By: Md. Elias, Ph.D, Senior Scientist

List Labs is one of the leading manufacturers of high quality adjuvants from bacterial sources. Our highly purified adjuvants for research and development are Tetanus Toxoid (Product #191), Cholera Toxin B Subunit (Product #104), Diphtheria Toxin CRM₁₉₇ Mutant (Product #149), Adenylate Cyclase Mutant, Cya-AC^– (Product #198L), Pertusis Toxin Mutant (Product #184), and LPS and its derivatives (Products #400, #401, #421, #423, #433, #434). GMP grade material is available by custom order.

In immunology, an adjuvant is a component that enhances and/or potentiates the immune responses (humoral and /or cell mediated) to an antigen and modulates it to achieve the desired immune responses. Adjuvants can be used for various reasons: (i) to enhance the immunogenicity of antigens; (ii) to reduce the amount of antigen or the number of immunizations needed for protective immunity; (iii) to improve the efficacy of vaccines in immune-compromised persons; (iv) to increase functional antibody titer; or (v) as antigen delivery systems for the uptake of antigens by the mucosa (1-3). Brief descriptions of List Labs products that have potential uses as vaccine adjuvants or immune modulators are provided below. For more details, please visit www.ListLabs.com.

Tetanus Toxoid (Product #191): Tetanus toxoid is prepared by formaldehyde inactivation of pure neurotoxin (Product #190). There are FDA approved vaccines that use a tetanus toxoid antigen to protect children and adult against tetanus such as DAPTACEL and Tripedia, and others that use it as a carrier in conjugate vaccines against various pathogens. For example, MenHibrix^® is an FDA approved vaccine where tetanus toxoid has been conjugated to Neisseria meningitidis serogroup C and Y capsular polysaccharides and Hib capsular polysaccharide. Several other tetanus toxoid conjugated vaccines are in research and investigation stages such as Type III group B streptococcal polysaccharide-tetanus toxoid conjugate vaccine (4). Information on our entire family of Tetanus products can be found at https://listlabs.com/products/tetanus-toxins-&-related-products/.

Cholera Toxin B subunit (Products #103B and #104): Cholera toxin B subunit (CTB) is the cell binding domain of cholera toxin protein complex. The holotoxin consists of a single A subunit bearing ADP-ribosyl-transferase activity surrounded by five B subunits that bind to GM1 ganglioside receptors on mammalian cell surfaces and facilitate entrance of the A subunit into cells. The non-toxic CTB has been shown to be an efficient mucosal adjuvant and carrier molecule for the generation of mucosal antibody responses and/or induction of systemic T-cell tolerance to linked antigens. Due to the ubiquitous presence of the GM1 ganglioside receptor on eukaryotic cell membranes, CTB has been extensively used as a conjugate and non-conjugate vaccine adjuvant in a wide variety of model systems.

A CTB-urease conjugated vaccine has been shown to prevent infection by Helicobacter pylori, a bacterium that infects greater than 50% of world population and can cause a variety of gastrointestinal diseases (5). A series of studies have been carried out to develop CTB carrier based vaccines to prevent HIV-1 (6) and West Nile Virus infections (7). CTB has been used as a component of a skin patch for transcutaneous immunization against hepatitis B virus in a mouse model (8). Besides the adjuvant activity, recent studies show that CTB can suppress immunopathological reactions in allergy and autoimmune diseases such as Crohn’s disease (9). Information on our entire family of Cholera products can be found at https://listlabs.com/products/cholera-toxins/.

Diphtheria Toxin CRM₁₉₇ Mutant (Product #149): CRM₁₉₇ is a non-toxic mutant of diphtheria toxin lacking the ADP-ribosylation activity (10). CRM₁₉₇ results from a naturally occuringsingle base change (glutamic acid to glycine) in the toxin gene which is immunologically indistinguishable from the native diphtheria toxin. CRM₁₉₇ functions as a carrier for polysaccharides and haptens making them immunogenic (11, 12). It is utilized as a carrier to develop conjugate vaccines for diseases such as pneumococcal and meningococcal infections. MenACWY-CRM is an approved vaccine to protect adults and adolescents against disease caused by meningococcal serogroups A, C, W-135 and Y. Information on our entire family of Diphtheria products can be found at https://listlabs.com/products/diphtheria-toxins/.

