayed or incorrect treatment can be fatal.
• Guidelines for the medical and public health management of plague were developed in 2001 to address the availability of the required drugs and the feasibility of mass distribution for treatment.

Pandemics and weaponry

"Plague is a disease of considerable historical importance," said Paul Mead of the Centers for Disease Control and Prevention in Fort Collins, Colorado. The first known natural pandemic, called the "Justinian Plague," began in 451 AD in the Byzantine empire. It killed an estimated 100 million people and up to 40% of the population in some cities.

The second pandemic, known as the "Black Death," began in 1346 AD. Within a few years, it spread throughout Europe, killing an estimated 20-30 million people. The third, or "Modern" pandemic arose in China in 1894, and quickly spread, Mead noted, resulting in 12 million deaths in India alone. "Thanks to the advent of rapid transcontinental transportation in the form of steamships, the third pandemic spread to all inhabited continents, officially arriving in the United States in 1900, in San Francisco. Currently, plague occurs throughout the world, with infection of rodents on every populated continent except Australia," he explained. Today, nearly 80% of reported cases are from Africa, 15% from Asia, and the remaining from the Americas.

Plague occurs throughout the world.

"Much of the current interest in plague, at least in the United States, comes not from its historical importance or global distribution but rather from its potential for use as a biologic weapon either for warfare or for terrorism," Mead continued. During WWII, a unit of the Japanese army reportedly dropped plague-infected fleas over populated areas of China. During the Cold War, both the US and the USSR bioweapons programs included plague, and both tried to develop effective ways of delivering infectious aerosols. The US defensive program was reportedly dismantled in the 1970s, whereas the Soviet program reportedly continued until the 1990s. More recently, said Mead, a microbiologist in Ohio was arrested for fraudulently acquiring Yersinia pestis—the Gram-negative bacillus that causes plague—by mail, presumably for nefarious purposes.

Spreading infection

Plague can be transmitted to humans by several routes, most commonly through the bites of infected rodent fleas, which pose a "serious threat" in some residential areas, said Mead. Humans also can become infected through direct contact with infected animals, including pets. "Cats, in particular, are susceptible to plague and have been the source of several primary pneumonic infections in humans." Lagamorphs (rabbits and hares) are occasionally a source of infection for hunters and others who handle their carcasses.

Inhaling respiratory droplets from plague-infected animals can also cause human disease. But although the inhalational route can also be exploited for purposes of bioterrorism, Mead observed, "it is not clear, to me at least, whether man-made aerosols have the same properties or behave in the same way as naturally generated aerosols. For example, man-made aerosols might travel over greater distances or maintain virulence for longer periods of time than would be expected with natural aerosols."

Three types of plague

The three main types of plague are bubonic, septicemic, and pneumonic. The latter two occur most often as secondary complications of bubonic plague, but may also be primary forms of infection. In the United States, bubonic plague accounts for 80%-85% of primary cases, septicemic for approximately 15%, and pneumonic for 1%-2%. Among patients who present with primary bubonic plague, approximately 20% will develop secondary sepsis and about 10% will develop secondary pneumonia.

Untreated, plague is fatal in over 50% of bubonic cases, and in nearly all cases of septicemic or pneumonic plague. The overall plague case fatality rate in the United States is about 15%. Fatalities are most often due to delay in seeking care or misdiagnosis with delayed or incorrect treatment.

Bubonic plague: tender nodules

The incubation period for bubonic plague is two to six days, occasionally longer. As with other forms of plague, illness begins with fever, headache, chills, muscle weakness, and a feeling of fatigue. At the same time or within 24 hours of symptom onset, the affected individual notices tenderness and pain in inguinal, axillary, or other regional lymph nodes.

Plague patients typically have white blood cell counts of 12,000-25,000 cells per cubic millimeter, but counts may go as high as 50,000, especially in children. The buboes are "exquisitely tender," said Mead, and often remain large and tender for a week or more after treatment has begun. In about 5% of cases, the area around the flea bite may become infected, and the resulting lesion may be confused with those caused by tularemia or anthrax.

White blood cell counts may go as high as 50,000.

Mild forms of bubonic plague, referred to as pestis minor, have been reported in South America and elsewhere. In these cases, the affected individuals typically have milder illness and subacute buboes. "Although the classic description of plague includes profound fatigue and listlessness, we recently saw a man who walked more than 20 kilometers to a local clinic to get treatment," Mead noted.

