By: Suzanne Canada, Ph.D.
Tanager Medical Writing

Concerns over outbreaks of potentially serious childhood diseases are in the news again in California with an increase in whooping cough infections in 2014, caused by the pathogen Bordetella pertussis, and a measles outbreak in December 2014 to February 2015.  Since outbreaks and epidemics may be infrequent, many of us do not realize that vaccine development is an ongoing process. Public health experts monitor changes in the predominant strains and pathogen virulence continuously.  Although the sudden upswing in cases is alarming for those with young children, fuller understanding of the cyclic nature of these outbreaks fosters refinements to public health practices as well as the vaccines.

Recent increases in the number of cases of pertussis infections are due to eroding immunity in the population of immunized individuals, as well as changes in the prevalent virulent strains.  Outbreaks of new strains or new agents occur every 6 months or so [1]. For example, every 4 or 5 years, a new increase in pertussis infections is observed [2]. However, development of safe and effective vaccines is a process that takes up to 15 years [1].  Therefore, health agency efforts must be ongoing and constant funding must be in place to have these vaccines ready when the public needs them.

Although we are observing outbreaks of whooping cough and measles in the past year, the rates of infection are still much lower than what was observed in the pre-vaccine era.  In the pre-vaccine era (prior to the 1940s in the USA), 157 serious pertussis infections occurred yearly per 100,000 cases, including ~15 infant deaths/100,000 cases [2].  The highest incidence recorded was 260,000 cases in 1934 [3]. Since the broad adoption of vaccinations, whooping cough is much rarer during most years; upswings were observed in California in 2010 and again in 2014.  In 2010, the most serious cases were seen among infants under one year old.  The experts then realized that they could best control the rate of serious infections in infants by immunizing mothers at G27-36 weeks or immediately after the birth [4], and by making sure everyone in the expectant family was also immunized.  Furthermore, the 2014 outbreak of whooping cough was highest among 15 year olds (137.8 cases/100,000) [2].  By gathering immunization records and analysis of blood samples for anti-pertussis antibodies among those who had an infection, the doctors deduced that eroding resistance to pertussis corresponded with the use of the acellular pertussis vaccine.

The acellular pertussis vaccine was developed in the 1990’s in response to a high rate of side effects (fever) in those receiving the whole cell vaccine.  The acellular vaccine uses a few especially prominent antigens (pertussis toxoid, filamentous heamagglutinin, and pertactin, among others) that are specific to all pertussis bacteria. In the late 1990s, the FDA recommended replacement of the whole cell vaccine with the acellular vaccine which doesn’t cause as much fever and discomfort following vaccination boosters.

Investigations of why the acellular pertussis vaccine is not conferring resistance as durable as others, such as the tetanus or diphtheria vaccines, are ongoing.  Some vaccine experts point to evidence that resistance to whooping cough isn’t as durable with the acellular vaccine [5, 6]; however, other analyses conclude that the genes encoding antigens targeted by acellular pertussis vaccines are changing at higher rates than other surface-protein encoding genes of the pathogen [7, 8].  A meta-analysis of different immunization schedules found that resistance depended on time since last immunization or exposure [9], with resistance dipping by 5 years after the last booster shot.  Some researchers have found that having more anti-pertussis antigens in the vaccine conferred a higher level of protection [10], and new antigens for the vaccine are under evaluation (e.g., LpxL 1 [11]).

Several virulence factors of B. pertussis are available for research purposes from List Labs including pertussis toxin (Products #179, #180 and #181), pertactin (Product #187), fimbriae 2/3 (Product #186), adenylate cyclase toxin (Product #188 and 188L),  filamentous heamagglutinin (#170) and lipopolysaccharide (Product #400). Inactivated toxins, known as toxoids, are frequently used in the vaccines.  List Labs offers toxoids of C. difficile toxins (Products #153 and #154), diphtheria toxin (Product #151), Staphylococcus aureus enterotoxin B (Product #123), tetanus toxin (Product #191) and botulinum neurotoxin types A and B (Product #133 and #139, respectively).  List Labs’ vaccine carrier proteins are provided for research use only; however, GMP material may be produced on a contract basis.  More information is available on our website: www.listlabs.com.

