From extending lifespan to bolstering the immune system, the drug’s effects are only just beginning to be understood.
Successful vaccines have been created to protect against pathogenic bacteria and viruses. Why aren’t there any for combating fungal infections?
June 1, 2011|
When fungal spores touch a moist patch of earth, they germinate and push hair-like hyphae deep into the soil, sucking up enough nutrients to feed the growing cells of the filaments. When a pathogenic fungal spore lands on human tissue under the right conditions, it too germinates and burrows deep into susceptible organs or multiplies like yeast, coating a tissue’s surface as it buds new offspring, colonizing and devouring the tissue beneath it.
Invasive fungal diseases often take hold when a person’s natural defenses are weakened. These infections frequently occur in hospital settings, after a patient’s normal bacterial flora is wiped out by antibiotics, or the skin and gut mucosa are breached by surgery or central venous catheters including for intravenous nutrition. In fact, candidiasis, an infection caused by one of several species of the yeast Candida, is now the fourth most common bloodstream infection in hospitalized patients both in the United States and in many European countries. And the death rate from such Candida infections remains about 30 to 40 percent, even after treatment with antifungal therapy. Given their increasing frequency and unacceptably high morbidity and mortality rates, prevention of invasive fungal infections has become of paramount importance. Vaccination is a promising strategy for prevention, since it has the potential to permanently protect individuals from fungal infection. In the immortal words of Ben Franklin, “An ounce of prevention is worth a pound of cure.”
For years the development of fungal vaccines has lagged behind that of vaccines formulated to attack viruses and bacteria. One barrier has been the widespread belief that most patients who develop life-threatening fungal infections have profound defects in immunity—for example, those whose immune systems have been impaired by cancer chemotherapy. Researchers always assumed that the immune systems of these patients would be too weak to respond vigorously to vaccination, thus limiting the usefulness of a vaccine in the hospital setting.
However, only some 10 to 20 percent of patients who develop bloodstream infection of Candida are seriously immunocompromised. The large majority of patients develop the infection because they become more susceptible while hospitalized, where use of broad spectrum antibiotics (which wipe out bacterial competitors), surgery, and intravenous catheters allow fungi to gain a foothold in tissues. Such patients have relatively intact immune systems and will generate an immune response to vaccination. In addition, there is extensive literature confirming the immunogenicity and efficacy of vaccines even in patients with extremely weakened immune systems—for example, those with active leukemia, HIV infections, or receiving immune-suppressing corticosteroids. Majorities of these groups have been shown to respond adequately to vaccination, if not as robustly as immunocompetent controls.[1. S. Santos et al., “Haemophilus influenzae type b immunization in adults infected with the human immunodeficiency virus,” AIDS Res Hum Retroviruses, 20:493-96, 2004.], [2. T. Nordoy et al., “Cancer patients undergoing chemotherapy show adequate serological response to vaccinations against influenza virus and Streptococcus pneumoniae,” Med Oncol, 19:71-78, 2002.]
In recent years a number of research groups around the world have begun to focus on creating vaccines against some of the most serious and deadly fungal infections. We are closer than ever to bringing a protective vaccine to the clinic, but a number of technical and economic barriers remain to be overcome before the first such vaccine is available for use in humans.
By far the most common culprits in invasive fungal infections are members of the genus Candida. Population-based surveys in the United States have reported that the annual incidence of systemic candidiasis is 60,000–70,000 cases per year.[3. J. Perlroth et al., “Nosocomial fungal infections: epidemiology, diagnosis, and treatment,” Med Mycol, 45:321-46, 2007.] It has been estimated that the health-care cost of treating bloodstream Candida infections is $2–4 billion/year in the US alone.[4. L.S. Wilson et al., “The direct cost and incidence of systemic fungal infections,” Value Health, 5:26-34, 2002.] A vaccine that could prevent or ameliorate these infections would clearly be a major health benefit and of significant value to national health-care systems.