The differential diagnosis for bubonic plague includes staphylococcal lymphadenitis (infection of the lymph nodes), tularemia, cat scratch disease, mycobacterial infection, chancroid and other sexually transmitted diseases that cause lymphadenitis, and strangulated inguinal hernias. The buboes in plague generally are distinguishable from lymphadenitis and most other causes by their rapid onset, extreme tenderness, and by the absence of cellulitis.

Septicemic plague: overwhelming infection

Septicemic plague is characterized by rapidly progressive, overwhelming bacterial infection, said Mead. Symptoms include fever, chills, physical exhaustion, and abdominal symptoms such as nausea, vomiting, and diarrhea. The course of illness is very rapid; patients develop signs of shock with low blood pressure and small red dots caused by bleeding into the skin.

Difficult-to-treat acute respiratory distress syndrome (ARDS) can occur in septic plague. Thrombi (clots) in the microvasculature, especially in areas such as the tips of the ears and fingers, may cause gangrene of the affected tissues, requiring amputation. The differential diagnosis for septicemic plague includes many other overwhelming systemic infections, including gram-negative sepsis and bacterial endocarditis. In some countries, the disease in its early stages may be confused with typhoid fever or with malaria.

Pneumonic plague: highly virulent

Pneumonic plague is a highly virulent form of plague. Primary pneumonic plague is caused by direct inhalation of infected respiratory droplets or aerosolized bacteria. The incubation period for this form of pneumonic plague is one to seven days, although most cases arise three to five days after exposure. Onset is usually sudden, with chills, fever, headache, body pain, weakness, dizziness, chest discomfort, and gastrointestinal symptoms. Cough, sputum production, increasing chest pain, and difficulty breathing typically predominate on the second day of illness, and may be accompanied by increasing respiratory distress. This is followed by cardiopulmonary insufficiency, cyanosis, and ultimately, circulatory collapse.

A patient with pneumonic plague requires intensive medical and nursing support.

Secondary pneumonic plague is caused by the spread of infection to the lungs from another part of the body, such as a bubo. Secondary pneumonic plague begins as an inflammation of the lungs with little sputum production. However, left untreated, it may progress to severe bronchial pneumonia with bloody sputum. Affected individuals occasionally develop pulmonary abscesses.

A patient with pneumonic plague requires intensive medical and nursing support, Mead emphasized. Death can occur quickly unless the person receives prompt treatment with antimicrobials. Health care providers who have close contact with pneumonic plague patients may be advised to take antibiotics prophylactically, usually doxycycline. Those who have less contact may watch for fever, taking their temperature several times a day for a week and getting immediate antibiotic treatment if they develop a fever.

The differential for pneumonic plague includes other bacterial pneumonias, Legionnaire's disease, streptococcal pneumonia, tularemia pneumonia, hantavirus pulmonary syndrome (particularly in the southwest United States) and acute respiratory syndrome of coronavirus.

Diagnosis and treatment

As with many diseases, a high index of clinical suspicion and careful history and physical exam are required to make a timely diagnosis of plague. As noted earlier, delayed or misdiagnosis is associated with a high case fatality rate. When plague is suspected, specimens should be obtained promptly for microbiologic study, Mead stressed. Appropriate diagnostic assessments include blood, lymph node aspirates in individuals with suspected buboes, sputum samples or tracheal bronchial aspirates in those suspected pneumonic plague, and cerebrospinal fluid in those with signs of meningitis.

Because of recent concerns about bioterrorism, CDC has worked with state and local health departments to develop the Laboratory Response Network, or LRN, a national network with the capability to identify plague, as well as other bioterrorism agents. Laboratory confirmation of plague depends on the isolation of Y. pestis from body fluids or tissues. When the organism can't be recovered, plague cases can still be confirmed by demonstrating a four-fold or greater change in antibodies to Y. pestis F1 antigen.

Recently, antigen capture, polymerase chain reaction assays, and hand-held dip sticks have been developed for rapid and early diagnoses. These are being further evaluated. The hand-held devices, in particular, allow for diagnosis at the bedside even in fairly primitive conditions. Mead's colleagues are currently studying the use of such devices in Madagascar and Uganda.

