Addressing challenges in the design of vaccines for the future

Little did Edward Jenner know, upon performing his „vaccination“ experiments in 18th century England, that his findings would change the course of medicine and lead to the first worldwide eradication of an infectious agent, the smallpox (Variola virus). A practice that was already known to the Chinese around the year 1000 AC, Jenner improved upon it by inoculating his gardener´s 8-year-old son in 1796 with a related virus that caused a milder form of the disease in cows and humans called „cowpox“. Jenner was able to achieve immunity against the smallpox virus in his research subjects and the term „vaccination“ was coined (from the Latin vaccinus, or cow-related).1

Airborn infection

The next milestone in the history of vaccines was the development of the rabies vaccine by Louis Pasteur in 1885 based on an attenuated („weakened“) virus obtained by drying out the nerve tissues of rabbits that had been used to propagate the virus.2 The first anthrax and cholera vaccines, likewise based on the principle of attenuation, were also developed by Pasteur. By the time World War I was over, devastating diseases such as typhoid fever, tuberculosis, and the plague had been tackled with either live or killed vaccines. Between 1930 and 1960, and thanks to major technological improvements such as the ability to culture viruses in embryonated chick eggs or mammalian cells, and continuous-flow-centrifugation for virus purification, new vaccines appeared on the radar, namely for yellow fever, Japanese B encephalitis, polio, and influenza. Bacterial vaccines were first developed after 1950, based on sub-unit capsular polysaccharide preparations for pneumococcus and meningococcus, followed by live attenuated paediatric vaccines for diseases such as measles, mumps, rubella, and varicella. 3, 4

Vaccines in the Genomic Era

Modern vaccines are much safer than their predecessors, as they undergo strict scrutiny by the authorities and are based on highly-purified microorganisms or antigenic proteins selected using techniques such as functional genomics and reverse genetics.5, 6 Designing vaccines that meet the high standards of the required safety and effectiveness continues to be a significant challenge to researchers.

One of the fronts where major progress has occurred in recent years is in the field of adjuvants.7 An adjuvant (from the Latin adiuvare, or „to help“) is any substance that potentiates the immune response to the antigen included in the vaccine. Aluminium salts were discovered to be effective adjuvants about 80 years ago, and have been included in numerous vaccines, due to their excellent safety record. „Alum“ is used in vaccines such as the combination diphteria-tetanus-pertusis (Dtap), Hepatitis A and B vaccines, and against TBE (tick-born encephalitis, or FSME, as it is known in Austria). A plethora of new adjuvants are being intensively researched and the immunological mechanism of action has been elucidated for some of these. MF59 (a squalene-based-oil-in-water emulsion developed by Novartis) has already being lisenced for seasonal (Fluad®) and pandemic Influenza vaccines (Aflunov® for H5N1 plus Focetria® and Celtura® for H1N1). Similarly AS03, another squalene emulsion developed by GSK is lisenced for Pandemrix® (H1N1). Its successor, AS04 is a mixture of Alum and monophosphoryl lipid A (MPL). AS04 is successfully being used as an adjuvant for Cervarix® (against Human Papilloma Virus infection, which can cause cervical cancer) and Hepatitis B (Fendrix®). While „Alum“ potentiates a Th2-biased immune response (generation of neutralizing antibodies), these new adjuvants are able to activate both a Th2 and a Th1 response (the latter causing activation of immune cells for destruction of intracellular pathogens). Virosomes, a lipid bilayer carrying active antigenic proteins (e.g. hemagglutinin and neuraminidase from the influenza virus) have been developed by Crucell and are also licensed as adjuvants in Inflexal® (influenza) and Epaxal® (Hepatitis A). Another example are ISCOMs (produced by Isconova) which are a mixture of cholesterol, phospholipids, and saponins, also capable of generating strong antibody and cell-based immune reponses.8 Recently, a Phase I clinical trial with an H5N1 vaccine showed that the inclusion of this potent adjuvant allows significant antigen-sparing (dose-sparing), which could be of critical importance during pandemic.9