Several recent vaccine approaches have shown promise in animal models. Many of them involve conjugating a fungal surface glycoprotein or polysaccharide—which generally does not activate the immune system well—to a nonfungal protein that is a strong immunogen. One vaccine, developed by Antonio Cassone, at the Istituto Superiore di Sanità in Rome, and colleagues, was protective not only against Candida infections in mice but those of Aspergillus and Cryptococcus—two other common and often fatal fungal infections. The vaccine is made of laminarin, a polysaccharide from a brown alga that is similar to a cell-wall component of many types of fungi, linked to a mutant diphtheria toxin carrier protein that is highly immunogenic but not toxic. In this case, the stimulation produced a strong antibody response that protected mice given an intravenously injected lethal load of fungal cells.[5. C. Bromuro et al., “Beta-glucan-CRM197 conjugates as candidates antifungal vaccines,” Vaccine, 28:2615-23, 2010.] More recently, Cassone established the efficacy of a related vaccine in mice using an oil-in-water adjuvant (MF59), which is acceptable for human use, making the laminarin vaccine a promising candidate for translation to clinical trials.
While the innate immune response can help keep some infections at bay, adaptive immunity controlled by B and T cells is necessary for lasting immunity. Vaccines are therefore generally designed to activate adaptive immunity against a pathogen, creating memory T and B cells that will rapidly and strongly respond when they encounter the pathogen a second time. While inflammation-inducing T cells (part of the Type 1 T-helper, or Th1, response) and antibody-producing B cells (activated by the Th2 response) can both be important in clearing a pathogenic infection, vaccines sometime stimulate only one of these types of adaptive immunity.
Although laminarin appears to activate B-cell mediated immunity, other fungal vaccines being developed activate cellular immunity as well. For example, a vaccine similar to Cassone’s employed the candidal surface polysaccharide mannan, conjugated to human serum albumin (HSA) to elicit a greater immune response than mannan alone. In rabbits, the mannan-HSA vaccine generated both antibodies and specific T cells. Other anticandidal vaccines in animal studies have focused on immunizing using heat-shock proteins derived from the Candida cell wall and surface, a method which also produced both antibodies and cell-mediated inflammatory responses. Our group demonstrated that the candidal cell wall protein HYR1 helps Candida escape phagocytosis by immune cells, and could itself serve as a vaccine target, resulting in impressive protection in a systemic infection model.[6. G. Luo et al., “Candida albicans Hyr1p confers resistance to neutrophil killing and is a potential vaccine target,” J Infect Dis, 201:1718-28, 2010.] In addition, it appears that the eukaryotic-cell model system, the yeast Saccharomyces cerevisiae, can act as a vaccine against many fungi after heat inactivation (effective against Candida, Aspergillus, or Coccidioides), due to its expression of carbohydrates shared across many fungal species.
With its entry into Phase I clinical trials, the Candida vaccine furthest along the development pathway is based on the agglutinin-like sequence (Als) family of proteins expressed on the surface of Candida albicans. The vaccine, developed in our lab, is made from the recombinant N-termini of the candidal agglutinin adhesion molecules Als1p or Als3p (rAls1p-N or rAls3p-N). Injection protected mice from otherwise lethal widespread candidiasis, and also reduced fungal burden in a model of vaginal infection and a steroid-treated—and thus immunocompromised—oral candidiasis model.[7. L. Lin et al., “Th1-Th17 cells mediate protective adaptive immunity against Staphylococcus aureus and Candida albicans infection in mice,” PLoS Pathog, 5(12):e1000703, 2009.]
The vaccine targeting Als proteins activates Th1 and Th17 CD4+ T helper cells, which then recruit and activate phagocytic cells that engulf and destroy the fungus in tissues.7 This vaccine did not require a Th2 response, characterized by activation of B cells and their subsequent production of antibodies, in order to be effective.
These results elucidate several critical concepts regarding vaccinations against fungal pathogens: 1) Vaccine efficacy against fungal pathogens likely requires enhancement of phagocytic host-defense mechanisms, whether by antibody-mediated or nonantibody-mediated methods; 2) Vaccine-responsive T cells can provide direct enhancement of phagocytic defense mechanisms in the absence of protective antibodies; and 3) Contrary to widely held assumption, it is not necessary to develop antibodies that neutralize virulence factors—the disease-causing proteins or toxins expressed by the pathogen—in order to achieve protection with a vaccine. These findings suggest that there may be a variety of antigens that will serve as excellent vaccine candidates even though they are not “virulence factors” for fungal pathogens.