Treatment of plague is complicated because some drugs are not widely available or FDA-approved.

"The antimicrobial treatment of plague is complicated by the fact that some of the drugs are not widely available and other ones are not FDA-approved," Mead noted. Because of its availability and ease of administration, gentamicin is replacing streptomycin as the treatment of choice for plague patients in the United States. Trials comparing the safety and efficacy of streptomycin with gentamicin in plague have not been completed. However, case reports indicate that gentamicin is an acceptable substitute, although it is not FDA-approved for this indication.

Chloramphenicol is indicated for conditions in which high tissue penetration is important, such as plague meningitis. The drug can be used on its own or in combination with aminoglycosides. Ciprofloxacin has also shown promise in vitro and in laboratory animal studies, but studies demonstrating its utility in human plague have not been widely reported.

Penicillin, cephalosporins, and macrolides have poor efficacy and should not be used, Mead cautioned. Although trimethoprim-sulfamethoxazol has been used successfully to treat bubonic plague, it is not considered a first-line choice, nor is it recommended for severe forms of the disease.

Y. pestis strains showing some degree of antimicrobial resistance have been occasionally isolated from humans, Mead said. However, in most instances they have not been associated with treatment failure.

Mead noted that guidelines for the medical and public health management of a potential bioterrorist attack involving plague have been developed and published in the Journal of the American Medical Association in 2000. The guidelines recommend streptomycin and gentamicin as first-line therapy in contained casualty settings, and oral doxycycline or ciprofloxacin as the preferred agent in mass casualty situations. "The guidelines were driven in part by the belief that pneumonic plague would be the most likely form of plague following an intentional release, and by the need to address the availability of effective drugs and the feasibility of mass distribution for treatment," he concluded.

Lassa Fever: a research perspective

Speaker:
Maria S. Salvato, University of Maryland Biotechnology Institute

Highlights

• Lassa virus is a bisegmented, negative-strand RNA virus with ambisense coding.
• The replication cycle has many targets for antivirals, although ribavirin is the only one presently in use.
• The virus has effects on host chemokines, mostly associated with suppression of innate immunity leading to suppression of acquired immunity.
• The monkey model for pathogenesis using LCMV-WE (lymphocytic choriomeningitis virus WE) has been useful for early diagnosis via transcriptome analysis, and for showing interferon-responsive genes, repair proteins, and translation factors that are affected at the transcription level.
• Vaccine efforts have demonstrated that live vaccines work, but non-infectious vaccines are in the pipeline.

Molecular structure

"Lassa fever virus was first identified as a member of the arenaviruses in the late 1960s," said Maria S. Salvato of the University of Maryland Biotechnology Institute as she laid the groundwork for her presentation on Lassa fever research. "The arenaviruses are named after the Latin word Arenosus, meaning 'sandy,' because of their granular appearance in electron micrographs." Lassa fever virus is closely related to the prototype arenavirus, lymphocytic choriomeningitis virus (LCMV). The benign relative of Lassa virus, Mopeia, is used in many studies comparing virulent and benign viruses.

Besides their granular appearance, the second unique feature of the arenaviruses is that their single-stranded RNA is in two genome segments, L (for large) and S (for small), and the four genes of the arenaviruses are encoded in an ambisense manner, Z [zinc-binding] and GP [glycoprotein] genes in the positive sense and the L and NP [nucleocapsid protein] genes in the negative sense, Salvato continued.

The Lassa fever virus is like other arenaviruses in its structure, with the most abundant molecule being the NP that associates with the viral RNA. It has a bimolecular genome structure, with the NP and polymerase forming the protein part of the ribonucleoprotein particle. The envelope glycoprotein spikes are comprised of membrane-embedded GP2 ionically bound to extracellular GP1. The Z protein is a small zinc-binding protein that functions as a matrix protein.

Viral life cycle and antiviral targets

Studies of the Lassa virus life cycle reveal potential antiviral targets at each step. The currently available treatment, ribavirin, is very effective (See Lassa fever: a clinical perspective). Salvato noted that IV ribavirin administered within three days of disease onset can reduce mortality from 50% to 2%-5%. "However, ribavirin treatments using sterile iv drips are not economical in West Africa."