 

References

1. Rappuoli, R., Vaccines, Emerging Viruses, and How to Avoid Disaster. BMC Biology, 2014. 12: p. 100. PMID: 25432510
2. Winter, K., et al., Pertussis epidemic–California, 2014. MMWR Morb Mortal Wkly Rep, 2014. 63(48): p. 1129-32. PMID: 25474033
3. CDC, Pertussis Vaccination: Use of Acellular Pertussis Vaccines Among Infants and Young Children Recommendations of the Advisory Committee on Immunization Practices (ACIP) Morbidity and Mortality Weekly Review, 1997. 46: p. 1-25. PMID: 9091780
4. Raya, B.A., et al., Immunization of Pregnant Women Against Pertussis: The Effect of Timing on Antibody Avidity. Vaccine, 2015. 33(16):1948-52 PMID: 25744227
5. Silfverdal, S.A., et al., Immunological Persistence in 5 y olds Previously Vaccinated with Hexavalent DTPa-HBV-IPV/Hib at 3, 5, and 11 Months of Age. Hum Vaccin Immunother, 2014. 10(10): p. 2795-8.
   PMID: 25483640
6. Hara, M., et al., Pertussis outbreak in university students and evaluation of acellular pertussis vaccine effectiveness in Japan. BMC Infect Dis, 2015. 15(1): p. 45. PMID: 25656486
7. Sealey, K.L., et al., Genomic Analysis of Isolates From the United Kingdom 2012 Pertussis Outbreak Reveals That Vaccine Antigen Genes Are Unusually Fast Evolving. J Infect Dis, 2014. 212(2): p. 294-301. PMID: 25489002
8. Torjesen, I., Proteins Targeted by Pertussis Vaccine Are Mutating Unusually Quickly, Study Finds. BMJ, 2014. 349: p. g7850. PMID: 25552634
9. McGirr, A. and D.N. Fisman, Duration of Pertussis Immunity After DTaP Immunization: A Meta-analysis. Pediatrics, 2015. 135(2): p. 331-343. PMID: 25560446
10. Tefon, B.E., E. Ozcengiz, and G. Ozcengiz, Pertussis Vaccines: State-Of-The-Art and Future Trends. Curr Top Med Chem, 2013. 13(20): p. 2581-96. PMID: 24066885
11. Brummelman, J., et al., Modulation of the CD4(+) T cell response after acellular pertussis vaccination in the presence of TLR4 ligation. Vaccine, 2015. 33(12): p. 1483-91. PMID: 25659267

By: Suzanne Canada, Ph.D.
Tanager Medical Writing

While you go about your day, you are surrounded by micro-organisms.  Although most of us spend a lot of time washing up and trying not to think about the propensity of creatures that share our personal space, scientists have been studying them.  Due to their great progress, we are reaching an understanding of how these bacteria and fungi affect our bodies’ functions [1]. The evidence indicates that this inner-ecosystem can not only cause disease if perturbed, but also influence our overall health!  Organisms including Escherichia coli, Helicobacter pylori, Streptococcus thermophilus, and species of Clostridia, Lactobacillus, and Bacterioides inhabit our gut. Corynebacterium jeikeium as well as Staphylococcus species live on our skin, and other Streptococci as well as Neisseria and Candida albicans inhabit our mouth and upper respiratory system [2].  The makeup and diversity of organisms has been found to be strongly influenced, not only by what you eat [3], but also by who you live with [4, 5].  With greater understanding of this rich soup of life that we carry with us, the microbiome has become the new frontier in cutting-edge drug development [6].