Modern Vaccine Delivery Systems

            Recently, new avenues for vaccine delivery have been explored. Mucosal-associated infections such as HIV, gastric ulcer, diahrrea, and sexually-transmited diseases could be tackled in the future by applying the vaccine directly on the entry route of the pathogen (e.g. orally, intranasally, vaginally)10,11. To date, oral vaccines are available only for polio and cholera and a nasal vaccine (FluMist® by Medimmune) is available for influenza. In addition to providing needle-free immunization, which might increase market-acceptance, these vaccines offer a first-line-of-defence against invading pathogens by stimulating generation of secretory antibody (IgA). A diverse array of molecules that function by activating „toll-like receptors“ (TLRs), and therefore innate immunity, are being studied intensively as a source of potent new adjuvants for traditionally-injected as well as for mucosal vaccines.12 MPL is the only TLR4 ligand approved to date and it is included in Cervarix®. Unmethylated CpG-oligonucleotides activate TLR9 and have given promising results in human clinical trials against hepatitis B, malaria, influenza, and anthrax.13 Flagellin activates TLR5 and has been tested in animal models as a fusion protein with the conserved M2e influenza protein with the aim of developing a future universal vaccination independent of seasonal-strain variations.12

To substitute the traditional, but unpopular „shot“ in the arm approach, new creative approaches have been employed to develop needle-free devices. Pharmajet has developed an intradermal-delivery needle-free „gun“ that has completed two successful clinical trials (rabies and influenza) and a massive immunization campaign in children against measles.14, 17 Vaginal delivery of HIV antigens is currently being explored by use of gels or intravaginal rings as vehicles.15, 16 Skin patches that can deliver the antigen of interest (e.g. influenza, rotavirus, measles, Herpex simple virus 2) directly to antigen-presenting cells in the skin by painless microabrasion are also being developed.17 This technology would seem ideally suited to treat skin-related disorders, such as psoriasis or acne. Furthermore, they would not require refrigeration and would therefore bypass the cold transport chain to countries in the southern hemisphere leading to a significant lowering of cost of transport and distribution. Vaccination through consumption of edible plants (for example by expressing the Hepatitis surface B antigen in maize) could be an ideal delivery system, although this technology is still in the very early stages.18

New Approaches and Applications

Increasingly vaccines are used not only to prevent acute diseases, but also to cure chronic infectious diseases and non-infectious diseases such as cancer. Moreover, many diseases which were traditionally thought to be caused by genetic and environmental factors, have been recently associated with underlying bacterial or viral infections that either initiate or exacerbate the conditions. Examples of such diseases are Chagas disease (caused by Trypanosoma cruzi), cervical cancer (human papilloma virus), and peptic ulcer (Helicobacter pylori). More and more evidence for the role of bacteria in other complex human diseases is gradually coming to surface. Likewise, acne vulgaris, a skin inflammatory condition that was traditionally attributed mostly to genetics and hormonal influences, has been recently re-defined as a complex chronic disease associated with bacterial infection.19-21

Just like Jenner, 200 years later, we are still faced with the challenge of developing safe, effective, and efficient ways of vaccinating the population. The road ahead is still long and winding, but never before has the field of vaccinology had so many tools at hand to tackle infectious diseases, as well as cancer, allergies, and age-related diseases.

 

References:

1 http://www.historyofvaccines.org/

2 Microbe Hunters“ by Paul de Kruif. Harcourt Brace & Company (1996).

3 Hilleman, M. R. Vaccines in Historic Evolution and Perspective. Journal of Human Virology 3, 63-76 (2000).

4 Plotkin S. A. Vaccines: past, present and future. Nature Medicine, vol. 11-4: S5-S11 (2005).

5 Scarselli M. et al. The impact of genomics on vaccine design. TRENDS in Biotechnology, vol. 23-2 (2005).

6 Sette A. and Rappuoli R. A. Reverse Vaccinology: developing vaccines in the era of genomics. Immunity, vol. 33-4:530-541 (2010).

7 Awate S. A. Mechanisms of action of adjuvants. Frontiers in Immunology, vol. 4-114: 1-10 (2013).

8 Hong-Xiang S. et al. ISCOMs and ISCOMATRIX. Vaccine, vol. 27: 4388-4401 (2009).

9 Pederson G. K. et al. T-Helper 1 Cells Elicited by H5N1 Vaccination Predict Seroprotection. The Journal of Infectious diseases, vol. 206: 158-166 (2012).

