The Intersection of Infection and Biodefense.

While in medical school at Columbia University, I spent six months in East Africa, working in clinics and studying public health. In that region of the world, infectious diseases have an especially large impact on human health, which influenced me to focus my career on infectious diseases. My initial studies, at The Rockefeller University, centered primarily on the biology and immunology of Legionella pneumophila, the agent of Legionnaires’ disease; early on, my mentor, Samuel Silverstein, and I discovered that L. pneumophila was an intracellular pathogen of human mononuclear phagocytes. Later, after joining the faculty at the University of California, Los Angeles, I turned toward studying tuberculosis (TB), caused by another intracellular pathogen, Mycobacterium tuberculosis. My interest in intracellular pathogens also naturally led me to study Mycobacterium leprae, the agent of leprosy, and later Francisella tularensis, the agent of tularemia. And that is where my work began to link in with biodefense – F. tularensis is considered a Tier 1 Select Agent of bioterrorism. When inhaled, F. tularensis can cause a severe and often fatal pneumonia. I was attracted to F. tularensis primarily because of the opportunity it presented to study an intracellular pathogen with a different intracellular lifecycle from others that I had previously worked on. In the wake of the anthrax attacks in the US in 2001, federal funding for research on bioterrorism agents, including F. tularensis, also increased dramatically making it possible to carry out both basic and applied research on this organism.

We successfully developed vaccines against Legionnaires’ disease, leprosy and TB, including the first TB vaccine that was more potent than the century-old BCG vaccine (1), all based on a new concept that I dubbed the “extracellular protein hypothesis for vaccines against intracellular pathogens.” In essence, my hypothesis held that extracellular proteins were key triggers of an immune system response against intracellular pathogens. The idea was that these proteins, released by pathogens inside infected host cells, would alert the immune system because their epitopes would be displayed on the surface of infected host macrophages as MHC-peptide complexes. Once alerted, T cells would mount an attack against the host cell by activating it with cytokines that would enable the host cell to inhibit the growth of the intracellular pathogen, or by lysing the host cell, depriving the pathogen of its intracellular niche for multiplication. 

The idea for the vaccine was to immunize with the extracellular proteins in such a way as to promote the development of T cells that would later recognize MHC-peptide complexes on the surface of infected host cells. The most abundantly secreted proteins were hypothesized to be the most protective because they would present the richest display of MHC-peptide complexes on the surface of the infected cell. The hypothesis culminated in a vaccine that yielded excellent protection against Legionnaire’s Disease, and subsequently leprosy and TB.

One shot; multiple targets

Based upon the success of the extracellular protein hypothesis, my laboratory applied it to developing a vaccine against tularemia. We developed several vaccines, of which the most potent was an attenuated version of a related pathogen that overexpressed parts of three key extracellular proteins of F. tularensis. 

We started with a previously developed but unlicensed vaccine called Live Vaccine Strain (LVS) that had been derived by the Russians by serial passage of a highly homologous but less pathogenic subspecies of F. tularensis – subsp. holarctica – in the mid part of the last century. We altered this vaccine to derive our vaccine vector.  Using the LVS vaccine as the basis for our vector had a significant practical advantage in that LVS had already been extensively tested in humans and was considered relatively safe. However, LVS still retained significant toxicity, and we, therefore, first rendered it safer by deleting a key gene.  The resultant vector, called LVS ΔcapB, was still highly immunogenic but over 10,000-fold less virulent than LVS.  We used this vector to overexpress parts of highly protective extracellular proteins of F. tularensis. You can read more details about the work in our study (2). 

Ultimately, the vaccine proved to be very safe, highly immunogenic, and highly potent in protecting against respiratory challenge with virulent F. tularensis subsp. tularensis. Once we had a vaccine able to protect well against tularemia, we considered the practical advantage of using the same vector to also express key immunoprotective antigens of other Tier 1 Select Agents. Hence, we engineered the vector to express key antigens of the bacteria that cause anthrax, plague, and later (since the original publication) melioidosis.

A single vector vaccine platform has tremendous practical advantages because it simplifies manufacture, regulatory approval, clinical evaluation, and vaccine administration by allowing for the concurrent administration of individual vaccines from a single vial. One of the major advantages of our vaccine platform is that the vaccine can be easily cultured in very large quantities in vats using conventional growth medium at very low cost. The vaccines directed against each of the various Tier 1 pathogens can be cultured under the same conditions. No purification is needed and no adjuvants are needed, markedly reducing the expense of production. The vaccines have also proven to be highly potent against challenge with highly lethal doses of pathogens by the respiratory route – the route of greatest concern in a bioterrorism attack. 

For several reasons, there are few vaccines against infections caused by intracellular pathogens. Generally, it is easier to develop vaccines targeting extracellular bacteria, viruses and toxins, against which antibodies play the dominant role in host defense. For intracellular pathogens, however, T cell immunity plays the dominant role, and it is more complicated to activate that type of immune response. Of the 23 pathogens/toxins targeted by vaccines available in the US, the agents of only two – TB and typhoid – are intracellular pathogens of mononuclear phagocytes.

Right now, there is considerable interest in medical countermeasures for bioterrorism and, since 2001, key vaccines, particularly for anthrax, have been stockpiled. But in some cases, pathogens that are considered a bioterrorism threat require effective vaccinations anyway. Consider melioidosis as just one example – this disease causes around 165,000 cases and 89,000 deaths annually, mostly in Southeast Asia and Australia. These are diseases that we have a duty to tackle regardless of their potential as bioterrorist weapons.