The first step in the virus life cycle is adsorption to the cell surface. Earlier studies showed that Lassa virus infectivity is inactivated by exposure of virions to acid pH or high salt, which explains why the infectious dose of Lassa virus is so much higher when given orally than intravenously, said Salvato. "Polysulfates such as dextransulfate and heparin have been shown to block viral adsorption for some South American arenaviruses, but these have not yet been explored for Old World arenaviruses such as Lassa."

The next step is receptor-mediated endocytosis. Early work showed that neutralizing antibodies bind the outer portion of the envelope glycoprotein (GP1). Later, an assay was developed to help identify the cellular receptor for arenaviruses. Subsequent studies showed that a soluble protein, a-dystroglycan (a-DG) could block virus entry into cells for certain strains of Lassa virus and LCMV. Although subsequent experiments with an a-DG peptide linked to an Fc receptor failed to block entry of LCMV or Lassa viruses, "this may still be a promising approach if a larger portion of a-DG is used," Salvato said.

Blocking virus entry into cells with an a-DG peptide is a promising approach.

Another way to block virus entry is to use neutralizing antibodies. Convalescent serum from individuals who survived Lassa fever should be useful for this purpose. However, Salvato pointed out that this approach has worked only on rare occasions; its failure has been attributed to low neutralizing titers as a result of the immunosuppressant activities of the Lassa virus. "If a proper reagent were made that could neutralize in high titers, it's possible that this could be a therapy for Lassa fever," she said.

In the second stage of endocytosis, virus particles are surrounded by endocytic vesicles, Salvato continued. A peptide within the GP2 glycoprotein is responsible for viral fusion with endosomal cell membranes, a step that was shown to be sensitive to lysomotropic compounds. These compounds, which include ammonium chloride, chloroquin, and carboxylic ionophores such as monensin work by raising the endosomal pH and preventing endosomal acidification, she explained. "The anesthetic protein procaine and the pinocytotic agent caffeine block this step for some South American viruses, but it's not yet known whether this would work for Lassa virus."

Virus particles are uncoated at the surface of the endosome, ejecting the ribonucleoprotein from the virion and beginning the task of virus transcription. It is not clear what type of antiviral can block the uncoating step. However, Salvato suspects that some of the zinc-reactive compounds that have been shown to block arenavirus replication in cell culture could be working by blocking the function of the viral zinc-binding proteins NP and Z in uncoating.

Several zinc-reactive antivirals also seem to inhibit arenavirus replication. These are thought to act on the zinc-binding regions of the Z protein, inhibiting its function, and there is also a zinc-binding region of NP that might be affected. "The biological consequence of inhibiting Z function is that you may abort virus budding and therefore slow down replication," she explained.

Another important step in the virus life cycle is the translation and maturation cleavage of the viral envelope glycoprotein. "Although there are no inhibitors of cleavage available, the current definition of the cleavage site and the knowledge that cleavage must occur to obtain infectious particles opens the door to rational design of cleavage inhibitors."

Finally, myristic acid analogues can act as antivirals by inhibiting viral assembly and budding, Salvato said.

Lassa fever pathogenesis

Salvato then described several aspects of Lassa fever pathogenesis. Defining the earliest events in the disease can help researchers link those events to outcomes, which could improve diagnosis, she said. Early gene expression changes might also be useful as diagnostics, in that they might identify a disease before its pathogen is detectable.

Studies have shown that in contrast to Ebola, Lassa virus seems to suppress, rather than induce, pro-inflammatory chemokines. In one study, for example, interleukin-8 (IL-8) was produced in uninfected cultures and in cultures infected with Mopeia virus, but was suppressed in cultures infected with Lassa fever virus. The finding, based on this and other studies, "contradicted the view that Lassa fever was a disease caused by raging pro-inflammatory cytokines and promoted the view that the pro-inflammatory response of the host is an early protective mechanism and that its suppression is a pathogenic event," said Salvato.

Lassa suppresses innate immune responses and acquired adaptive immunity.

The finding was corroborated by the work of the Centers for Disease Control team in West Africa looking at Lassa patients who survived or succumbed to fatal Lassa fever. "The fatalities had lower levels of IL-8 and IP-10 (another chemokine) than the survivors so they probably succumbed due to an absence of neutrophils and lymphocytes for systemic defense."

