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The book presents invaluable insights into the latest advancements, challenges, and research on vaccine adjuvants, which are key to developing more effective and safer vaccines essential for tackling pressing global health challenges.
Emerging Pathways of Vaccine Adjuvants: A Nonspecific Stimulant of the Immune System aims to drive progress in vaccine research, paving the way for the development of more potent and safer vaccines to address global health threats. This volume provides a comprehensive overview of the evolving landscape of vaccine adjuvants, encompassing a wide range of topics critical to their design, development, and application. Adjuvants play a crucial role in vaccine formulations by boosting the immunogenicity of antigens, thereby enhancing vaccine efficacy. While antigens can initiate immune responses independently, adjuvants amplify these responses. Extensive research efforts are focused on the formulation of adjuvants to establish accurate, efficient, and safe manufacturing techniques. This book provides a clear explanation of the strict regulatory issues, making it an essential resource for students, businesspeople, and academics across the globe.
Readers will find the book:
- Encompasses current adjuvant usage and possible tactics to ensure effective production and delivery of the active constituent;
- Presents challenges and innovations with implications to provide cheaper, more efficient solutions in the industry;
- Prepares students for work in the industry, refining their skills for the production of critical medications.
Audience
Researchers and pharmacy students in biomedical engineering and chemical engineering, biotechnology, as well as pharmaceutical and biopharmaceutical industry engineers working in drug discovery, chemical biology, computational chemistry, medicinal chemistry, and bioinformatics.
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Veröffentlichungsjahr: 2025
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Table of Contents
Cover
Table of Contents
Series Page
Title Page
Copyright Page
Preface
1 Adjuvants Boosting Vaccine Effectiveness
1.1 Vaccines Over the Years
1.2 Adjuvants in the Modern Era
1.3 Conventional Adjuvants
1.4 Particulate Adjuvants
1.5 Immunostimulatory Adjuvants
1.6 Approved Adjuvants for Human Use
1.7 Conclusion
References
2 In Silico Adjuvant Design and Validation for Vaccines
2.1 Introduction
2.2 In Silico Techniques for Adjuvant Discovery
2.3 Case Studies: Successful Applications of In Silico Adjuvant Design
2.4 Challenges and Future Directions of In Silico Adjuvant Design
2.5 Conclusion
References
3 Adjuvant and Immunity
3.1 Introduction
3.2 Immune Response to Vaccines
3.3 Mechanisms of Adjuvants in Modulating Immunity
3.4 Immunogenicity According to the Types of Adjuvants
3.5 Adjuvants and Humoral Immunity
3.6 Adjuvants and Cellular Immunity
3.7 Adjuvants and Innate Immunity
3.8 Adjuvants and Mucosal Immunity
3.9 Adjuvants and Vaccine Efficacy in Specific Populations
3.10 Conclusion
References
4 Antigen Selection and Design
4.1 Introduction
4.2 Types of Antigens Used in Vaccines
4.3 Antigen Design Strategies
4.4 Adjuvants: Mechanism of Action and Types
4.5 Novel Formulation Strategies for Improved Vaccine Efficacy
4.6 Future Directions and Challenges
4.7 Conclusion
5 Adjuvants in Licensed Vaccines
5.1 Introduction
5.2 Adjuvants Included in Vaccines
5.3 Cellular and Molecular Targets for Adjuvant
5.4 Endogenous Adjuvants in Live Vaccines
5.5 Vaccine Adjuvants in COVID-19 Vaccines
5.6 Adjuvant-Related Toxicities
5.7 Conclusion
References