In the last three years, research into the molecular basis of microbial influence has blossomed.  The first and most obvious application for this information was in treating C. difficile infections; which result from overgrowth of the opportunistic pathogen after an antibiotic regimen or hospital stay.  Researchers found that fecal transplants from a healthy individual were an effective way to treat this potentially fatal infection [7, 8]. The role of intestinal microbes in Inflammatory Bowel Disease (IBD) has been established [9] in the last year or two.  Based on this knowledge, possible treatments for IBD, such as ulcerative colitis and Crohn’s disease, are in development. Other publications point to microbes’ role in inflammation of the skin and respiratory tract, including acne [10]and asthma[11].  More excitement has been generated as investigators have found links to other chronic diseases including diabetes [12, 13], hypertension [14, 15], and chronic liver disease [16].  Preliminary investigations suggest a connection between overall gut microbial composition and obesity [17].  Some studies in mouse models have even linked the microbiome to the neurological conditions of Alzheimer’s [18] and autism [19, 20].

With all this research going on, you need a great resource like LIST Biological Laboratories, with experience and expertise with microbial products spanning over 25 years. LIST has several products available that can serve as positive controls for your microbial research.  Potent toxins from C. difficile are available (LIST products #157, #158), as well as antibodies that aid in their detection (LIST products #753, #754). Lipopolysaccharides are also available, which cause inflammation and activation of immune signaling cascades, and are extracted from bacterial cell walls of E. coli O111:B4, O55:B5, O157:H7, J5 and K12; Salmonella typhimurium, Salmonella minnesota and Bordetella pertussis. Other acute immune system activators such as Staphylococcal toxins (LIST products #120, #122) and Shiga toxins (LIST products #161 & #162) are also available.

In case the assortment of purified bacterial products on hand are insufficient for your research needs, LIST also provides contract manufacturing for biotherapeutics, as well as microbial purification services.

References

1. Human Microbiome Project, C., A framework for human microbiome research. Nature, 2012. 486(7402): p. 215-21. PMID: 22699610
2. Human Microbiome Project, C., Structure, function and diversity of the healthy human microbiome. Nature, 2012. 486(7402): p. 207-14. PMID: 22699609
3. David, L.A., et al., Diet rapidly and reproducibly alters the human gut microbiome. Nature, 2014. 505(7484): p. 559-63. PMID: 24336217
4. Yatsunenko, T., et al., Human gut microbiome viewed across age and geography. Nature, 2012. 486(7402): p. 222-7. PMID: 22699611
5. La Rosa, P.S., et al., Patterned progression of bacterial populations in the premature infant gut. Proc Natl Acad Sci U S A, 2014. 111(34): p. 12522-7. PMID: 25114261
6. Donia, M.S., et al., A Systematic Analysis of Biosynthetic Gene Clusters in the Human Microbiome Reveals a Common Family of Antibiotics. Cell, 2014. 158(6) p1402 – 1414. PMID: 25215495
7. Seekatz, A.M., et al., Recovery of the gut microbiome following fecal microbiota transplantation. MBio, 2014. 5(3): p. e00893-14. PMID: 24939885
8. Scott, K.P., et al., Manipulating the gut microbiota to maintain health and treat disease. Microb Ecol Health Dis, 2015. 26: p. 25877. PMID: 25651995
9. Huttenhower, C., A.D. Kostic, and R.J. Xavier, Inflammatory bowel disease as a model for translating the microbiome. Immunity, 2014. 40(6): p. 843-54. PMID: 24950204
10. Christensen, G.J. and H. Bruggemann, Bacterial skin commensals and their role as host guardians. Benef Microbes, 2014. 5(2): p. 201-15. PMID: 24322878
11. Martin, C., et al., Host-microbe interactions in distal airways: relevance to chronic airway diseases. Eur Respir Rev, 2015. 24(135): p. 78-91. PMID: 25726559
12. Tang, D., et al., Comparative investigation of in vitro biotransformation of 14 components in Ginkgo biloba extract in normal, diabetes and diabetic nephropathy rat intestinal bacteria matrix. J Pharm Biomed Anal, 2014. 100: p. 1-10. PMID: 25117949
13. Sato, J., et al., Gut dysbiosis and detection of “live gut bacteria” in blood of Japanese patients with type 2 diabetes. Diabetes Care, 2014. 37(8): p. 2343-50. PMID: 24824547
14. Pluznick, J., A novel SCFA receptor, the microbiota, and blood pressure regulation. Gut Microbes, 2014. 5(2): p. 202-7. PMID: 24429443
15. Pluznick, J.L., Renal and cardiovascular sensory receptors and blood pressure regulation. Am J Physiol Renal Physiol, 2013. 305(4): p. F439-44. PMID: 23761671
16. Minemura, M. and Y. Shimizu, Gut microbiota and liver diseases. World J Gastroenterol, 2015. 21(6): p. 1691-702. PMID: 25684933
17. Al-Ghalith, G.A., P. Vangay, and D. Knights, The guts of obesity: progress and challenges in linking gut microbes to obesity. Discov Med, 2015. 19(103): p. 81-8. PMID: 25725222
18. Bibi, F., et al., Link between chronic bacterial inflammation and Alzheimer disease. CNS Neurol Disord Drug Targets, 2014. 13(7): p. 1140-7. PMID: 25230225
19. De Angelis, M., et al., Fecal microbiota and metabolome of children with autism and pervasive developmental disorder not otherwise specified. PLoS One, 2013. 8(10): p. e76993. PMID: 24130822
20. Pequegnat, B., et al., A vaccine and diagnostic target for Clostridium bolteae, an autism-associated bacterium. Vaccine, 2013. 31(26): p. 2787-90. PMID: 23602537