"The view that Lassa suppressed some host cytokine responses evolved to the view that Lassa suppressed innate immune responses that were essential for the development of acquired adaptive immunity," Salvato continued. "This is probably an accurate view of the pathogenesis of Lassa fever. We've shown in the monkey model (macaques infected with LCMV-WE) that surviving animals develop high cell-mediated immune responses earlier than animals that succumb, so an adequate cell-mediated immune response is probably critical for survival.

Genetic studies are also helping to elucidate Lassa fever pathogenesis. Recent experiments suggest that of the 40,000 human genes, roughly 3% are differentially regulated during the course of the disease-approximately 600 in the liver, 446 in the spleen, and 185 in the blood. Eighteen of the genes are regulated in the same way in all three tissues. "We're proceeding to validate these genes by real-time PCR and other types of experiments," Salvato noted.

A gene that is down-regulated by the second day of infection is the proline-rich homeobox (PRH) gene, which is needed for repair of hematopoietic and hepatic systems. This gene expresses a transcription factor that ordinarily resides in the nucleus, but is in the cytoplasm when it is inactive. The effects on this gene by the virulent viral infection are obvious both at the transcription level and at the level of subcellular localization. In benign infection the PRH protein does not change its subcellular distribution, but in the virulent infection the nuclei become depleted of PRH.

Vaccine efforts

Salvato concluded her presentation with an overview of studies by numerous groups involved in Lassa fever vaccine research. Key points included the following:

• Monkeys inoculated with live Mopeia virus (a Lassa fever-related non-pathogenic virus) were fully protected from Lassa virus.
• An inactivated Lassa fever virus induced a high titer of antibodies against viral proteins in vaccinated macaques, but did not protect against a lethal challenge.
• VV NPLFV protected guinea pigs from a challenge by the Lassa fever virus, but failed to protect macaques.
• An attenuated reassortant vaccine (MOP/LAS) protected mice and guinea pigs from Lassa fever challenge. "In a rather remarkable experiment with the guinea pigs, it was shown that this vaccine does not need a great deal of time to develop protection before the lethal challenge," Salvato commented. "It can be delivered on the day of lethal challenge and still manages to protect seven of nine guinea pigs. This indicates that the MOP vaccine is probably eliciting protective innate responses and that innate immunity is an essential component of the protective mechanism."
• A recombinant Salmonella (delivered orally) expressing the NP of Lassa fever virus protected 30% of mice from heterologous lethal intracerebral challenge with LCMV
• Vaccinia expressing the GP genes of the Lassa fever virus protected both guinea pigs and macaques. Both GP1 and GP2 were necessary to induce protection; however, immune protection was not correlated with antibody levels, suggesting that the cell-mediated immune response is critical in protecting against Lassa fever

In summary, the critical point for Lassa vaccines is that innate and cell-mediated immunity are most important for protection, said Salvato. "So far, only live vaccines are protective. But non-infectious vaccines are in the pipeline," she concluded.

Hantaviruses: a research perspective

Speaker:
Erich R. Mackow, Stony Brook University

Highlights

• Hantaviruses are carried by small mammal hosts such as rodents, persistently infecting them, with little or no deleterious effect on the animals.
• Hantaviruses are transmitted to humans by means of aerosolized excreted virus.
• Humans are not viral hosts but are accidentally and transiently infected.
• Hantaviruses, of which there are several pathogenic and non-pathogenic types, cause both hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome; the latter has a 40% mortality rate.
• Hantaviruses primarily infect animal and human endothelial cells, although the cells are not destroyed by the infection; how the cells are altered is under investigation.
• Pathogenic hantaviruses use b3 integrins as cellular receptors, although non-pathogenic ones do not; the importance of b3 integrins in platelet and endothelial cell functions suggests that integrin usage may contribute to pulmonary edema and hemorrhage caused by pathogenic hantaviruses.
• The virus may interact with host cells in an RGD-independent manner, which is a novel means of interaction with b3 integrins.

B3 integrins: key to hantavirus pathogenesis?