6 Nanomaterial-Based Vaccine Adjuvants
6.1 Introduction
6.2 Vaccine Adjuvants and Their Role in Enhancing Immune Responses
6.3 Overview of Nanotechnology and Introduction to Innovative Applications in Medicines
6.4 Exploring the Nano Realm: Properties and Varied Types of Nanomaterials in Vaccines or Exploring Nanomaterials in Vaccines: Properties, Types, and Implications for Immunization
6.5 Engineered Nanomaterials as Vaccine Adjuvants
6.6 Challenges in the Development of ENM-Based Adjuvants
6.7 Mechanism of Action and Data
6.8 Case Studies: Examples of Nanomaterial-Based Adjuvants
6.9 Design and Development Considerations
6.10 Future Perspectives and Challenge
6.11 Conclusion
References
7 Adjuvants for Non-Invasive Routes of Vaccine Delivery
7.1 Introduction
7.2 Vaccine Delivery Through Non-Invasive Routes: Scopes and Challenges
7.3 Conventional and Novel Adjuvants
7.4 Toxicity and Adverse Events
7.5 Regulatory Approval for Adjuvants and Adjuvanted Vaccines
7.6 Prospects
7.7 Conclusion
References
8 Regulatory Guidelines for Vaccine Adjuvants
8.1 Introduction
8.2 Vaccine Adjuvants
8.3 Mechanism of Action
8.4 Adjuvant Platforms
8.5 Regulatory Guidelines for Vaccine Adjuvants
8.6 Conclusion
Acknowledgments
References
9 Adjuvant and Vaccine Safety
9.1 Introduction
9.2 Method and Mechanism of Action
9.3 Approaches and Perceptions of Adjuvant Safety in Public Health
9.4 Guidelines and Regulatory Considerations
9.5 Adjuvant Safety Testing With Emerging Technologies
9.6 Conclusion
References
10 Shortcomings of Current Adjuvants and Future Prospects
10.1 Introduction
10.2 Limitations
10.3 Advancements
10.4 This Book
10.5 The Future
References
Index
Wiley End User License Agreement
List of Tables
Chapter 2
Table 2.1 A list of the docking software and scoring function.
Chapter 5
Table 5.1 Summary table of adjuvants and their utilization.
Table 5.2 Adjuvants in licensed vaccines.
Table 5.3 Advanced vaccine designs under review.
Chapter 7
Table 7.1 Conventional adjuvants and licensed vaccines [39].
Table 7.2 Nanocarriers for various non-invasive routes of immunization.
Chapter 8
Table 8.1 Regulatory bodies and their approved vaccines.
Chapter 9
Table 9.1 Adjuvanted vaccines currently licensed by FDA and EMA for human use...
List of Figures
Chapter 1
Figure 1.1 A timeline of vaccine adjuvant development over the last 100 years...
Chapter 4
Figure 4.1 Pictorial representation of antigens used in the development of...
Figure 4.2 Flow diagram showing protocols for the selection of antigens.
Figure 4.3 Description of the mechanism of reverse vaccinology in the design...
Figure 4.4 Induction of both innate and adaptive immunity via VLP. Adopted un...
Chapter 5
Figure 5.1 Molecular targets of adjuvants [12].
Chapter 6
Figure 6.1 Engineered nanomaterials as vaccine adjuvants are capable of...
Figure 6.2 Engineered aluminum oxyhydroxide (A100H) nanorods as vaccine...
Figure 6.3 Immune activation mechanism by ENMs.
Chapter 7
Figure 7.1 Timeline of non-invasive vaccine development [3].
Figure 7.2 An infographic depicting number of research studies being pursued...
Figure 7.3 Modes of non-invasive immunization [3].
Figure 7.4 Barriers/challenges to immunization and strategies to overcome them.
Figure 7.5 Novel adjuvants for immunization.
Chapter 8
Figure 8.1 Overview of the mechanism of vaccine adjuvant.
Figure 8.2 A schematic diagram representing the mechanism of action of vaccine...
Chapter 9
Figure 9.1 Characteristic properties of vaccine adjuvant and its benefits.
Figure 9.2 Using aluminum salts as a vaccination adjuvant using a mechanical...
Figure 9.3 Innate and adaptive immune system mechanism of action.
