NDV-3: The First Cross-Kingdom Vaccine for Candida albicans and Staphylococcus aureus

By: Jenny Vitcak

Figure 1. Image of a patient afflicted
with oral candidiasis. 

             Fungal pathogens are an increasing problem in healthcare due to a rising number of immunocompromised individuals as well as an increasing resistance to antifungal drugs (1).  One of these opportunistic fungal pathogens is Candida albicans. In the United States alone, 60,000 cases of bloodstream Candida infections are reported yearly, which is a 15 to 20-fold increase compared to the 1990s (2). This fungus is able to cause superficial oral and vaginal infections in healthy individuals as well as systemic infections in immunocompromised patients (3). To demonstrate the scope of these infections, 70% of all women will experience at least one episode of vaginitis caused by Candida within their lifetime while 70% of patients with AIDS will experience oropharyngeal candidiasis (4).

Figure 2. Yeast (round) and hyphal
(filamentous) forms of C. albicans.    

             Despite this high occurrence of infection, C. albicans is generally non-pathogenic. It is a normal microbe found at many different tissue sites, including the oral cavity, vagina, and on the skin. However, it is capable of causing disease in both healthy patients and those with a compromised immune system when it turns from its yeast form into filamentous hyphal cells. In addition to this morphogenesis, the fungus also requires adhesion to the host tissue for infection (4). The genome for C. albicans contains an eight-member family of proteins called ALS. The proteins made from these genes function as adhesins, which allows the cell that expresses them to adhere to a variety of surfaces. Als3 is a member of the ALS family and this specific protein is expressed on the cell surface during hyphal development, which helps the fungus adhere to epithelial cells (3, 5). Once the pathogen is stuck to host tissue, Als3 functions as an invasion. This function causes the host cell to produce protrusions around the hyphal cell and subsequently engulfs it, resulting in infection (6).
             Researchers have been trying to develop a vaccine that combats Candida infections since it is the third-leading cause of hospital-acquired infections in the United States (7). The ALS family seemed like a good place to start, considering that ALS1 and ALS3 are highly expressed in clinical specimens (8). In 2005, a preclinical trial was performed using a vaccine for Als1 that showed significant improvement in survival for both healthy and immunocompromised mice after infection with C. albicans (9).  Despite these promising results, more work went into developing a vaccine that targeted Als3 since this protein shows greater adherence to cells compared to Als1 (10).
             In 2005, another preclinical trial was performed on mice that compared the Als3 vaccine to the Als1 vaccine. Both of these developmental vaccines were made using the N-terminal portion of the protein (7). This is the part of the protein that is furthest away from the cell membrane of C. albicans and comes in contact with immune cells. This contact allows the immune cell to become primed to a specific protein (known as an antigen) on the pathogen, which allows the cell to remember it for future infections. Mice were given either the Als1 or the Als3 vaccine and infected with C. albicans directly in the bloodstream, orally, or vaginally.  Overall, mice who received either vaccination had a 40% higher survival rate compared to mice who did not receive any treatment. Orally or vaginally infected mice showed a lower number of lesions caused by C. albicans when they were given the Als3 vaccine compared to the Als1 vaccine (7). These results suggest that the Als3 vaccine offers more protection against oropharyngeal and vaginal candidiasis; therefore, research continued with Als3 as a vaccine candidate.

Figure 3. Patient afflicted with a MRSA
infection caused by S. aureus in a burn wound.    

