Carbohydrate and peptide – based baccines: the way forward

Abstract

Existing treatments and therapies have supported a huge variety of diseases and infections, a significant example being antibiotics. However the increasing presence of multi-resistant bacteria, as well as increased changes observed in the mechanisms responsible for variation in viruses, involving accumulation of mutations within the genes that code for antibody-binding sites (known as antigenic drift), has resulted in these new strains not being inhibited as effectively by those treatments that originally targeted them (Reche, Fernandez-Caldas, Flower, Fridkis-Hareli and Hoshino, 2014). The knock-on effect has been that the bacteria or virus is able to spread more easily, and therapeutic treatments (used after a person contracts a disease), become less effective, unable to work by boosting the host’s own immune system. As a result, it has been recognised that the vaccine offers the advantage of preventing the anticipation of disease occurrence, using advance action to counteract infection and chronic illness. Prophylactic, and to a lesser extent therapeutic, vaccines are the most cost-effective and efficient alternative to other treatments and prevention of infectious and chronic diseases. They work by causing changes to the T- and B-cells of the adaptive immune system to eliminate or prevent pathogen growth (Plotkin, Orenstein, and Offit, 2013). Going back to the introduction of vaccines more than 200 years ago, these were initially composed of killed pathogens, which although successful, also caused unacceptably high levels of adverse reactions. During the years of research that have since followed, as with the changes observed with antibiotics and other treatments becoming less effective, the need for safer and more effective vaccines has also been acknowledged. In addition, an improved understanding of antigen presentation and subsequent recognition has supported the development of newer vaccine types (Flower, 2013). Equally, whilst many diseases and infections are controlled by vaccines, for some, no vaccines have been developed, including Streptococcus pyogenes, human immunodeficiency virus (HIV) and hepatitis C virus (HCV) (Wang and Walfield, 2005; Barrett and Stanberry, 2009). Efforts to develop new vaccines are discussed in more details, with a focus on peptide-based and carbohydrate-based vaccines. Challenges are also discussed, leading to a summary of the potential direction of vaccination and research, which describes a promising future.

Peptide-based vaccines

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An example of a newer category of vaccine is peptide-based vaccines. Peptides are short sequences of proteins, and diseases/infections use these proteins as part of their attack on the immune system. In many cases, the immune system has the ability to recognise the proteins associated with an attack by disease or infectious causing pathogens and can respond effectively. However as observed with many cancers, HIV, HCV and other conditions, an effective immune response is not triggered, hence the need for newer vaccine developments including those based on peptides, which encompass single proteins or synthetic peptides encompassing many antigenic determinants (B- and T-cell epitopes) (Flower, 2013). Peptide vaccines are a type of subunit vaccine, which presents an antigen to the immune system, using the peptide of the original pathogen, supporting immunity. Such peptide-based vaccines avoid the adverse effects described with traditional whole-organism vaccines (Moisa and Kolesanova, 2012) with additional benefits also noted (Ben-Yedidia and Arnon, 1997), including:

• The absence of infectious material
• An immune response that is specific, focusing only on the targeted epitope, with the induction of site-specific antibodies
• No risk of an immune attack or cross-reactivity with the host tissues
• Flexibility, with an ability to modify products accordingly
• Improved effectiveness in relation to manufacturing on a large scale, and long-term storage where necessary e.g. a pandemic.

However, a number of difficulties have been encountered during the development of such vaccines (Simerska, Moyle and Toth, 2011; Dudek, Perlmutter, Aguilar, Croft and Purcell, 2010) including:

• A short biological activity of peptides due to degradation by enzymes
• The trigger of a weak immune response when used alone i.e. single peptides