Guide
Cover Page
Table of Contents
Series Page
Title Page
Copyright Page
Preface
Begin Reading
Index
WILEY END USER LICENSE AGREEMENT
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Scrivener Publishing100 Cummings Center, Suite 541JBeverly, MA 01915-6106
Publishers at ScrivenerMartin Scrivener ([email protected])Phillip Carmical ([email protected])
Emerging Pathways of Vaccine Adjuvants
A Nonspecific Stimulant of the Immune System
Edited by
Vivek P. Chavda
Dept. of Pharmaceutics and Pharmaceutical Technology, LM College of Pharmacy, Ahmedabad, Gujarat, India
and
Vasso Apostolopoulos
School of Health and Biomedical Sciences, RMIT University, Melbourne VIC, Australia
This edition first published 2025 by John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, USA and Scrivener Publishing LLC, 100 Cummings Center, Suite 541J, Beverly, MA 01915, USA© 2025 Scrivener Publishing LLCFor more information about Scrivener publications please visit www.scrivenerpublishing.com.
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Library of Congress Cataloging-in-Publication Data
ISBN 978-1-394-23761-6
Front cover image courtesy of Adobe FireflyCover design by Russell Richardson
Preface
Adjuvants play a crucial role in vaccine formulations by boosting the immunogenicity of antigens, thereby enhancing vaccine efficacy. While antigens can initiate immune responses independently, adjuvants amplify these responses. They do so by stimulating antigen-presenting cells and facilitating the maturation of these cells to effectively present antigenic peptides to both T and B cells. The main objective of this book is to provide readers with an in-depth understanding of the latest advancements in adjuvant technology. Ultimately, the book aims to drive progress in vaccine research, paving the way for the development of more potent and safer vaccines to address global health threats.
This book provides a comprehensive overview of the evolving landscape of vaccine adjuvants, encompassing a wide range of topics critical to their design, development, and application. Chapter 1 is an introductory chapter on adjuvants and Chapter 2 presents the cutting-edge field of in silico adjuvant design and validation, shedding light on computational approaches to optimize adjuvant properties. The chapter explores the intricate relationship between adjuvants and immunity (Chapter 3), elucidating how these immunomodulators enhance vaccine responses. Novel formulation strategies for vaccines incorporating adjuvants are discussed (Chapter 4), along with detailed characterization methods to ensure their quality and performance. The book also highlights the role of adjuvants in licensed vaccines (Chapter 5), emphasizing their contribution to vaccine efficacy. Emerging nanomaterial-based adjuvants (Chapter 6) and innovative non-invasive routes of vaccine delivery (Chapter 7) are explored as promising avenues for future vaccine development. Regulatory guidelines governing vaccine adjuvants are outlined to navigate the complex landscape of vaccine approval and licensing (Chapter 8). Importantly, the book addresses vaccine safety concerns associated with adjuvants (Chapter 9), discussing strategies to mitigate risks. Chapter 10 highlights the limitations of adjuvants and explores future directions to advance the field of vaccinology.
The Editors
Vivek P. Chavda
Vasso Apostolopoulos
November 2024
1Adjuvants Boosting Vaccine Effectiveness
Vasso Apostolopoulos
School of Health and Biomedical Sciences, RMIT University, Melbourne VIC, Australia
Abstract
Vaccine development has evolved significantly with the identification and isolation of specific antigens, leading to subunit vaccines. Adjuvants, crucial in modern vaccine design, enhance antigen immunogenicity, allowing for more effective vaccines that stimulate both humoral and cell-mediated immunity. Conventional adjuvants, including aluminum salts, SAF-1, QS-21, and squalene-based adjuvants such as MF59 and AS03, play pivotal roles in enhancing vaccine efficacy. Particulate adjuvants, including liposomes, immunostimulatory complexes, and emulsions like MF59 and AS03, offer improved antigen stability and targeted delivery. Additionally, immunostimulatory adjuvants like Toll-like receptor agonists, monophosphoryl lipid A, cytokines, and CpG oligodeoxynucleotides directly activate immune responses. Approved adjuvants, AS01, AS03, AS04, MF59, Matrix-M, and virosomes are key adjuvants in approved human vaccines, enhancing immune responses and vaccine efficacy. Despite advancements, ongoing research is required to optimize adjuvant safety and efficacy in order to develop safer and more effective vaccines against infectious diseases and cancers.