10 Rhee J. H. et al. Mucosal Vaccine adjuvants update. Clinical and Experimental Vaccine Research, vol. 1: 50-63 (2012).

11 Gebril A. et al. Optimizing efficacy of mucosal vaccines. Expert Rev. Vaccines, vol. 11-9: 1139-1155 (2012).

12 Steinhagen F. et al. TLR-based immune adjuvants. Vaccine, vol. 29-17: 3341-3355 (2011).

13 Bode C. et al. CpG DNA as vaccine adjuvant. Expert Rev. Vaccines, vol. 10-4: 499-511 (2011).

14 http://www.pharmajet.com/

15 Yu M. and Vajdy M . Mucosal HIV transmission and vaccination strategies through oral compared to vaginal and rectal routes. Expert Opin Biol Ther., vol. 10-8: 1181-1195 (2010).

16 Ho E. A. Intravaginal rings as a novel platform for mucosal vaccination. Molecular Pharmeceutics & organic process research,  vol. 1-2: (2013).

17 Williams S. C. P. Under the skin of intradermal vaccines. News feature PNAS, vol. 110-25: 10049-10051 (2013).

18 Haydn C. A. et al. Bioencapsulation of the hepatitis B surface antigen and its use as an effective oral immunogen. Vaccine, vol. 30-19: 2937-2942 (2012).

19Dessinioti, C. & Katsambas, A.D. The Role of Proprionibacterium acnes in acne pathogenesis: facts and controversies. Clinics in Dermatology 28, 2-7 (2010).

20Lomholt, H.B. & Kilian, M. Population genetic analysis of Propionibacterium acnes identifies a subpopulation and epidemic clones associated with acne. PloS one 5, e12277 (2010).

21 McDowell, A. et al. An expanded multilocus sequence typing scheme for propionibacterium acnes: investigation of “pathogenic”, “commensal” and antibiotic resistant strains. PloS one 7, e41480 (2012).

The role of P. acnes in the pathogenesis of acne vulgaris

Acne vulgaris continues to burden every generation, and despite the multitude of the treatments and products on the market, there is still no magic formula that can guarantee a long-term benefit to all acne patients. Antibiotics are most common treatment. However they cannot be given long term. Furthermore, widespread use of antibiotics is thought to be a factor in the rise of antibiotic-resistant bacteria, possibly leading to greater harm than benefit. Vitamin A derivatives (isotretinoins) represent the second widely used treatment option. Oral isotretinoin suppresses sebaceous gland activity and so indirectly reduces inflammatory lesions; nevertheless, isotretinoin is not a curative drug and discontinuation of the treatment is frequently followed by recurrence in the absence of appropriate maintenance treatment.1 Besides, due to potentially serious side effects (birth defects, vision problems, mental health problems, hair loss, vision problems, etc.), isotretinoin is not routinely prescribed to patients. Therefore, acne patients would welcome an alternative, safer, and long-term effective treatment. Devising an alternative prophylactic and curative treatment may soon be possible, thanks to the recent research findings that have uncovered the pathogenic role of the long-suspected culprit, a bacterium named Propionibacterium acnes (P. acnes).

Acne vulgaris affects >85% of teenagers and >10% of adults and has recently been re-defined as a complex chronic disease associated with P. acnes, which takes advantage of immune susceptible host.2–5 In the past, the pathogenic role of P. acnes has been debated due to the fact that this bacterium is found not only on the skin of acne patients, but also on the skin of healthy individuals. The theory that P. acnes does not cause the acne per se, but rather contributes to inflammation once the acne lesion has been formed, has been challenged by the newest epidemiological studies using highly sensitive, improved methods for genetic profiling.

These and other studies have uncovered important new facts about acne formation:

  • Significant differences between the P. acnes strains isolated from the patients with severe acne compared to healthy controls;4,6
  • Striking functional differences among strains from different phylogenetic clusters.7,8
  • P. acnes secretion of a glycocalyx biofilm that acts as an “adhesive glue” for the corneocytes, thereby inducing comedonal acne.9,10
  • Bacterial biofilms acting as a protective barrier to antibactericidal agents used in acne therapy.11,12

P. acnes resides on the surface of our skin and in the most individuals it poses no problem. In fact, it is even considered the “gate keeper”, protecting our skin from colonization by pathogens that can cause more severe infections. However, not all P. acnes strains are harmless skin protectors. Each of us carries a distinct population of P. acnes strains, and if we happen to be colonized by a more virulent type, the strain may take the advantage of the skin’s unique environment, and in the absence of effective immune defenses, spread to the deeper skin layers (dermis) where it will induce inflammation.