Adenylate Cyclase Toxoid, Cya-AC^– (Product #198L): A genetically modified adenylate cyclase toxin (ACT) lacking adenylate cyclase activity (CyaA-AC^–) has been produced (13). Although the catalytic activity is destroyed, CyaA-AC^– is still cell invasive and able to induce an immune response to co-administered pertussis antigens (14, 15).  CyaA-AC^– has been shown to promote delivering of vaccine antigens into the cytosol of major histocompatibility complex (MHC) class I antigen-presenting cells (16). CyaA-AC^– has been used as a tool to deliver antigens to T-cells in anti-cancer immunotherapeutic vaccines (17, 18).

Pertussis Toxin Mutant (Product #184): List Labs produces Pertussis Toxin Mutant, a genetically inactivated form of pertussis toxin where mutations were introduced to abolish the catalytic activity of the S1 subunit while the toxin complex still retains the cell binding ability (19). A pertusis toxin mutant has been used as an adjuvant or as a carrier to promote an immune response. These studies indicated that pertussis toxin mutant possesses adjuvant properties with the ability to encourage both local and systemic responses, to promote T helper cell responses to co-administered antigens and to favor the production of Th1/Th17 cells, important in mediating host immunity to infectious pathogens (20). Pertusis toxin binds to the cell receptor, TLR4 which activates Rac and subsequently causes various effects depending on the type of cell treated (21). The toxin or binding oligomer induces dendritic cell maturation in a TLR4-dependent manner (22). Information on our entire family of Pertussis products can be found at https://listlabs.com/products/pertussis-toxins-&-virulence-factors/.

LPS and its derivatives: List Labs provides LPS and various derivatives: highly purified HPT^TM LPS from Escherichia coli O113 (Product #433); Ultar Pure Escherichia coli O111:B4 LPS (Product #421); Escherichia coli O55:B5 LPS (Product #423); Ultra pure LPS from Salmonella Minnesota R595 (Product #434); Lipid A Monophosphoryl from Salmonella Minnesota R595 (Product #401) and highly purified HPT^TM LPS from Bordetella pertusis strain 165 (Product #400). For other LPS products please go to our product website. These LPS products are widely used as vaccine adjuvants and immune stimulators.

LPS is a potent stimulator of the vertebrate innate immune system mediated by macrophages and dendritic cells and generates a rapid response to infectious agents. Structural patterns common to diverse LPS molecules are recognized by Toll-like receptors (TLR) and accessory proteins in serum.  LPS released from bacterial membranes is bound to LPS binding protein (LBP) in serum, transferred to CD-14, an LPS receptor glycoprotein, and presented to the TLR-4-MD-2 complex, stimulating production of cytokines. LPS has a wide range of uses in research and drug development.  It may be used to stimulate immune cells and investigate the innate immune responses.  In drug development, structurally modified LPS forms, such as monophophoryl lipid A (MPLA) have been used as adjuvants in a wide range of vaccine formulations. MPLA, a TLR4 agonist has been formulated with liposomes, oil emulsions, or aluminium salts for several vaccines such as malaria vaccine (known as RTS,S) that is comprised of MPLA and a detoxified saponin derivative, QS-21 (3). Information on our entire family of Lipopolysaccharides can be found at https://listlabs.com/products/lipopolysaccharides/.

List Labs specializes in producing high quality adjuvants for vaccine development and is interested in partnering with others on new projects.  See some of our special projects or contact us for more information.