Keywords: Adjuvants, vaccination, AS01, MF59, Matrix-M, virosomes, SAF-1, QS-21
1.1 Vaccines Over the Years
The history of vaccination spans over a millennium, with early attempts to prevent infectious diseases dating back to 1000 A.D. in China, where smallpox vesicles were used for inoculation. Edward Jenner’s work in the late 1700s marked a significant advancement when he observed that individuals who had contracted cowpox were protected against smallpox. By 1796, Jenner successfully immunized a young boy with cowpox, confirming protection against smallpox. Louis Pasteur furthered the field by demonstrating the use of attenuated pathogens as vaccines in the late 19th century. He attenuated Pasteurella septica to develop a vaccine against fowl cholera and later applied a similar approach to Bacillus anthrax, achieving remarkable success in protecting farm animals. Additionally, Pasteur’s work with rabies marked a significant milestone in the development of live virus vaccines. In the realm of dead organism vaccines, the Salk vaccine against poliomyelitis, developed in 1960, had a profound impact on disease incidence before being succeeded by the Sabin vaccine. Challenges persisted in producing killed vaccines due to potential destruction of important antigenic components.
The identification and isolation of specific antigens responsible for protection paved the way for “subunit” and “extract” vaccines. For instance, diphtheria and tetanus toxoids were purified and inactivated using formalin, retaining their antigenicity but reducing adverse reactions. Despite these advancements, the history of vaccine development is not without setbacks. Disasters such as the Lubeck Disaster in 1932, where infants were mistakenly given Mycobacterium tuberculosis instead of BCG vaccine, and the Cutter Disaster in 1955, where a faulty polio vaccine led to cases of poliomyelitis, highlighted the need for stringent quality control and safety measures. As public awareness and standards for vaccine safety have increased, modern vaccinology has embraced advancements in genetics, chemistry, peptide synthesis, protein production methods, DNA, mRNA, x-ray crystal structures, molecular biology, and immunology, allowing for the development of safer and more efficient vaccines [1]. However, there are still many obstacles for their clinical use, and the limited immunogenicity of many of these candidates has hindered their development as potential vaccines. Strategies to enhance the immunogenicity of candidate vaccines are therefore critical. As such, adjuvants have been developed to enhance immunogenicity of vaccines, aiming to overcome their limited efficacy. These advancements are critical for optimizing the clinical potential of novel vaccine candidates.
1.2 Adjuvants in the Modern Era
Adjuvants play a pivotal role in modern vaccine development, enhancing the immune response to antigens and thereby improving vaccine efficacy [2–4]. While antigens alone can stimulate the immune system to some extent, adjuvants amplify this response, making vaccines more effective at inducing both humoral and cell-mediated immunity. This is particularly crucial for subunit vaccines, which consist of purified antigens and often require adjuvants to boost their immunogenicity. Additionally, adjuvants can help reduce the amount of antigen needed per dose, which is beneficial for both vaccine production and delivery. Adjuvants enable the use of novel vaccine technologies, such as synthetic peptides and recombinant proteins, which may otherwise lack sufficient immunogenicity to elicit a protective immune response [5]. Despite their importance, the development and use of adjuvants in human vaccines have been limited by safety concerns, requiring the need for rigorous testing and evaluation. As the area of vaccine development continues to advance, the discovery and optimization of safe and effective adjuvants remain a critical area of research, holding the potential to revolutionize vaccine design and contribute to global health by combating infectious diseases more effectively [6].
1.3 Conventional Adjuvants
Adjuvants play a crucial role in enhancing antigen immunogenicity, amplifying both humoral and cell-mediated immune responses. A widely used adjuvant in experimental animals is complete Freund’s adjuvant (CFA), a water-in-oil emulsion containing killed M. tuberculosis. Despite its effectiveness and long sustained immune responses, CFA is not suitable for human use due to its propensity to induce granulomas, fever, and inflammation. Incomplete Freund’s adjuvant, which lacks the mycobacterial component, has been evaluated, which does not induce granulomas and is safer than CFA, but it is still not approved for human vaccines due to other safety concerns. However, aluminum salts approved for human use in the 1930s, being either as aluminum hydroxide or aluminum phosphate, are the most widely used adjuvants in human vaccines. They enhance the immune response by forming a depot at the injection site, facilitating antigen uptake by antigen-presenting cells and stimulating cytokine secretion [7–9]. Alum primarily stimulates humoral immune responses and is used in vaccines against diphtheria, tetanus, and hepatitis B. However, aluminum-based adjuvants primarily stimulate humoral immune responses and are limited in cell-mediated immune stimulation (Figure 1.1). Emerging conventional adjuvants include the following:
SAF-1:
Comprising squalene oil, threonyl-MDP, and non-ionic block polymers, SAF-1’s block polymers act as adhesive molecules, enhancing antigen presentation and have been used in malaria and influenza vaccine studies [
10
–
12
].