             In 2008, the Als3 vaccine demonstrated cross-kingdom protection against both fungal and bacterial pathogens (11). The idea that this vaccine could provide protection against two different types of organisms came through the discovery that both Als1 and Als3 have structural similarity to clumping factor (ClfA) in Staphylococcus aureus. ClfA is a protein that is expressed on the bacterium’s surface and helps the organism with adherence (10), which is a similar function of the Als family in C. albicans. Based upon this finding, mice were immunized with the N-terminal portion of Als3 and exposed to S. aureus two weeks later. 60% of the mice that received the vaccine were alive after 28 days compared with 5% of the mice who did not receive the drug (11).
             This study also determined that T cells are the portion of the immune system that are primed to recognize Als3 (11). Once a T cell is able to recognize a specific antigen, it will remember what that antigen looks like to help destroy it when another infection occurs. B cells also play a similar role in immunity, except they produce antibodies that recognize and kill the pathogen. The researchers determined which immune cell type was responsible for protection by infecting mice that were deficient in either T cells or B cells with S. aureus. Mice that lacked T cells perished quickly after infection with S. aureus, while mice without B cells had similar survival rates compared to mice with a fully functional immune system (11).
             The promising results from the preclinical trials using Als3 led to the first in-human trial in 2012 that used a vaccine called NDV-3.  The vaccine was developed using the N terminal portion of Als3. The Phase I clinical trial was a double-blind, placebo-controlled study performed on healthy individuals age 19-47 from a single study site.  The study looked at the safety and ability to produce an immune response for both a low dose and high dose of the vaccine.  Volunteers were given the low dose vaccine, high dose vaccine or saline control.  Participants who were receiveing the vaccine were also given a booster shot after three months and were followed for a total of nine months (2).
             After vaccination, all participants had a rapid rise in antibody levels, which suggests that only one dose would need to be administered rather than a vaccine with a booster (2). This rise in antibody levels, which come from B cell activity, is a different result than was seen in the preclinical trials with mice. The mice did not have any benefit from B cell activity (11) whereas humans do (2). With regards to T cell activation, robust activity was seen in both treatment groups with the higher dose having a more rapid response. Since this is a Phase I clinical trial, safety of the drug was monitored. NDV-3 was shown to be well tolerated in healthy adults with only mild or moderate adverse events at both doses. The most common adverse event was pain at the injection site that resolved within one to two days (2). A second Phase I trial was conducted in May 2012 using three different vaccine formulations and two routes of administration. Once again, NDV-3 was safe, well-tolerated and produced a robust immune response (Results not published) (12).
             Based upon the experiments performed in mice using an Als3 vaccine as well as the Phase I clinical trial, NDV-3 looks promising. The results have shown a highly immunogenic response as well as increased survival in mice after infection with either C. albicans or S. aureus. It has also proved to be safe in humans with little side effects. As of September 2013, NovaDigm Therapeutics announced the beginning of a Phase Ib/IIa clinical trial for NDV-3. This trial will specifically look at preventing vaginal candidiasis in patients with recurrent vaginal infections (13). Hopefully, this trial will have successful results and research can continue with this vaccine to help the ever growing number of people who are afflicted with either candidiasis or MRSA skin infections.

References

1.     Morschhauser, J. (2010). Regulation of multidrug resistance in pathogenic fungi. Fungal Genetics and Biology 47, 94-106.

2.     Schmidt, C.S., White, C.J., Ibrahim, A.S., Filler, S.G., Fu, Y.,  Yeaman, M.R., Edwards, J.E. Jr., & Hennessey, J.P. Jr. (2012). NDV-3, a recombinant alum-adjuvanted vaccine for Candida and Staphylococcus aureus, is safe and immunogenic in healthy adults. Vaccine 30(52), 7594-7600.

3.     Argimόn, S., Wishart, J.A., Leng, R., Macaskill, S., Mavor, A., Alexandris, T., Nicholls, S., Knight, A.W., Enjalbert, B., Walmsley, R., Odds, F.C., Gow, N.A.R., & Brown, A.J.P. (2007). Developmental regulation of an adhesion gene during cellular morphogenesis in the fungal pathogen Candida albicans. Eukaryotic Cell 6(4), 682-692.

4.     Calderone, R.A., & Fonzi, W.A. (2001). Virulence factors of Candida albicans. TRENDS in Microbiology 9(7), 327-335.

5.     Liu, Y. & Filler, S.G. (2011). Candida albicans Als3, a multifunctional adhesion and invasion. Eukaryotic Cell 10(2), 168-173.

6.     Rotrosen, D., Edwards, J.E. Jr., Gibson, T.R., Moore, J.C., Cohen, A.H., & Green, I. (1985). Adherence of Candida to cultured vascular endothelial cells: mechanisms of attachment and endothelial cell penetration. J. Infect Dis. 152(6), 1264-1274.

7.     Spellberg, B.J., Ibrahim, A.S., Avanesian, V., Fu, Y., Myers, C., Phan, Q.T., Filler, S.G., Yeaman, M.R., & Edwards, J.E. Jr. (2006). Efficacy of the anti-Candida rAls3p-N or rAls1p-N vaccines against disseminated and mucosal candidiasis. JID 194, 256-260.

8.     Cheng, G., Wozniak, K., Wallig, M.A., Fidel, P.L., Trupin, S.R., & Hoyer, L.L. (2005). Comparison between Candida albicans agglutinin-like sequence gene expression patterns in human clinical specimens and models of vaginal candidiasis. Infect. Immun. 73, 1656-1663.