Erich R. Mackow's talk focused on his laboratory's efforts to understand hantavirus pathogenesis by analyzing hantavirus interactions with key cellular receptors that regulate vascular permeability, studying the function of hantavirus proteins, and defining cellular responses that contribute to disease. Specifically, the team is investigating the unique use of b3 integrins by pathogenic—but not non-pathogenic-hantaviruses, and defining a means by which such integrin usage dysregulates normal endothelial cell functions.

"The importance of b3 integrins in platelet and endothelial cell functions suggests that integrin usage may contribute to pulmonary edema and hemorrhage caused by pathogenic hantaviruses," Mackow said. "Recently, we demonstrated that endothelial cell migration on b3 but not b1 integrin ligands was blocked by only pathogenic hantaviruses. This defines a means by which hantavirus-integrin interactions selectively alter endothelial cell functions that maintain capillary integrity."

B3 integrins may contribute to the pulmonary edema and hemorrhage caused by hantaviruses

For viruses such as Ebola, this mechanism isn't a concern, Mackow continued: "Ebola lyses endothelial cells, causing bleeding by a completely different mechanism from that of the hantaviruses. Since hantaviruses are not lytic infections, the question is, what is the mechanism by which hemorrhage or vascular leakage may occur? The b3 integrin that pathogenic hantaviruses use, dysregulate, and may direct antibodies to, is the perfect way by which hantavirus might cause disease."

In fact, pathogenic hantaviruses interact with a very small, 53 amino-acid domain present at the apex of the bent conformation of avb3 integrins, Mackow said. This affects b3 integrin function in both platelets and capillaries, setting the stage for vascular disorders, including hemorrhage and edema. By contrast, non-pathogenic hantaviruses—for example, Prospect Hill virus and Tula virus—interact with b1, not b3, integrins.

Moreover, pathogenic hantaviruses interact with b3 integrins in an RGD-independent way. "That's a novel means by which substances interact with b3 integrins, and that has also been part of the problem in looking at how the virus binds. Most substances that bind to b3 integrins are ligands that bind to the extended (not bent) conformation and via RGD binding to the integrin," Mackow explained. "The real key that pulled all the data together is that we showed the interaction was RGD-independent-there was no RGD motif in the virus-and that calcium and manganese work opposite to what activates the integrins. Calcium activates viral infection and manganese inhibits infection. That's counterintuitive to what is normally the state of ligand binding to the integrin."

Another key is the structural data showing that two conformations of the integrins exist: bent (in the presence of calcium) and extended (in the presence of magnesium). "If we didn't have that information, we'd be hard-pressed to explain how the virus is binding," Mackow said. "That revelation is what made this come together and permitted us to test a number of interesting things about the binding of hantaviruses to a protein 53 amino-acid piece N-terminal domain of b3 that we discovered. This structure shows that the b3 integrin is dynamically regulated into two different conformations that are either active or inactive. The inactive bent conformer provides a means by which viral binding interactions can inactivate b3 and maintain the integrin in an inactive ligand binding conformation. Viral binding to the bent integrin may act very much like an antibody would act by binding to that integrin to dysregulate its function."

Next steps

"One possibility is that the 53 amino-acid N-terminus of b3 we've identified, which interacts with hantaviruses, is important not only for hantavirus binding but for other antibody responses that direct hemorrhagic disease. This small domain also provides a viable target for devising a means to block hantavirus infection," Mackow said. "Can we design antibodies that are directed at this protein? Can we develop other ligands that block it, thereby blocking hantavirus infection? This is very interesting for potential therapeutics."

The focus of future work will be on the types of therapeutics that can be used to block hantavirus infection—for example, antibodies that can be directed to the b3-integrin domain and ways of using that domain to identify binding partners for the viral glycoprotein.

In addition, said Mackow, "there are more viruses to look at. We've looked at many of the pathogenic hantaviruses, but not all of them. We need to determine whether the other pathogenic hantaviruses use the b3 integrin and whether blocking hantavirus interactions with b3 integrins inhibits disease in an animal model."

"We have a target, and so we have the potential to define therapeutics."

Work on therapeutics is also progressing. "We've been defining even smaller pieces of the target," Mackow noted. "We have three small targets for use in recombinatorial libraries, and we're setting up screens for drugs that might block binding to b3 polypeptides, and then testing substances that bind for function. First, we'll find out if they bind, and second, we'll see if they block hantavirus infection or disease. We have a target, and so we have the potential to define therapeutics," he concluded.