The success of the Als vaccine when combined with an aluminum-based adjuvant was a critical milestone for this vaccine’s development, as aluminum derivatives have been widely used in US Food and Drug Administration (FDA)-approved vaccines. Hence, a dosing schedule, route of administration, and adjuvant have now been identified for rAls3p-N, which helped support the granting of Investigational New Drug (IND) status enabling clinical testing to begin in 2011.
Aspergillus is the second most common cause of hospital-acquired invasive fungal infections, with an incidence of approximately five per 100,000 in the US.4 The infection usually takes hold in the lungs, and can cause invasive pneumonia in some individuals, but it can spread to other parts of the body, especially when immune defenses are compromised. Both the advantages of and the barriers to developing a vaccine against aspergillosis are magnified when compared with invasive candidiasis. Despite the use of antifungal therapy, the mortality rate is extremely high—between 45 and 80 percent—underscoring the failure of current therapies.3 However, a particular barrier for development of such a vaccine is that virtually all patients with invasive aspergillosis are highly immunocompromised, which makes development of a vaccine for these infections particularly challenging.
The risk factors for aspergillosis are well understood. They include the severe depletion of white blood cell levels from cancer chemotherapy, leukemia, or bone-marrow transplantation, as well as the necessity for high doses of corticosteroids or other immunosuppressants in patients receiving organ transplants or with severe rheumatic or other autoimmune diseases. On the other hand, the infection tends to occur after multiple weeks in at-risk situations, suggesting that clinicians could vaccinate well before infection sets in.
In 2002, Luigina Romani at the University of Perugia, in Italy, and colleagues found that recombinant protein antigens from Aspergillus induced a Th1, cell-mediated immune response that protected mice against invasive aspergillosis. The mice received an intranasal administration of the allergen Asp f 16, delivered together with CpG oligonucleotides—an adjuvant that promotes a Th1-type response. The pretreated mice, whose immune systems were compromised using cancer chemotherapy, showed improved survival when they were subsequently infected with A. fumigatus via inhalation.[8. S. Bozza et al., “Vaccination of mice against invasive aspergillosis with recombinant Aspergillus proteins and CpG oligodeoxynucleotides as adjuvants,” Microbes and Infection, 4:1281-90, 2002.] The same research group also tested a dendritic-cell vaccine approach. Dendritic cells are crucial to the natural antifungal response, as they activate both innate immunity, mediated by phagocytic cells, and the adaptive immune response, mediated by T cells and antibody-producing B cells. When Romani’s team cultured dendritic cells with Aspergillus or with fungal RNA in a test tube and then added lymphocytes to the mix, they noticed lymphocyte activation and release of cytokines associated with the Th1, or cellular, immune response. These primed dendritic cells could confer antifungal protection once reinjected into the mouse. Such an adoptive-transfer vaccination method could be extremely useful in bone-marrow transplant recipients. Dendritic cells could be pulsed with the vaccine antigen and administered along with bone marrow, to reduce the risk of aspergillosis in this highly susceptible population.[9. S. Bozza et al., “A dendritic cell vaccine against invasive aspergillosis in allogeneic hematopoietic transplantation,” Blood, 102:3807-14, 2003.]
Although experimental vaccines using crude antigen extracts prepared from killed A. fumigatus were also effective in protecting mice from infection, it would be impossible to produce such mixtures according to good manufacturing practices (GMP), which require stringent consistency and reproducibility between batches. Along these lines, Markus Kalkum and James Ito, at City of Hope’s Beckman Research Institute in Duarte, California, and colleagues determined that the active component of their previously published Aspergillus crude extract was the fungal surface antigen Asp f 3.[10. J.I. Ito et al., “Vaccinations with recombinant variants of Aspergillus fumigatus allergen Asp f 3 protect mice against invasive aspergillosis,” Infect Immun, 74:5075-84, 2006.] Vaccination with recombinant Asp f 3 protected mice from a lethal inhaled challenge with A. fumigatus. While protection required the use of TiterMax adjuvant, which is too toxic for use in humans, the investigators also demonstrated that a protein-precipitate form of the vaccine, administered as a suspension in methylcellulose carrier, was also protective. Hence, Kalkum and colleagues have identified a potential practical vaccine that could be made GMP-compliant using a carrier agent that is safe in humans.