Figure 1.1 A timeline of vaccine adjuvant development over the last 100 years and used in humans.
Figure adapted by content experts Iwasaki, A., Lee, J-H. and Omer, S.B at Biorender.com as Figure 1.1 in https://www.cell.com/cell/pdf/S0092-8674(20)31327-X.pdf
QS-21:
QS-21 is a potent vaccine adjuvant sourced by extraction from the Chilean soapbark tree (
Quillaja saponaria
). QS-21 exhibits freeze-thaw stability and has shown promise as an adjuvant for inducing specific CD8+ T-cell responses and exhibits minimal toxicity [
13
]. Quil A is also derived from
Quillaja saponaria
tree.
Monophosphoryl Lipid A:
A derivative of lipopolysaccharide has been used as an adjuvant in vaccines to enhance antibody and T-cell immune response to antigens. Monophosphoryl lipid A binds to Toll-like receptor 4 (TLR4) on antigenpresenting cells stimulating pro-inflammatory cytokines, maturation, and activation of antigen-presenting cells [
14
,
15
]. AS04 is the best known formulation, which incorporates both monophosphoryl lipid A and aluminum hydroxide.
Ribi Formulation:
Incorporating mycobacterial cell walls and monophosphoryl lipid A, this formulation has demonstrated superior antibody titers and both humoral and cellular immune responses compared to aluminum hydroxide adjuvants [
14
,
16
,
17
].
Squalene-Based Adjuvants:
MF59 by Novartis approved in 1997 and AS03 by GlaxoSmithKline approved in 2013 are oil-in-water emulsions containing squalene, a naturally occurring lipid. MF59 and AS03 adjuvants enhance antigen uptake by antigen-presenting cells and stimulate immune cells at the injection site, resulting in activation of both humoral and cell-mediated immune responses. MF59 is used in seasonal influenza vaccines for older adults, whereas AS03 is used in some pre-pandemic (H5N1) and pandemic influenza vaccines [
5
,
18
–
20
].
Bacterial Toxoids:
Toxoids, such as detoxified forms of tetanus and diphtheria toxins, can serve as adjuvants when co-administered with antigens [
21
]. They provide T cell help and can enhance the immune response to the co-administered antigen as were shown to be effective in anti-cancer peptide based vaccines [
22
–
27
].
Mineral Salts:
Besides aluminum salts, other mineral salts like calcium phosphate and calcium carbonate have been used as adjuvants to stabilize antigens and enhance their immunogenicity. These salts can adsorb antigens and facilitate their uptake by antigen-presenting cells.
Conventional adjuvants have been instrumental in the success of several vaccines by improving their efficacy and durability. However, they often have limitations, such as inducing primarily humoral immune responses or having reactogenicity concerns, which have driven the search for novel adjuvants with improved safety profiles and broader immunostimulatory capabilities.
1.4 Particulate Adjuvants
Particulate adjuvants are a class of adjuvants that consist of particles designed to enhance the immune response to co-administered antigens. These adjuvants are often formulated as nanoparticles, liposomes, or other particulate structures to improve antigen delivery, uptake by antigenpresenting cells and subsequent activation of the immune system. Some notable types of particulate adjuvants include the following:
Liposomes:
Liposomes, phospholipid-based vesicles, have been extensively studied since the 1970s for targeted drug delivery and immunoadjuvant applications [
28
]. They offer a versatile platform for adjuvant design, with the ability to entrap antigens, cytokines, and other immunomodulators [
29
]. As such, enhanced immune responses with liposome-entrapped antigens compared to free antigens have been shown, including some of the original studies 30 years ago against influenza virus A/PR/8 envelope proteins [
30
,
31
]. Liposomal-based vaccines hold promise for more effective and tailored approaches in the design of vaccines against infectious diseases, cancer, and other health challenges.