Finding optimal delivery systems

As a result, and to overcome the difficulties mentioned above, synthetic peptide vaccines have been developed, on the basis that a greater more accurately targeted immune response will be achieved. Peptide antigens are not immunogenic by themselves, so this has led to investigations into co-administration of subunit peptide antigens with adjuvants (immunostimulants) to increase the peptide-induced responses to corresponding antigens. Appropriate delivery systems and often toxic adjuvants have demonstrated effective immunity, however, although many adjuvants are described in the literature, only a few have been approved for use with vaccines for delivery in humans due to their toxicity and include water/oil emulsions, liposomes, and bacterial lipophilic compounds to offer a few examples (Heegaard et. al., 2010). Incomplete Freund’s adjuvant (IFA) and Montanide ISA (both oil-based) have been used in clinical trials. Focusing on liposomes as another example, researchers have demonstrated that use of lipid core peptide (LCP) technology (lipidation of peptides) improves the effectiveness of a self-adjuvanting vaccine delivery system, targeting a specific disease and triggering an effective immune response. This system provides a promising platform for human vaccine development (Zhong, Skwarczynski and Toth, 2009; Moyle and Toth, 2008).
In animal models, peptide vaccines have been effective in generating the required immune response, and during recent years, peptide-based vaccines have advanced from animal models and pre-clinical studies, to human clinical trials (Yang et al., 2001). Although currently, all known peptide vaccines under development for humans remain at the stage of clinical trials, these trials should build on the promising evidence resulting from research to date of the potential application of vaccine candidates based on a LCP system, as well as other strategies. Prevention of not only many infectious diseases including hepatitis C virus, malaria, human immunodeficiency virus and group A streptococci), but also for cancer immunotherapy and improved allergen specific tolerance, remains an exciting, and very real possibility.

Carbohydrate-based vaccines

The development of vaccines based on carbohydrates not only has quite a history, but is also an area that is fast moving in the current research world. The literature provides evidence as far back as the early 1900s where researchers discovered a connection between type-specific polysaccharides and the induction of antibodies being developed against certain types of pneumococci (Francis and Tillett, 1930). This was confirmed by evidence of pneumococcal capsular polysaccharides being used as vaccines, providing effective and long lasting immunity (Heidelberger, Dilapi, Siegel and Walter, 1950). However despite these early findings, the discovery and success of other treatments such as antibiotics and chemotherapeutics led to this area of research being put on hold. As mentioned earlier however, due to increased resistance to existing treatments such as antibiotics, coupled with the recognition for a need of newer treatments including improved vaccines, renewed interest into preventive vaccines has resulted in novel approaches, which include carbohydrate vaccines.

Vaccines are commonly made from weakened pathogens, or, as we now know, other approaches also use immunogenic proteins or polysaccharides. Carbohydrates have been the centre of attention in the research field of vaccination because not only do they exhibit more stability than proteins, but they have roles in both physiology and pathophysiology, including cell interaction and signalling, inflammation, pathogen host adhesion/recognition, to name a few examples (Doshi, Shanbhag, Aggarwal, Shahare and Martis, 2011). During the last ten years or so, they have been used as adjuvants, as carriers for protein antigens to aid immunotherapy, and as targets for vaccines against bacteria. Additionally, as observed with DNA and proteins, carbohydrates are now recognised as biopolymers also, playing a role in many molecular and biological activities (Doshi et. al., 2011). These discoveries, partnered by an improved understanding of the immune system and the identification of specific and relevant carbohydrate structures, led to the development of glycoconjugates, which in turn led to carbohydrate vaccine development (Holemann and Seeberger, 2004). Glycoconjugates are present in the surfaces of cells, as well as in the surrounding extracellular matrices and connective tissue. Therefore both the identified structure and presence of glyconjugates, plus the role they play, means they are a suitable basis for the development of new vaccines. Induction of protective antibodies is key to an effective immune response as a result of a vaccine, and as with peptide vaccines, challenges have been evident in the research to develop effective carbohydrate vaccines, including the following:

• Glycans struggle to effectively induce protective antibodies
• Carbohydrates have a low immunogenic impact by themselves (as observed with peptides).

There are two main carbohydrate vaccine types:

1. Natural carbohydrate vaccines: these include small amounts of impurities
   Synthetic carbohydrate vaccines: these are produced with no contaminants, and are cost-effective due large-scale production.

Synthetic carbohydrate antigens used to develop vaccines have triggered immune responses in clinical studies and are favourable given the risk of adverse effects with natural vaccines. Four crucial aspects need to be considered for the design of carbohydrate-based vaccines (Astronomo and Burton, 2010):

1. The antigen source: glycan antigens are diverse, ranging from large polysaccharide capsules, to small monosaccharides, to oligosaccharides, all of which have been shown to be adequate for preparation of vaccines.
2. The carrier: this is most often proteins, although other materials have been investigated, with the aim of ensuring that the link between the antigen and the carrier is specific.
3. The method of conjugation (or ligation): protein conjugates, lipid conjugates and polyvalent scaffold conjugates have been developed. The success of a conjugate vaccine depends partly on the method of conjugation employed. This should be simple and efficient, as well as causing minimal distortion to the individual components involved, with many differing techniques used (Zou & Jennings, 2009; Ada and Isaacs, 2003).
4. The choice of adjuvant: required to improve immunogenicity of the carbohydrate antigens being targeted, with a limited choice approved for use in humans.