9.     Ibrahim, A.S., Spellberg, B.J., Avenissian, V., Fu, Y., Filler, S.G., & Edwards, J.E. Jr. (2005). Vaccination with recombinant N-terminal domain of Als1p improves survival during murine disseminated candidiasis by enhancing cell-mediated, not humoral, immunity. Infect Immun 73, 999-1005.

10.  Sheppard, D.C., Yeaman, M.R., Welch, W.H., Phan, Q.T., Fu, Y., Ibrahim, A.S., Filler, S.G., Zhang, M., Waring, A.J., & Edwards, J.E. Jr. (2004). Functional and structural diversity in the Als protein family of Candida albicans. J Biol Chem 279, 30480-30489.

11. Spellberg, B., Ibrahim, A.S., Yeaman, M.R., Lin, L., Fu, Y., Avanesian, V., Bayer, A.S., Filler, S.G., Lipke, P., Otoo, H., & Edwards J.E. Jr. (2008). The antifungal vaccine derived from the recombinant N terminus of Als3p protects mice against the bacterium Staphylococcus aureus. Infection and Immunity 76(10), 4574-4580.

12. Business Wire (2012). Results from positive Phase I study of NovaDigm Therapeutics’ NDV-3 vaccine for Candida and Staph infections published in Vaccine. Business Wire. Retrieved from http://www.businesswire.com/news/home/20121213005244/en/Results-Positive-Phase-1-Study-NovaDigm-Therapeutics%E2%80%99.

13. Business Wire (2013). NovaDigm Therapeutics initiates Phase 1b/2a clinical trial with NDV-3 vaccine in recurrent vulvovaginal candidiasis (RVVC). Business Wire. Retrieved from http://www.businesswire.com/news/home/20130926005382/en/NovaDigm-Therapeutics-Initiates-Phase-1b2a-Clinical-Trial.

Iron Wars: Hand-to-Hand Combat for Vital Nutrients

            You have likely inhaled several fungal spores while reading this sentence. Fungi are ubiquitous members of our environments, present almost everywhere humans live. In order to reproduce, these organisms create airborne spores that can disperse and spread. Luckily, inhalation of these fungal spores typically has no effect on the person that inhales them. However, in the case of an immunocompromised person, fungi can cause harmful infections in people (1). Aspergillus fumigatus is an example of such a fungus, a common organism found in close association with humans. This fungus can cause a disease, known as aspergillosis, in immunocompromised individuals, when the fungus infects various parts of the body (1). However, in rare cases, a healthy person can be infected with Aspergillus in the cornea of the eye. This disease, called fungal keratitis, causes eye pain, blurred vision, and clouding of the cornea. If left untreated, this infection can lead to severe impairment of vision or even blindness (2).

 

Fungal keratitis in human cornea

            When Aspergillus infects another organism, like a human or another susceptible mammal, it needs to find all the food and nutrients it needs from sources inside that organism’s body. Like most other organisms, Aspergillus requires iron in order to survive and since other organisms need iron as well, the fungus can usually find a source of this iron in the host organism’s blood and fluids. Here, iron can exist in a free-floating soluble form or attached to proteins that transport or store the molecule. During an Apsergillus infection, since both the fungus and the host need iron, the two organisms enter into a heated battle for the iron present in the host’s body. Both sides employ different weapons and tactics in order to win over the iron source and use it for themselves.

            To pick up iron from its environment, Aspergillus uses a molecule called a siderophore. On the outside of the cell, these structures act like iron magnets for the fungus, attracting and binding soluble iron in the fungus’ surroundings. On the inside of the cell, siderophores help store iron for the cells to use later (3). This action makes extracellular siderophores in particular very important when iron is low in the surroundings. Without the ability to bind iron and bring it in to the cell, Aspergillus becomes less virulent (3).
           Humans have their own iron-carrying and iron-storing molecules to do battle with, called transferrin and ferritin, respectively (4). However, the iron-binding strength of the siderophore gives the Aspergillus an advantage in hand-to-hand combat for iron. This is demonstrated in mice infected with Aspergillus, when levels of iron in the mouse blood decreased when the fungus was introduced. Additionally, when iron is added to the mouse’s blood, the severity of the Aspergillus infection increased, suggesting that the fungus thrived on this increase in iron (5). With the fungus stealing iron so efficiently, the human body needs to find a way to level the playing field, and it does so by targeting siderophores.