ISCOMs (Immunostimulatory Complexes):
ISCOMs, composed of Quil A adjuvant and peptides, achieve enhanced antigen immunogenicity with reduced adjuvant concentrations. These complexes induce both humoral and cell-mediated immune responses and have demonstrated promise in various animal models, including vaccines against hepatitis B, hepatitis C, influenza virus, malaria, human immunodeficiency, and certain veterinary vaccines [
32
–
37
].
Emulsions:
Emulsion-based adjuvants, such as MF59 and AS03, consist of oil-in-water or water-in-oil formulations. They can stabilize antigens, promote their uptake by antigen-presenting cells, and enhance immune responses, particularly in elderly individuals. Indeed, MF59 is used in seasonal influenza vaccines for elderly individuals who have not responded to standard influenza vaccines [
19
,
38
,
39
]. In addition, MF59 has been evaluated in vaccines against meningococcus B, SARS-CoV-2, and malaria [
40
].
Virosomes:
Virosomes are reconstituted viral envelopes devoid of viral genetic material. They can encapsulate antigens and fuse with cell membranes, facilitating antigen delivery and uptake by antigen-presenting cells. Virosomes are used in vaccines like Inflexal V for influenza [
41
].
Nanoparticles:
Nanoparticles made of biodegradable polymers or inorganic materials can be used to encapsulate antigens and/or adjuvants [
42
]. These nanoparticles can protect antigens from degradation, target them to specific cell types, and promote antigen uptake and presentation by antigen-presenting cells [
43
–
45
].
Microparticles:
They are larger than nanoparticles but smaller than cells and can be made from various materials, including polymers and proteins. They can be designed to secrete antigens and adjuvants in a controlled manner, enhancing antigen presentation and immune stimulation. Key findings 30 years ago noted that size of the particle was important to stimulate different arms of the immune system [
46
].
Nanogels:
Nanogels are hydrogel-based nanoparticles, which encapsulate antigens and adjuvants. They provide sustained release of encapsulated components, improve stability, and enhance antigen uptake and presentation by antigen-presenting cells.
Particulate adjuvants offer several advantages, including improved antigen stability, targeted delivery, and enhanced immune stimulation. They are being studied for use in various vaccines against infectious diseases, cancers, and other conditions to improve vaccine efficacy and facilitate the development of novel vaccine formulations.
1.5 Immunostimulatory Adjuvants
Immunostimulatory adjuvants enhance the immune response by directly activating immune cells or signaling pathways. Such examples include the following:
TLR Agonists:
TLR agonists, such as Poly I:C (TLR3) and R848, mimic pathogen-associated molecular patterns to stimulate innate immune responses [
47
]. In addition, imiquimod, a synthetic imidazoquinolinone compound, binds to TLR7 stimulating pro-inflammatory immune responses. Alum Plus is an improved adjuvant, which combines aluminum salts with TLR agonists.
Monophosphoryl Lipid:
Stimulates immune cells via binding to TLR4.
Cytokines:
Incorporation of cytokines into adjuvants and vaccine formulations has been shown to enhance immune responses, such as interleukin (IL)-1, IL-2, interferon (IFN)-gamma, and granulocyte-macrophage colony-stimulating factor (GM-CSF).
CpG Oligodeoxynucleotides:
These synthetic DNA sequences stimulate TLR9, promoting a Th1-biased immune response. CpG adjuvants are being investigated for various vaccines, including those against infectious diseases and cancer [
48
–
51
].
1.6 Approved Adjuvants for Human Use
Improved adjuvants are essential for enhancing the efficacy of vaccines by boosting the immune response to antigens. While aluminum salts have been key in human vaccines, their limitations, such as variable efficacy and lack of cell-mediated immune stimulation, have directed research into novel adjuvant formulations (Figure 1.1). Several adjuvants have been approved in human vaccines:
AS01:
The adjuvant used in the Shingrix vaccine against shingles is a combination of monophosphoryl lipid A and QS21 inducing robust immune responses even in older adults [
52
–
54
].