What factors facilitate the P. acnes invasion of deeper skin layers?
P. acnes colonizes and grows optimally in the presence of lipids and in the absence of oxygen. Sebum secretion is intensified during puberty and other hormonally active periods (e.g. premenstrual period or pregnancy). Increased sebum production provides an ideal environment for P. acnes and attracts the bacteria into the hair follicles, which are located deeper within the skin (Figure 1). Additional genetic factors may be also at play. However, regardless of the signal that attracts them, our immune system must be able to recognize the bacteria and rapidly eliminate them, in order to prevent the formation of acne lesions.

If the immune system is not effective enough, it can be trained to respond more efficiently through vaccination.

A vaccine against P. acnes could be designed to induce a stronger immune response against specifically targeted virulent P. acnes strains. Such a vaccine could provide the benefits that are not available from other types of therapies including the following:

  • Rapid and effective cure
  • Long-term protective effect
  • Preservation of normal bacterial skin flora
  • Does not contribute to antibiotic resistance of bacteria
  • Avoids unwanted side-effects, since the vaccine would induce the immune response against foreign, and not host cells
  • Prophylactic application would be also possible, before acne scars are formed
The role of P. acnes in the pathogenesis of acne vulgaris
Click to enlarge

Figure 1. The role of P. acnes in acne pathogenesis.

  1. Sebum secretion and clogged pores limit access to oxygen.
  2. High lipid content and low oxygen concentration creates optimal growth environment for P. acnes.
  3. P. acnes residing on the skin surface is attracted to lipid-rich, optimal environment inside the hair follicles. Here, the bacteria rapidly multiply inducing a local inflammatory response. If the immune system is not able to efficiently kill and remove the bacteria, the inflammatory reaction persists leading to the creation of cysts and pustules, ultimately leading to the creation of scars.

REFERENCES

  1. Kurokawa, I. et al. New developments in our understanding of acne pathogenesis and treatment. Experimental dermatology 18, 821-32 (2009).
  2. Grange, P. a, Weill, B., Dupin, N. & Batteux, F. Does inflammatory acne result from imbalance in the keratinocyte innate immune response? Microbes and infection / Institut Pasteur 12, 1085-90 (2010).
  3. Dessinioti, C. & Katsambas, A.D. The Role of Proprionibacterium acnes in acne pathogenesis: facts and controversies. Clinics in Dermatology 28, 2-7 (2010).
  4. Lomholt, H.B. & Kilian, M. Population Genetic Analysis of Propionibacterium acnes Identifies a Subpopulation and Epidemic Clones Associated with Acne. PLoS ONE 5, e12277 (2010).
  5. Hu, J. et al. Comparative Genomics and Transcriptomics of Propionibacterium acnes. PLoS ONE 6, e21581 (2011).
  6. McDowell, A. et al. An expanded multilocus sequence typing scheme for Propionibacterium acnes: investigation of “pathogenic”, “commensal” and antibiotic resistant strains. PloS one 7, e41480 (2012).
  7. Holland, C. et al. Proteomic identification of secreted proteins of Propionibacterium acnes. BMC microbiology 10, 230 (2010).
  8. Nagy, I. et al. Distinct strains of Propionibacterium acnes induce selective human beta-defensin-2 and interleukin-8 expression in human keratinocytes through toll-like receptors. The Journal of investigative dermatology 124, 931-8 (2005).
  9. Burkhart, C.N. & Burkhart, C.G. Microbiology’s principle of biofilms as a major factor in the pathogenesis of acne vulgaris. International Journal of Dermatology 42, 925-927 (2003).
  10. Burkhart, C.G. & Burkhart, C.N. Expanding the microcomedone theory and acne therapeutics: Propionibacterium acnes biofilm produces biological glue that holds corneocytes together to form plug. Journal of the American Academy of Dermatology 57, 722-4 (2007).
  11. Bellew, S., Thiboutot, D. & Del Rosso, J.Q. Pathogenesis of acne vulgaris: what’s new, what’s interesting and what may be clinically relevant. Journal of drugs in dermatology : JDD 10, 582-5 (2011).
  12. Coenye, T., Peeters, E. & Nelis, H.J. Biofilm formation by Propionibacterium acnes is associated with increased resistance to antimicrobial agents and increased production of putative virulence factors. Research in Microbiology 158, 386-392 (2007).