Examples of diseases targeted by carbohydrate-based vaccines

The discussion will now move on to the use of carbohydrate-based vaccines in three disease areas: Group A Streptococcus (GAS), HIV/AIDS and Haemophilus influenza type b.

GAS

The need for a safe, effective, affordable and practical vaccine against GAS (also known as Streptococcus pyogenes), has been recognised for many years, as has the research into a vaccine against this disease, given the global burden on health that this disease causes in particular in less developed countries. More than 500,000 deaths result from the GAS each year, with the bacteria causing a range of both less complicated and life-threatening illnesses (Carapetis, Steer, Mulholland and Weber, 2005). The diversity of GAS strains is the major challenge for the development of an anti-GAS vaccine, with more than 100 different strains identified, of which the genetic sequence for several different strains have been determined (Johnson and Pinto, 2002). Research has identified that GAS bacteria contain a surface polysaccharide made up of long, repetitive polysaccharide chains. The conserved and constant arrangement of these chains suggests conjugate vaccines to be an attractive and achievable option, with animal models supporting this theory (Cunningham, 2000). Synthetic carbohydrate vaccines, although only studied in a limited set of GAS infections, have demonstrated a protective immune response (Robbins et al., 2009). In addition, some areas of research have focused on the molecular analysis of a surface protein labelled the M protein, which is encoded by the emm gene. This particular gene has been found to be the major cause of GAS related clinical manifestations (Smeesters, McMillan and Sriprakash, 2010). These findings have allowed a greater understanding of the functioning of specific proteins responsible for the virulence of the disease, which in turn, supports the development of potential GAS vaccines. Vaccine prevention of GAS and the resulting symptoms and complications has been a goal of researchers for many years. A number of vaccines have been in research development to offer protection against GAS, with the research vaccine strategies focusing on either M protein, or non-M protein antigens (Smeesters, 2014). However only those vaccines that use the M protein as the antigen have progressed to clinical trials (McNeil et. al., 2005), and have included conserved antigens coverage across the many strains of GAS, a type-specific vaccine based on the N-terminal portion of the M protein, and a recombinant vaccine that reached phase II clinical trials (Pandey, Wykes, Hartas, Good and Batzloff, 2013; Bauer, 2012). However no vaccine has currently reached licensing and so the diseases caused remain uncontrolled in many areas, with reviews covering the research suggesting that even those vaccines developed with the aim of providing large coverage of GAS strains, these vaccine might achieve acceptable coverage in developed countries, but in less developed countries where the disease burden is much greater, the positive impact of the vaccines would be much lower due to a greater strain diversity (Smeesters, McMillan, Sriprakash, and Georgousakis, 2009; Steer, Law, Matatolu, Beall and Carapetis, 2009; McMillan and Sanderson, 2013). Equally, antibiotic treatment is either impractical with regards to implementation (specifically in less developed countries) or ineffective. One research group targeted the bacteria by synthesising a new self-adjuvanting vaccine candidate, incorporating a carbohydrate carrier and an amino acid-based adjuvant, resulting in successful synthesis and characterisation of the vaccine candidate. This may contribute to the identification of a safe and effective vaccine against GAS in the future (Simerska et. al., 2008; Simerska, Lu and Toth, 2009).

HIV/AIDS

One of the main challenges researchers have faced within the field of vaccine development against HIV/AIDS, is that the virus surface is covered with layers of glycans, which conceal underlying viral antigens that are potential good targets in the production of vaccines (Scanlan, Offer, Zitzmann, and Dwek, 2007). They are produced by the host cell, which makes the virus appear as “self” resulting in no attack being triggered by the host immune system. The layers of carbohydrate also contain mannose residues, making these another potential target for a vaccine aimed at preventing HIV infection, whereby lectins preferentially bind to ? 1-2 linked mannose residues. Such lectins are being investigated as possible therapeutic tools (Tsai et al., 2004) although the fact that lectins are often toxic needs to be researched further to avoid the host immune system damaging host cells. Indeed, other drugs that are known to inhibit synthesis of carbohydrates only have this effect at often toxic concentrations to cause antiviral activity. Another strategy based on the same principle of developing a carbohydrate vaccine, is the identification of antibodies that again recognise and bind to glycans. (Scanlan et al., 2002, Scanlan et al., 2007). The antibody appears to recognize these glycans because although they belong to the host, they are arranged in a “non-self” manner (Scanlan et al., 2002; Scanlan et al., 2007), making the production of effective ant-HIV vaccines a real possibility, in addition to vaccines for other diseases such as cancer (Galonic and Gin, 2007). Studies have also been described using immune enhancing adjuvants, carrier peptides such as keyhole limpet hemocyanin and altered glycan structure constructs that support immune recognition in the development of vaccines against cancer (Galonic and Gin, 2007). These same strategies are being used in development of possible HIV vaccines, where antibodies target self-carbohydrates arranged slightly differently on cancer cells and HIV-infected cells, in comparison to healthy cells. (Galonic and Gin, 2007). These approaches have not as yet led to clinically effective vaccines, but it is clear that antibodies that strongly bind to carbohydrate antigens on, for example, prostate cancer cells, have been generated (Slovin et al., 2003) and this appears to be a highly promising approach. Further exploration is required based on the carbohydrate coat of the virus, which may lead to improved prevention treatment of HIV.