1. Lee S.,Nguyen M.T. Recent advances of vaccine adjuvants for infectious diseases. Immune Netw. 2015, 15(2): 51-7. PMID: 25922593
2. Petrovsky N., Aguilar J.C. Vaccine adjuvants: current state and future trends. Immunol Cell Biol.2004, 82(5): 488-96. PMID: 15479434 
3. Alving C.R., Peachman K.K.,Rao M., Reed S.G. Adjuvants for human vaccines. Curr Opin Immunol. 2012, 24 (3):310-5. PMID: 22521140 
4. Baker C.J., Rench M.A., McInnes P. Immunization of pregnant women with group B streptococcal type III capsular polysaccharide-tetanus toxoid conjugate vaccine. 2003. 21(24)3468-72. PMID: 12850362
5. Guo L., Li X., Tang F., He Y., Xing Y., Deng X., Xi T. Immunological features and the ability of inhibitory effects on enzymatic activity of an epitope vaccine composed of cholera toxin B subunit and B cell epitope from Helicobacter pylori urease A subunit. Appl Microbiol Biotechnol. 2012, 93(5):1937-45. PMID: 22134639
6. Matoba N., Kajiura H., Cherni I., Doran J.D., Bomsel M., Fujiyama K., Mor T.S. Biochemical and immunological characterization of the plant-derived candidate human immunodeficiency virus type 1 mucosal vaccine CTB-MPR. Plant Biotechnol J.2009, 7(2):129-45. PMID: 19037902
7. Tinker J.K., Yan J., Knippel R.J., Anayiotou P., Ornell K.A. Immunogenicity of a West Nile virus DIII-cholera toxin A2/B chimera after intranasal delivery. Toxins (Basel).2014, 6(4):1397-418. PMID: 24759174
8. Anjuere F., George-Chandy A., Audant F., Rousseau D., Holmgren J., Czerkinsky C. Transcutaneous immunization with cholera toxin B subunit adjuvant suppresses IgE antibody responses via selective induction of Th1 immune responses. J Immunol.2003, 170(3):1586-92. PMID: 12538724
9. Sun J.B., Czerkinsky C.,Holmgren J. Mucosally induced immunological tolerance, regulatory T cells and the adjuvant effect by cholera toxin B subunit. Scand J Immunol. 2010, 71(1):1-11. PMID: 20017804
10. Pappenheimer Jr. A.M., Uchida T., Harper A.A. An immunological study of the diphtheria toxin molecule. 1972, 9(9):891-906. PMID: 4116339
11. Gupta R.K., Siber G.R. Reappraisal of existing methods for potency testing of vaccines against tetanus and diphtheria. 1995, 13(11): 965-6. PMID: 8525688
12. Benaissa-Trouw B., Lefeber D.J, Kamerling J.P., Vliegenthart J.F., Kraaijeveld K., Snippe H. Synthetic polysaccharide type 3-related di-, tri-, and tetrasaccharide-CRM (197) conjugates induce protection against Streptococcus pneumoniae type 3 in mice. Infect Immun.2001, 69(7):4698-701. PMID: 11402020
13. Simsova M., Sebo P., Leclerc C. The adenylate cyclase toxin from Bordetella pertussis–a novel promising vehicle for antigen delivery to dendritic cells. Int J Med Microbiol. 2004, 293(7-8):571-6. PMID: 15149033
14. Macdonald-Fyall J., Xing D., Corbel M., Baillie S., Parton R., Coote J. Adjuvanticity of native and detoxified adenylate cyclase toxin of Bordetella pertussistowards co-administered antigens. 2004, 22(31-32):4270-81. PMID: 15474718
15. Cheung G.Y., Xing D., Prior S., Corbel M.J., Parton R., Coote J.G. 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.2006, 74(12):6797-805. PMID: 16982827
16. Osicka R., Osicková A., Basar T., Guermonprez P., Rojas M., Leclerc C., Sebo P. 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, 2000, 68(1): 247-256. PMID: 10603395
17. Dadaglio G., Morel S., Bauche C.,  Moukrim Z., Lemonnier F.A., Van Den Eynde B.J., Ladant D., Leclerc C.  Recombinant adenylate cyclase toxin of Bordetella pertussisinduces cytotoxic T lymphocyte responses against HLA*0201-restricted melanoma epitopes. Int Immunol. 2003 15(12):1423-30. PMID: 14645151
18. Fayolle C., Ladant D., Karimova G., Ullmann A., Leclerc C. Therapy of murine tumors with recombinant  Bordetella pertussisadenylate cyclase carrying a cytotoxic T cell epitope. J Immunol. 1999, 162(7):4157-62. PMID: 10201941
19. Brown D.R.,Keith J.M., Sato H., Sato Y. Construction and characterization of genetically inactivated pertussis toxin. Dev Biol Stand. 1991, 73:63-73. PMID: 1778335
20. Nasso M., Fedele G., Spensieri F., Palazzo R., Costantino P., Rappuoli R., Ausiello C.M. Genetically detoxified pertussis toxin induces Th1/Th17 immune response through MAPKs and IL-10-dependent mechanisms. J Immunol. 2009, 183(3):1892-9. PMID: 19596995
21. Nishida M.,Suda R., Nagamatsu Y., Tanabe S., Onohara N., Nakaya M., Kanaho Y., Shibata T., Uchida K., Sumimoto H., Sato Y., Kurose H. Pertussis toxin up-regulates angiotensin type 1 receptors through Toll-like receptor 4-mediated Rac activation. J Biol Chem. 2010, 285(20):15268-77. PMID: 20231290
22. Wang ZY., Yang D., Chen Q., Leifer C.A., Segal D.M., Su S.B., Caspi R.R., Howard Z.O., Oppenheim J.J. Induction of dendritic cell maturation by pertussis toxin and its B subunit differentially initiate Toll-like receptor 4-dependent signal transduction pathways. Exp Hematol. 2006, 34(8):1115-24. PMID: 16863919

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

 

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