Strategies to Improve Vaccines for the Neonate

ABSTRACT

Aside from clean water, vaccination has proven to be the most effective means of controlling infectious diseases. Current vaccines technologies include live attenuated vaccines, inactivated vaccines, vectored vaccines, and DNA vaccines. Many challenges still remain including immunization of the neonate, delivery of the vaccine via the mucosal surfaces, and vaccination against chronic infections. Some of the novel concepts to overcome these challenges are based on the use of adjuvants that can induce both innate and acquired immunity or that can target the vaccine to specific immune cells, such as dendritic cells. This article highlights some of these strategies to develop novel vaccine formulations for the neonate.

Background image: Vaccine & Infectious Disease Organization

The use of vaccines dates back to Jenner and Pasteur, who first described the concept of vaccination more than 200 years ago. Since then, vaccines have been used successfully in humans and animals, and aside from clean water represent the most effective means of controlling systemic, respiratory, and gastrointestinal infections in both humans and animals.

Adjuvants: Important Components of Vaccines

Adjuvants are important components of vaccines. They provide an additional stimulus for the immune system, which typically results in stronger immune responses, and can assist in delivering or even targeting of the vaccine. Adjuvants represent a wide variety of molecules and formulations including cell-wall components, alum, QuilA, immunostimulatory complexes (ISCOMs), carbomers, liposomes, and oil-in water emulsions, to name a few. Many of these adjuvants have been developed empirically without an exact understanding of their mechanisms of action. Recent research, however, has provided insight into the activation of the immune system by pathogen recognition receptors, including toll like receptors, C-type lectins, mannose-receptors, and nucleotide-binding oligomerization domain (NOD)-like receptors. These receptors typically recognize highly conserved molecular pattern in pathogens such as bacterial DNA, lipopolysaccharide, or flagellin^1,2 and activation often leads to increased NF-κB and/or type I interferon (IFN) production, which can result in up-regulation of chemokines and cytokines needed for a variety of immune functions including recruitment of immune cells to the site of immunization, activation and maturation of dendritic cells, and improved presentation of the antigen.^3–5 These events constitute the innate immune response phase, which serves to 1.) contain and eradicate pathogens, and 2.) induce an appropriate adaptive immune response to eliminate infection and prevent reccurrence. Thus, by signaling through pathogen recognition receptors, adjuvants can stimulate both innate and acquired immunity resulting in stronger and longer lasting immune responses to vaccination.

Immune responses elicited by vaccines can be divided into innate and acquired immune responses. Innate immune responses occur within hours of infection or vaccination and are characterized by upregulation of chemokines and cytokines, typically providing a pro-inflammatory environment at the site of immunization. Other innate immune molecules include host defense peptides, also called cationic antimicrobial peptides, complement, and acute phase proteins, which together can recruit and activate dendritic cells to provide a link with the acquired immune response.⁵ The latter is characterized by the presence of antibodies and cytotoxic T cells, both of which are highly antigen-specific and which recognize very specific structures of the pathogen.

While vaccination in the past traditionally has focused on the induction of the acquired immune response, more recent approaches are focused on stimulating both innate and acquired immunity. In fact, it has become clear now that the innate immune response sets the stage for the acquired immune response, and thus, novel immunomodulators are being used as vaccine adjuvants that can link both innate and specific immunity to enhance the immune responses following vaccination.

Vaccination of the Newborn

Enhancing and modulating immune responses is particularly important in neonates, as infants and young children are highly susceptible to many diseases. When compared to the adult immune system, the neonatal immune system displays a number of functional differences, including its bias towards a Th2-type immune response, delayed germinal center development, shorter lifespan of plasma cells, fewer T-helper cells, and functionally impaired antigen-presenting cells. Together, these functional differences represent a great challenge for the development of effective vaccines for the newborn. Furthermore, potential interference with maternally-derived antibodies makes most vaccines less effective in the neonate.

We recently developed a novel disease model for pertussis in neonatal pigs.⁶ Using this model we are currently developing novel vaccine formulations against pertussis, also called whooping cough. Pertussis is an important disease of infants and young children, causing more than 50 million cases and about 300,000 deaths every year.⁷ Current vaccines are based on recombinant proteins (acellular vaccine) or inactivated bacteria (whole cell vaccines), and require multiple immunizations between 2–60 months of age. While this is possible in developed countries, access to multiple immunizations is limited in developing countries resulting in lower vaccine coverage and less effective protection in many regions of the world. Funded by the Bill & Melinda Gates Foundation, the Krembil Foundation, and the Canadian Institutes of Health Research, our project aims at developing a variety of immunomodulators that link innate and acquired immunity and therefore can provide more effective immune protection in the neonate after fewer immunizations.