AS03:
AS03 adjuvant is an oil-in-water emulsion based on squalene, a natural lipid, polysorbate 80, and a-tocopherol, a form of vitamin E. It was approved in 1997 by Novartis to be used in influenza vaccines. AS03 was also present in the Pandemrix influenza vaccine of the H1N1 influenza pandemic 2009-2010. Even though Pandemrix showed strong immune responses and protection against H1N1 infections, there were increased risks of narcolepsy. As such, the use of Pandemrix was discontinued in several countries, and alternative vaccines without the AS03 adjuvant were used for subsequent influenza seasonal and pandemic response vaccines.
AS04:
This adjuvant combines aluminum hydroxide with monophosphoryl lipid A, a detoxified derivative of bacterial lipopolysaccharide, which has been shown to enhance both humoral and cell-mediated immune responses especially in human papilloma virus (HPV) vaccines. Indeed, Cervarix vaccine against HPV types 16 and 18; the Gardasil vaccine against HPV types 6, 11, 16, and 18; and Gardasil 9 against HPV types 11, 16, 18, 31, 33, 45, 52, and 58 comprise the adjuvant AS04 [
55
–
58
].
MF59:
This is composed of squalene oil, polysorbate 80, and sorbitan trioleate and has been used in seasonal influenza vaccines. MF59 stimulates immune response, particularly in older individuals who have reduced response to standard influenza vaccines.
Matrix-M:
A saponin-based adjuvant, purified from
Quillaja saponaria
Molina tree, combined with cholesterol and phospholipids to form 40-nm-like nanoparticles. Matrix-M enhances Th1 and cellular immune responses to several antigens and has a favorable safety profile. In fact, the Novavax (NVX-CoV2373) COVID-19 vaccine includes matrix-M adjuvant [
59
].
Virosomes:
These are reconstituted viral envelopes containing no viral genetic material but capable of fusing with cell membranes and antigen uptake. Virosomal adjuvants are used in the influenza vaccine, Inflexal V, to enhance immune responses [
41
,
60
–
63
].
1.7 Conclusion
The history of vaccination, spanning over a millennium, has witnessed remarkable advancements, from early inoculations in China to the modern era of sophisticated vaccine technologies. Edward Jenner and Louis Pasteur laid the foundation for vaccine development, whereas the identification of specific antigens enabled the creation of subunit and extract vaccines. Despite these strides, challenges such as safety concerns and limited immunogenicity persist, driving the need for innovative solutions like adjuvants. Adjuvants, crucial in modern vaccine development, enhance immune responses, making vaccines more effective and enabling the use of novel technologies. While conventional adjuvants like aluminum salts have been foundational, their limitations have spurred research into safer and more efficient options. Particulate adjuvants, such as liposomes and ISCOMs, offer improved antigen stability and targeted delivery, whereas immunostimulatory adjuvants like TLR agonists and cytokines directly activate immune cells, enhancing vaccine efficacy. Approved adjuvants, AS01, AS03, AS04, Matrix-M, MF59, and virosomes, have revolutionized vaccine formulations, enhancing immune responses against diseases like shingles, influenza, and HPV. Matrix-M™ in the Novavax COVID-19 vaccine exemplifies the potential of innovative adjuvants in pandemic responses. In conclusion, the ongoing evolution of vaccine technologies and adjuvants holds promise for safer and more effective vaccines. In the face of persistent global health challenges, enhancing vaccine design and delivery is essential to ensure the well-being of people worldwide.
References
1. Chavda, V.P.
et al.