By: Suzanne Canada, Ph.D.
Tanager Medical Writing

 

Vaccines have been used to help control diseases for more than 200 years and are the common practice for children and adults. Childhood vaccination has substantially reduced the morbidity and mortality from infectious diseases in much of the developed world, and influenza vaccinations have reduced the impact of seasonal influenza infections.¹ However, medical researchers are constantly looking for ways to improve the vaccines that are already used, and develop new ones.

Opportunities for improvement of vaccines abound. For example, although much attention is given to child vaccinations, a reservoir of infection could be eliminated through promotion of adult booster shots such as pertussis booster shots for expectant mothers and close family members, to help protect susceptible newborns. In addition, some diseases that have vaccines currently available still flourish in areas of the world where infrastructures for vaccination are poor and are too costly or cannot be delivered in their current forms.¹ Researchers are still trying to develop vaccines for other important diseases, such as HIV/AIDS, malaria and leishmaniasis. Vaccines are also being developed for bacterial pathogens, such as Vibrio cholerae O1 and enterotoxigenic Escherichia coli (ETEC) that are responsible for a high proportion of diarrheal disease and death in adults and children in many countries in Africa and Asia.²

By modifying the factors included in the vaccine, researchers balance the effectiveness of the immune response with the side effects. Previously, whole cell vaccines containing whole organisms that had been chemically inactivated were the norm, but the side effects of fever and discomfort following injections were much more common. Many of the vaccines used today, including those for measles and some influenza vaccines, use live, attenuated viruses. Others use killed forms of viruses, pieces of bacteria (lipopolysaccharides), or inactivated forms of bacterial toxins, known as “toxoids.” Killed viruses, lipopolysaccharides and toxoids can evoke an immune response that protects against future infection.³ Acellular vaccines were introduced in the late 1990’s that contain either three or five key bacterial proteins and have been quite effective in protecting infants and children under four with a much lower rate of side effects.

List Labs offers several virulence factors which are used in vaccine testing.  For testing C. difficile vaccines; available reagents are C. difficile Toxin A (Product #152), C. difficile Toxin B (Product #155), C. difficile Toxoid A (Product #153), C. difficile Toxoid B (Product #154), and both subunits of the Binary Toxin (Products #157 and #158).  Numerous Bordetella pertussis virulence factors are available for use in testing including: Products #179, #180 or #181 Pertussis Toxin, Product #170 FHA, Product #186 Fimbriae, Product #187 Pertactin, Product #188 and #189 Adenylate Cyclase and Product #400 Highly Purified B. pertussis LPS.  Anthrax vaccine testing maybe carried out using Protective Antigen (Product #171) with Lethal Factor (Product #172) in a toxin neutralization assay.  Although these factors are not suitable for testing on humans, they are excellent research tools.