Innate Immune Stimulators: CpG DNA

Innate immune stimulators function as adjuvants that can enhance both innate and acquired immunity. The most often used stimulator of the innate immune system is bacterial DNA or synthetic DNA containing CpG motifs (CpG DNA). CpG DNA activates the immune system by signaling via toll like receptor 9, which typically results in activation of both innate and acquired immunity. Treatment with CpG oligonucleotides (ODNs) has been successfully used in a variety of experimental infectious and non-infectious diseases and clinical studies are in various stages to evaluate CpG ODN therapy against infectious disease, cancer, asthma, and allergy.^8–11 As an adjuvant, CpG ODNs promote predominantly Th1-type immune responses, a quality needed for optimal protection against intracellular pathogens. Because of the Th2 bias in neonates, however, CpG ODN also represent potential adjuvants for balancing the immune response in the neonate or skewing it toward a Th1 response, which is needed for a number of diseases including pertussis.^12,13 In this regard, neonatal mice immunized with HBsAg in the presence of CpG developed a predominantly Th1-type immune response, while immunization with only HBsAg alone induced a Th2-type response.⁹ Contradicting results have been obtained from in vitro stimulation studies with CpG using neonatal peripheral blood mononuclear cells or dendritic cells. Some studies demonstrated that stimulation with CpG DNA could elicit Th1 responses in the neonate, high levels of IgM 16 upregulation of co-stimulatory markers on dendritic cells, and proliferation of cord blood B cells. Other studies, however, showed that innate responses in the neonate including the production of IFN-α by plasmacytoid dendritic cells were impaired and Th-2 responses to allergens were increased following addition of CpG DNA to house dust mite allergens.^14–19 Despite the controversy, encouraging results have been obtained in our own laboratory, which have confirmed that CpG DNA is highly active in cord blood cells and can induce similar levels of interferons when compared to adults (Gong, et al.,unpublished results). However, more research is necessary to fully evaluate the potential of CpG DNA for the use in neonates.

Host Defense Peptides

Host defense peptides (HDPs), also called cationic antimicrobial peptides, are another example of innate immune stimulators. These molecules are found in virtually every life form and can be grouped as defensins and cathelicidins. HDPs are fundamental components of the innate immune response.^5,20 Their wide spectrum of functions includes direct antimicrobial activities, immunostimulatory functions of both innate and acquired immunity, and involvement in wound healing, cell trafficking, and vascular growth.^21–23 The antimicrobial activities of HDPs have been known for a long time, but recent evidence suggests that under physiological concentrations the immunomodulatory functions of mammalian HDPs outbalance the direct antimicrobial activities. These functions can include the recruitment of immature dendritic cells and T-cells, glucocorticoid production, macrophage phagocytosis, mast cell degranulation, complement activation, and interleukin-8 production by epithelial cells.^24–26 HDPs also have been shown to up-regulate gene expression in epithelial cells and monocytes, and neutralization of pro-inflammatory cytokine induction and lethality in response to lipopolysaccharide/endotoxin.^{5, 27–34} Moreover, recent studies have shown that host defense peptides can also adjuvant vaccines.^35–39 For example, ovalbumin-specific immune responses were enhanced after intranasal co-administration of ovalbumin and the human neutrophil peptides 1–3. The fusion of the gene encoding the murine beta-defensin 2 to the gene encoding the human immunodeficiency virus glycoprotein 120 resulted in not only stronger but also mucosal immune responses in immunized mice.^35,38,39 Other examples include B-cell lymphoma vaccines, which were enhanced by fusion of the tumor epitope with genes encoding murine beta defensins.³⁶ These examples illustrate that HDPs have been successfully used as adjuvants to enhance vaccine-specific immunity.

Polyphosphazenes

Polyphosphazenes are synthetic polymers, which are water soluble and biodegradable. They can function both as innate immune stimulators as well as delivery vehicles for vaccines. Polyphosphazenes are made from polymers with alternating nitrogen and phosphorus atoms, with side groups attached to each phosphorus.⁴⁰ Polyphosphazenes are inexpensive to produce, can be lyophilized and stored for a long time at room temperature, and are safe to use. Polyphosphazenes have been used to enhance the magnitude of the immune response against viral and bacterial antigens in mice and to modulate the quality of immune responses, resulting in a more balanced or even Th1-type of immunity.^41–44 For example, poly[di(sodium carboxylato-ethylphenoxy)phosphazene] (PCEP) altered the magnitude and quality of influenza antigen X:31-specific immune responses in mice resulting overall in higher and more balanced immune responses.⁴⁵ Similarly, PCEP induced a balanced Th1/Th2-type immune response with hepatitis B surface antigen (HBsAg), and compared to the conventional adjuvant alum induced much higher immune responses (Mutwiri, et al., unpublished results). Moreover, polyphosphazenes can be formulated in microparticles, which facilitate immunization by the mucosal routes, an important feature of modern vaccines.