Cryptococcus causes life-threatening infections in patients with substantially compromised T-cell-mediated immunity resulting from HIV infection, congenital causes, or the use of immune-suppressing corticosteroids for transplantation, arthritis, or other conditions. Estimates of the prevalence of invasive Cryptococcus place this fungal infection third, behind Candida and Aspergillus.4 As with aspergillosis, a vaccine against cryptococcal infection must be effective in patients who have substantial immune deficiencies.
Cryptococcus is covered with a capsule carbohydrate called glucuronoxylomannan (GXM), which is a known virulence factor that suppresses the host inflammatory response and prevents antibody-mediated phagocytosis of the fungus. In fact, natural infection may induce the immune system to produce nonprotective antibodies.[11. O. Zaragoza, A. Casadevall, “Antibodies produced in response to Cryptococcus neoformans pulmonary infection in mice have characteristics of nonprotective antibodies,” Infect Immun, 72:4271-74, 2004.] However, several antigens have been found to induce protective immunity against cryptococcal infection in mice, including the laminarin vaccine developed in the Cassone laboratory, which cross-reacts with a number of different fungal genera. Although the vaccine wasn’t tested in T-cell-deficient or steroid-treated mice, it appeared to induce specific antibodies against Cryptococcus and reduced fungal burden in healthy mice as well as those lacking white blood cells. In addition, vaccinating with a synthetic peptide mimic of the GXM virulence factor as well as other mixtures of surface antigens shows tentative promise.
An important result of much of the antifungal vaccine work that has occurred over the past decade is the realization that fungal sugars, such as mannan, when oxidatively coupled to protein antigens, can act as an adjuvant for vaccines by helping to stimulate potent immune responses. Stuart Levitz, at the University of Massachusetts Medical School, and colleagues have conducted much of the seminal work in this area. They grew fungal proteins in bacteria, which do not add mannose to the proteins, or in yeast, which do cover proteins in mannose.[13. C.A. Specht et al., “Contribution of glycosylation to T cell responses stimulated by recombinant Cryptococcus neoformans mannoprotein,” J Infect Dis, 196:796-800, 2007.] Only the sugar-coated mannoproteins produced in yeast generated a strong pro-inflammatory T-cell-mediated immune response to the protein; the protein grown in bacteria generated a much weaker immune response. The addition of the sugar units to the protein served to boost the immune response to that protein. In another study, the same group found that addition of mannose groups to the protein stimulated not only T-cell proliferation, but also the secretion of pro-inflammatory cytokines such as TNF and IL-12. Collectively, these results demonstrate the fundamental potential of incorporating mannosylation into vaccine protein design, either by growing the proteins in yeast, or, if E. coli is used to grow the vaccine proteins, by conjugating fungal mannans in order to boost immunity and protection.
Given the aging global and US populations, and the increasingly intensive medical treatment of critical illnesses, the incidence of invasive fungal infections will continue to rise over the coming decades. Due to enhanced understanding of the host defenses and pathogenic mechanisms that underlie invasive fungal infections, we are now in a position to begin developing such vaccines. The concept of niche vaccination of acutely at-risk patients, or patients in restricted geographical areas, is a new idea in vaccinology. Furthermore, the novel immunological concept of stimulating Th1 or Th17 responses, instead of relying on antibody-based responses, opens new avenues to explore in vaccinology against such infections.
The lack of complete understanding of the market potential for such vaccines has created significant impediments to the availability of the capital to develop such vaccines. Continued education about the economic importance of vaccines for invasive fungal infections, combined with the development of well-defined antigens and effective adjuvants with a track record of safety, should enable these vaccines to enter clinical testing in the coming decade.
The costs of preparation for an Investigational New Drug (IND) application supporting the Phase I trial are significant, and include several million dollars required to develop GMP-compliant manufacturing, as well as additional costs for preclinical toxicity studies using GMP-compliant material. This represents a major barrier to development of vaccines for invasive fungal infections in general, and an even greater barrier for infections caused by organisms other than Candida, which have smaller perceived markets to drive the investment of capital.
Brad Spellberg is at the Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center. He is a cofounder of NovaDigm Therapeutics, which is developing candidal vaccine technology.
This article is adapted from an upcoming review in F1000 Medicine Reports. It will be available for citation at f1000.com/reports (open access).