Inactive toxins are quite useful in making antibodies or in capturing antibodies from a vaccinated population on ELISA plates.  Three of our inactivated toxins, which carry mutations in the toxin active site, are B. pertussis Adenylate Cyclase Toxoid, Pertussis Toxin Mutant, Product #184 and CRM197, a non-toxic Mutant Diphtheria Toxin, Product #149.  Toxoids made by formaldehyde treatment of toxins include versions of C difficile Toxins (Products #153 and #154), Diphtheria Toxoid (Product #151), Staphylococcus aureus Enterotoxoid B (Product #123), Tetanus Toxoid (Product #191) and Toxoids of Botulinum Neurotoxins A and B (Product #133 and #139, respectively).

 

1. Hammond B., Sipics M., Youngdhal K., (2013). From the History of Vaccines, a project of the college of physician of Philadelphia. ISBN: 9780988623101
2. Svennerholm AM., (2011) From Cholera to Enterotoxigenic Escherichia coli (ETEC) vaccine development.  Indian J Med Res. 133(2): 188–194. PMID: 21415493
3. Leitner DR., Feichter S., Schild-Prüfert K., Rechberger GN., Reidl J., Schild S., (2013) Lipopolysaccharide modifications of a cholera vaccine candidate based on outer membrane vesicles reduce endotoxicity and reveal the major protective antigen. Infect Immun 81(7):2379-93. PMID: 23630951

Adenylate cyclase toxin-hemolysin (ACT, AC-Hly, or CyaA) is an important virulence factor for Bordetella pertussis.  Adenylate cyclase toxin is a large (178 kDa), 1,706-residue-long, toxin consisting of an amino-terminal adenylate cyclase (AC) domain of 400 residues and a repeat toxin (RTX) moiety of 1,306 residues.  Sequences within the RTX domain are responsible for target binding, pore-formation and calcium binding.  Also located within this domain are two lysine residues which undergo posttranslational modified by acylation (1).

Adenylate cyclase toxin targets sentinel cells of the host’s innate immune defense. It penetrates a variety of immune cells and, when activated by calmodulin, catalyzes conversion of cellular ATP to cyclic AMP (cAMP), interfering with cell signaling and with the anti-bacterial activity of phagocytes. ACT acts on phagocytes limiting the phagocytes ability to produce oxidative burst and kill bacterial through complement or antibodies (2, 3).  The entire AC-Hly protein is necessary for adenylate cyclase delivery into cells (4).

In the intoxication process, the Hly portion of the toxin binds to CR3 receptor on target cells (CD11b⁺) and allows translocation of the AC enzyme into the cell.  Within the target cell, adenylate cyclase rapidly produces extremely high levels of cAMP, disabling the immune cell (5, 6).  At a lower efficiency, adenylate cyclase-hemolysin can penetrate cells lacking the CR3 receptor and create cAMP (7). In addition to binding target cells, CyaA is able to form small cation-selective pores in cytoplasm membranes, causing hemolysis in erythrocytes (8).

A non-enzymatic, genetically detoxified CyaA-AC^– toxoid has been produced (9). Although the catalytic activity in this AC toxoid is destroyed, it is still cell invasive and able to induce an immune response to co-administered pertussis antigens (10, 11, 12).  This toxoid has been shown to be capable of delivering vaccine antigens into the cytosol of major Histocompatibility complex (MHC) class I antigen-presenting cells (13).CyaA-AC^– toxoid has been used as a tool to deliver antigens to T cells in anti-cancer immunotherapeutic vaccines. (9, 14, 15)

List Labs provides several variations of the B. pertussis Adenylate Cyclase Toxin.  Most recently we have prepared a new formulation of the lyophilized Adenylate Cyclase Toxin Product #188.  This product may be obtained, upon request, as a liquid (Product #188L).  Additionally we have samples available of the Genetically Detoxified CyaA-AC Toxoid, request Product #188X, and samples of an especially low endotoxin preparation of Adenylate Cyclase Toxin Product #188U.  Finally, you will find in our product offering Product #189, Adenylate Cyclase Antigen, Native from Bordetella pertussis.