            Lipocalin-1 is a molecule produced by human cells that can bind fungal siderophores. Blocking the siderophores with this molecule renders them useless in iron binding, impairing Aspergillus’ ability to take up iron. This effect was shown in mice that were treated with human Lipocalin-1 during Aspergillus infection. Mice that received topical treatment of the human produced molecule had a decrease in the amount of fungus that grew in their cornea (5). This suggest that Lipocalin-1 is an effective weapon in combating siderophore use by Aspergillus during infection.

Some interactions between siderophores, iron,and host cell molecules

            As a tactical fighter, Aspergillus needs ways to deploy its siderophores only when it needs them. When iron is scarce in the environment, the fungus wants to produce an abundance of siderophores to give itself every advantage in trapping what little iron there is. However, when iron is common and the fungus doesn’t need to waste its energy trying to find iron, Aspergillus makes fewer siderophores. In addition, too much iron uptake can be toxic to a cell, so it is important to regulate this process.To do this, the fungus utilizes a system to sense the iron in the environment and respond to those levels. For Aspergillus, this system hinges on a transcriptional regulator called SreA. This gene set is activated by high iron levels in the surroundings and represses the uptake and storage of iron by the fungus. When SreA is deleted, iron levels in the cell build up under high-iron conditions, creating harmful circumstances for the fungus (3). SreA presents a potential target for warfare against Aspergillus; inactivating SreA could turn this fungus’ own weapons against it and make iron acquisition toxic for fungal cells.

            Aspergillus also needs specific equipment to build its siderophores. The biosynthetic pathway that makes extracellular siderophores in this fungus incorporates the molecule mevalonate at a crucial step in the process. Without the inclusion of this molecule in its siderophores, the fungus seems to be unable to carry out infection. This was demonstrated by testing in mice. Deleting genes which encode the enzymes that incorporate mevalonate into siderophores resulted in an Aspergillus strain that does not infect the corneas of mice, suggesting that mevalonate plays a crucial role in the virulence of this fungus (5).

            Realizing the importance of siderophores and their biosynthesis as factors in Aspergillus infection can help identify treatments for these infections. Humans naturally produce one compound, Lipocalin-1, which targets siderophores and helps fight infection. Other compounds with similar actions can also be used to target siderophores and treat Aspergillus infections. Perhaps the most powerful weapons at our disposal are statins and iron chelator compounds. Statins work to block the enzymes that incorporate mevalonate into extracellular siderophores. When these compounds are applied topically to mice that are infected with Aspergillus, the severity of the infection is significantly decreased (5). Iron chelators act in a similar fashion to siderophores, binding iron with high affinity and sequestering it. These compounds can compete with fungal siderophores for iron and prevent the siderophores from binding it. Topical treatment with iron chelator compounds in Aspergillus infected mice also decreased the severity of these infections. Furthermore, when these two treatments were combined, they seemed to have additive effects, decreasing the severity of Aspergillus infection more so than either did separately (5).

            Understanding the balance of iron use between the mammalian host and an infecting fungus gives crucial information needed to develop treatments for these infections. Identifying how fungi use and regulate siderophores during infection presents clear targets for the next strike against these invaders. This war tactic is not limited solely to Aspergillus either, but can have effects in fighting related adversaries such as Fusarium oxysporum, a common plant pathogen (plos). With this inside information, we can stage an attack to win the war against these fungal pathogens, starting with the battle for iron.

References 

1.       "Aspergillosis." Centers for Disease Control and Prevention. Centers for Disease Control and Prevention, 09 Jan. 2012. Web. 02 Dec. 2013

2.       “Fungal Keratitis.”Centers for Disease Control and Prevention. Centers for Disease Control and Prevention, 09 Jan. 2012. Web. 02 Dec. 2013 

3.       Schrettl, M., Kim, H. S., Eisendle, M., Kragl, C., Nierman, W. C., Heinekamp, T., Werner, E. R., Jacobsen, I., Illmer, P., Yi, H., Brakhage, A. A. and Haas, H. (2008), SreA-mediated iron regulation in Aspergillus fumigatus. Molecular Microbiology, 70: 27–43

4.       Hentze MW, Muckenthaler MU, Galy B, Camaschella C (2010) Two to tango: regulation of Mammalian Iron Metabolism. Cell 142: 24-38 

5.       Leal SM Jr, Roy S, Vareechon C, Carrion SdJ, Clark H, et al. (2013) Targeting Iron Acquisition Blocks Infection with the Fungal Pathogens Aspergillus fumigatus and Fusarium oxysporum. PLoS Pathog 9(7): e1003436. doi:10.1371/journal.ppat.1003436