Vaccine industry challenges

Infectious diseases are still the leading global cause of morbidity and mortality. Vaccine-preventable diseases are still responsible for about 25% of the 10 million deaths occurring annually among children under five years of age, and ~25% of adult (15–59 years) deaths are still attributed to infectious diseases.1 Moreover, infectious diseases continue to rise due to an alarming rate of antibiotic resistance among bacteria, and due to the trends of pathogen spreading driven by globalization and enlarged mobility.

Despite the great need to develop novel and more efficient vaccines, the progress has been slow. Current approaches to vaccine antigen discovery are inefficient, costly, and frequently lead to the selection of ineffective targets: only 15% of vaccines that enter clinical trials get market approval.2 It has been estimated that even a 10-percent improvement in predicting failures before embarking on clinical trials could save a pharmaceutical company $100 million in development costs per product.3

Therefore, it is of crucial significance to develop methods, which would predict the efficacy of vaccine candidates in clinical trials with greater certainty.

The reason behind such low success in selecting optimal vaccine candidates lie primarily in the challenges associated with the vaccine antigen selection. Most widely used methods in antigen discovery are:

  • reverse vaccinology,
  • bioinformatics analysis,
  • proteomics, and
  • antigenome technology.

Reverse vaccinology as an approach was validated for the first time on a Gram-negative bacteria, Neisseria meningitidis serogroup B (MenB); although it lead to identification of several antigens (the vaccine has successfully completed Phase III clinical trials), the screening process required large resources and several years to complete.4

Other approaches are similarly laborious and frequently biased. Proteomic analysis of cellular fractions or peptides digested from the bacterial surface has the limitations that not all proteins are equally abundant and prone to digestion, and it is difficult to control enzymatic digestion to preserve the integrity of the cell while maximizing the amount of surface digested proteins. Moreover, the proteomic analysis gives no information about the functional significance and protective potential of the identified antigens.

Antigenome approach relies on the use of sera from the patients who overcame infection or healthy individuals expected to contain protective antibodies. However, this approach too is biased towards antigens that are immunogenic but not necessarily protective, and is not suitable for identifying the antigens containing large conformational epitopes or epitopes created by post-transcriptional modifications (due to the use of a non-native antigen expression system which can display only a limited number of amino acids from a native, pathogen-derived antigen).

Besides the challenges encountered during initial antigen screening and selection, a successful vaccine development is further complicated by the biased experimental methods used during pre-clinical validation of antigen efficacy. For example, relying heavily on the animal models and in vitro testing under artificial laboratory culture conditions are not best practices for evaluating protective potential of the vaccines against strictly human pathogens. Such pathogens are highly adapted to the human tissue environment and have developed multiple mechanisms for evading human immune responses (e.g. binding of components of human complement and fibrinogen, escaping or interfering with phagocytosis, binding human immunoglobulins). These immune evasion mechanisms do not operate in other species and therefore, data obtained from such studies cannot be extrapolated to humans. The current requirement by regulatory authorities for extensive data demonstrating the protective efficacy of vaccine candidates in animal models compounds this problem, whereby those engaged in vaccine development continue to utilize animal models that are not relevant for investigating strictly human pathogens.

For each year of delay in vaccine discovery and development, millions human lives are being lost. Overcoming these challenges should be the highest priority of the scientific community and industry: this is the most powerful motivation behind our research efforts at Origimm.

REFERENCES:

  1. Adamczyk-Poplawska, M., Markowicz, S. & Jagusztyn-Krynicka, E. K. Proteomics for development of vaccine. Journal of proteomics 8, 1–21 (2011).
  2. Kola, I. & Landis, J. Can the pharmaceutical industry reduce attrition rates? Nature reviews. Drug discovery 3, 711-5 (2004).
  3. Innovation or Stagnation. Challenge and Opportunity on the Critical Path to New Medical Products. (Critical white paper). U.S. Department of Health and Human Services. Food and Drug Administration. (2004).
  4. Kaushik, D.K. & Sehgal, D. Developing antibacterial vaccines in genomics and proteomics era. Scandinavian journal of immunology 67, 544-52 (2008).