1. Sebo P., Osicka R., Masin J., (2014) Adenylate cyclase toxin-hemolysin relevance for pertussis vaccines. Expert Review of Vaccines 13(10): 1215-1227. PMID: 25090574
2. Confer DL. and Eaton JW., (1982) Phagocyte impotence caused by an invasive bacterial adenylate cyclase. Science 217:948–950. PMID: 6287574
3. Mock M. and Ullmann A., (1993) Calmodulin-activated bacterial adenylate cyclases as virulence factors. Trends Microbiol 1:187–192. PMID: 8143137
4. Sakamoto H., Bellalou J., Sebo P., Ladant D., (1992) Bordetella pertussis adenylate cyclase toxin. Structural and functional independence of the catalyticand hemolytic activities. J Biol Chem 267:13598–13602. PMID: 1618862
5. Wolff J., Cook GH., Goldhammer AR., Berkowitz SA., (1980) Calmodulin activates prokaryotic adenylate cyclase. Proc Natl Acad Sci USA 77(7):3841-4. PMID: 6253992
6. Hanski E. and Farfel Z., (1985) Bordetella pertussis invasive adenylate cyclase. Partial resolution and properties of its cellular penetration. J Biol Chem 260(9):5526-32. PMID: 2859287
7. Vojtova J., Kamanova J., Sebo P., (2006) Bordetella adenylate cyclase toxin: a swift saboteur of host defense. Curr Opin Microbiol 9(1):69-75. PMID: 16406775
8. Szabo G., Gray MC., Hewlett EL., (1994) Adenylate cyclase toxin from Bordetella pertussis produces ion conductance across artificial lipid bilayers in a calcium and polarity-dependent manner. J Biol Chem 269(36):22496–22499. PMID: 8077197
9. Simsova M., Sebo P., Leclerc C., (2004) The adenylate cyclase toxin from Bordetella pertussis–a novel promising vehicle for antigen delivery to dendritic cells. Int J Med Microbiol 293(7-8):571-6. PMID: 15149033
10. Macdonald-Fyall J., Xing D., Corbel M., Baillie S., Parton R., Coote J., (2004) Adjuvanticity of native and detoxified adenylate cyclase toxin of Bordetella pertussis towards co-administered antigens. Vaccine 22(31-32):4270-81. PMID: 15474718
11. Orr B., Douce G., Baillie S., Parton R., Coote J., (2007) Adjuvant effects of adenylate cyclase toxin of Bordetella pertussis after intranasal immunisation of mice. Vaccine 25(1):64-71. PMID: 16916566
12. Cheung GY., Xing D., Prior S., Corbel MJ., Parton R., Coote JG., (2006) Effect of different forms of adenylate cyclase toxin of Bordetella pertussis on protection afforded by an acellular pertussis vaccine in a murine model. Infect Immun 74(12):6797-805. PMID: 16982827
13. Osicka R., Osicková A., Basar T., Guermonprez P., Rojas M., Leclerc C., Sebo P., (2000) Delivery of CD8(+) T-cell epitopes into major histocompatibility complex class I antigen presentation pathway by Bordetella pertussis adenylate cyclase: delineation of cell invasive structures and permissive insertion sites. Infection Immunity 68: 247-256. PMID: 10603395
14. Dadaglio G., Morel S., Bauche C.,  Moukrim Z., Lemonnier FA., Van Den Eynde BJ., Ladant D., Leclerc C., (2003) Recombinant adenylate cyclase toxin of Bordetella pertussis induces cytotoxic T lymphocyte responses against HLA*0201-restricted melanoma epitopes. Int Immunol 15(12):1423-30. PMID: 14645151
15. Fayolle C., Ladant D., Karimova G., Ullmann A., Leclerc C., (1999) Therapy of murine tumors with recombinant Bordetella pertussis adenylate cyclase carrying a cytotoxic T cell epitope. J Immunol 162(7):4157-62. PMID: 10201941