Particulate Delivery Systems

Particle-based delivery of vaccine antigens has proven to be a very effective way of delivery, especially when compared to the delivery of soluble protein antigens. Microparticles can protect the vaccine antigen from degradation while increasing uptake by specialized immune cells such as dendritic cells.⁴⁶ They therefore represent effective ways of delivering vaccines, and large efforts are currently underway to develop effective particulate delivery systems. Microparticles including polymers such as PLGA particles, polyphosphazenes, liposomes, and ISCOMs facilitate phagocytosis by antigen-presenting cells.⁴⁷ Once inside the cell, they release the antigen into the phago-endosome, where it is processed and subsequently loaded onto MHC molecules for antigen-presentation. Pathogen recognition receptors such as TLR9 are present in the same compartment, suggesting the incorporation of CpG DNA as an effective method of stimulating antigen presenting cells. Furthermore, microparticles also facilitate uptake across the mucosal surfaces, which predominantly occurs by M cells which are overlying the Peyer's patches.^48,49 Dendritic cells have been shown to extend their dendrites between epithelial cells to sample antigen in the lumen, but the significance of this pathway for uptake of particles is unclear.⁵⁰ The use of microparticles in the delivery of mucosal vaccines has been modestly successful. However, novel approaches such as targeting to specific cells as well as addition of adjuvants such as CpG ODN or HDPS have the potential to improve the potency of microparticles as delivery vehicles for the induction of mucosal immune responses.⁵¹

Mucosal Vaccination

Because most invading pathogens enter the host via the mucosal surfaces, induction of strong mucosal immunity is often crucial for disease protection.⁵² Prime examples of such infections include gastrointestinal infections with enterotoxigenic E. coli (ETEC), rotavirus, or calicivirus, and respiratory infections with Mycoplasma, influenza virus, or respiratory syncytial virus. Effective vaccines therefore need to target the mucosa-associated lymphoid tissues (MALT), ideally at the potential site of infection. The MALT represent the largest immune compartment in the body and is required to (1) protect the mucosal membranes against infection; (2) to tolerate antigens derived from ingested food, airborne matter, and commensal microorganisms; and (3) to prevent the development of any potentially harmful immune responses against these antigens in case they breach the mucosal lining.⁵² To induce strong mucosal immune responses vaccines must be delivered to the mucosal site, where the vaccine antigen is taken up by either M cells, dendritic cells, or epithelial cells and subsequently passed on to antigen presenting cells. Subsequently, these cells migrate into specialized tissues such as Peyer's patches, lymph nodes, and tonsils, in which the antigen is presented to effector cells of the immune system. During this process the effector cells are imprinted to home back to the mucosal surfaces, a process mediated by interaction between locally expressed chemokines and integrins and chemokine receptors found on the surface of the effector cells. It is this interaction that allows effector cells to specifically home to the site of induction. For example, expression of the chemokine CCL25 and recognition by the chemokine receptor CCR9 allow specific homing to the small intestinal mucosa. This demonstrates the importance of delivering future vaccines by the mucosal surfaces. Delivery systems and effective mucosal adjuvants are needed that not only facilitate delivery of the vaccine to mucosal sites, but also enhance antigen uptake and provide a pro-inflammatory environment resulting in recruitment and activation of immune cells.

Conclusions

Vaccines are the most effective means of controlling infectious diseases. Novel approaches have been developed to improve existing vaccines and to develop novel vaccines. In this regard, adding of potent adjuvants that link innate and acquired immunity as well as delivery systems for mucosal delivery that eliminate the risk of needles, are promising approaches to make vaccines more effective in humans and animals. The development of vaccines for the newborn will greatly benefit from these novel technologies, as immunization of the newborn has proven to be a major challenge for today's vaccines.

Acknowledgements

Funding in the investigators' laboratories was provided by a grant through the Grand Challenges in Global Health Initiative by the Bill and Melinda Gates Foundation, the Krembil Foundation, the Canadian Institutes for Health Research (CIHR), the Natural Sciences and Engineering Research Council of Canada (NSERC), the Saskatchewan Health Research Foundation (SHRF), and the Agriculture Development Fund Saskatchewan (ADF Saskatchewan). Published with permission of the Director of the Vaccine & Infectious Disease Organization as article number 480.

VOLKER GERDTS is an associate director and GEORGE MUTWIRI is a program manager at Vaccine & Infectious Disease Organization Saskatoon, Canada, +1.306.966.1513, volker.gerdts@usask.ca

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