Haemophilus influenza type b

The first synthetic vaccine for human application was developed in 2003 for protection against Haemophilus influenza type b vaccine, not only providing protection against this bacterium, but also against all the associated diseases it causes ranging from meningitis, septicaemia, pneumonia and arthritis (Doshi, Shanbhag, Aggarwal, Shahare and Martis, 2011). Indeed this bacterium is the leading cause of serious illnesses in children under 5 years worldwide. The majority of strains of Haemophilus influenza are non-encapsulated, and are lacking in any carbohydrate polysaccharide protective structure, as opposed to the GAS bacteria and HIV virus described earlier. This structural information armed researchers with the knowledge that carbohydrate polysaccharide conjugate vaccines would be required to ensure the development of an effective vaccine (Verez-Bencomo et. al., 2004). As a result, carbohydrate-based vaccines have been licensed for protection in humans against haemophilus influenza type b, using oligomerization and a carrier protein (Doshi et. al., 2011).

Evidence of progress

To end this section of the discussion, several conjugate polysaccharide carbohydrate vaccines are now well into pre-clinical/clinical development, or have been licensed and are now commercially available. Examples of licensed vaccines include the following (Astronomo and Burton, 2010):

• Haemophilus influenza type b (Hib) – 4 carbohydrate-based vaccines are licensed via 3 different pharmaceutical companies: ActHIB and Hiberix; Pentacel; PedvaxHIB; and Comvax
• Neisseria meningitides A, C, Y and W-135 – 2 carbohydrate-based vaccines are licensed via the same pharmaceutical company: Menactra; and Menomune-A/C/Y/W-135
• Salmonella typhi – 1 carbohydrate- based vaccine is licensed: TYPHIM Vi
• Streptococcus pneumonia variants – 2 carbohydrate-based vaccines are licensed via 2 different pharmaceutical companies: Prevnar; and Pneumovax 23.

Examples of carbohydrate-based vaccines in development include the following, where the disease is described in addition to the phase of development (Astronomo and Burton, 2010):

• Breast cancer – with 1 vaccine at the preclinical phase and a second at phase I
• Prostate cancer – 4 vaccines are in development at the preclinical, phase I and phase II stages
• HIV-1 – 1 vaccine at the preclinical phase
• Group A streptococcus – 1 vaccine at the preclinical phase
• Group B streptococcus – 1 vaccine at phase II.

Conclusion

It is fact that vaccines have had a major role to play in the success of preventing and treating many diseases, however many challenges remain. Diseases exist for which no effective vaccines have yet been discovered, including HIV/AIDs. In addition, diseases that have been controlled by vaccines in some parts of the world continue to affect the lives of people adversely in other areas where infrastructures for vaccination are poor/non-existent. Continued research is necessary to develop vaccines not only for those diseases with no vaccine available, but also to improve the effectiveness of existing vaccines. In addition to research focusing on novel and promising approaches such as carbohydrate and peptide based vaccines, efforts also need to concentrate on areas such as lower cost, more convenient delivery of vaccines, and longer-term protection. The future direction of research in this field has become focused with the help of new evidence-based information and promising data. The advent of synthetic peptide-based and carbohydrate-based vaccines signified a new era for vaccines, over-taking traditional treatments and vaccines which have become either ineffective or only offer short term protection. As the discussion demonstrates, a number of vaccines are already successfully protecting humans against some pathogens and disease, with the potential for further vaccines to follow. Finally, and perhaps most importantly, it should be remembered that unlike drug-based medicines, vaccines primarily offer a cure, a goal all aim to achieve.

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