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Note
:
2In Silico Adjuvant Design and Validation for Vaccines
Vivek P. Chavda1*, Anjali P. Bedse2, Pankti C. Balar3, Bedanta Bhattacharjee4, Shilpa S. Raut2 and Vasso Apostolopoulos5†
1Department of Pharmaceutics and Pharmaceutical Technology, LM College of Pharmacy, Ahmedabad, Gujarat, India
2Department of Pharmaceutics, K K Wagh College of Pharmacy, Nashik, Affiliated to Dr. Babasaheb Ambedkar Technological University, Lonere, Raigad, Maharashtra, India
3Pharmacy Section, LM College of Pharmacy, Ahmedabad, Gujarat, India
4Department of Pharmacology and Toxicology, Girijananda Chowdhury Institute of Pharmaceutical Science-Tezpur, Sonitpur, Assam, India
5School of Health and Biomedical Sciences, RMIT University, Melbourne VIC, Australia
Abstract
Vaccines are pivotal in reducing disease burden and mortality, particularly in vulnerable populations like children, the immunocompromized, and the elderly. While vaccines provide individual protection, they also establish herd immunity, protecting the broader community. In an attempt to increase immunogenicity of vaccines, adjuvants are designed and incorporated into vaccines. Adjuvants amplify immune responses through various mechanisms, enhancing antigen presentation and immune cell stimulation. Traditional adjuvant discovery is time-consuming, involving target receptor identification, high-throughput screening, and stringent safety evaluations, with predicting human reactogenicity remaining a challenge. In contrast, in silico methods, employing computational models, artificial intelligence, and machine learning, expedite adjuvant discovery by predicting efficacy and safety. Immunoinformatics tools, including epitope prediction for B-cell and T-cell recognition and molecular docking simulations, are instrumental in these computational approaches. Next-generation sequencing and high-content imaging further streamline data analysis, offering insights into adjuvant mechanisms and interactions. A multidisciplinary approach combining computational research, in vitro characterization, animal model evaluations, and toxicology studies is crucial. These partnerships validate, refine, and optimize in silico predictions, enhancing the predictive accuracy and applicability of computational methods in adjuvant discovery and design.
Keywords: Adjuvant selection, in-silico, vaccination, epitope, immunology
2.1 Introduction
2.1.1 Importance of Vaccines and Adjuvants in Immunology
Vaccines are useful to reduce the morbidity and mortality from serious illnesses, particularly those that influence children, immune compromised, and elderly. The majority of individuals who get vaccinated are primarily focused on their immediate protection. From a public health perspective, the benefit of vaccination is the establishment of herd immunity, which helps protect the community at large [1, 2]. Adjuvants boost the immune response to antigens through mechanisms like targeting specific cells, immunomodulation, stimulation of T cells, and depot formation. They can act through multiple mechanisms to enhance immunity against antigens. Adjuvants stimulate receptors such as Toll-like receptors (TLRs), nucleotide binding oligomerization (NOD)- like receptors, and retinoic acid-inducible gene (RIG)-I-like receptors and can stimulate cytokines and chemokines to skew immune responses toward specific pathways. For example, Interleukein (IL)-12 promotes Th1 responses, whereas IL-4 promotes Th2 responses. Adjuvants can also be designed to enhance antigen uptake and presentation by antigen-presenting cells (APCs), such as dendritic cells. Adjuvants, together with small molecules or biologics, directly influence immune cell activity, including immune checkpoint inhibitors or regulatory T-cell agonists [3, 4].
2.1.2 Limitations of Traditional Adjuvant Discovery Methods
The process of adjuvant discovery is labor-intensive. First, a target pattern recognition receptor, like TLR7/8, is identified in the standard adjuvant discovery. Millions of molecules are screened for their ability to agonize the target receptor using a process known as high-throughput screening. The top “hits” of activity are determined to be lead compounds after numerous iterative screening rounds. To optimize these lead compounds, modifications can be made based on structure-activity relationships. Before advancing to thorough testing, potential lead compounds are combined with antigens in vaccines. Both adjuvants and vaccines are subject to rigorous safety standards. As such, before human clinical trials can begin, extensive in vivo studies using mice and non-human primates are essential. Despite advancements in adjuvant development, accurately predicting human reactogenicity and tolerability while achieving the desired immune response remains challenging [5].
2.1.3 Introduction to In Silico Approaches for Adjuvant Design
In silico experiments, using computational models, can accelerate adjuvant discovery, often enhanced by machine learning (ML) for predictive algorithms. In silico protein modeling has identified adjuvants and used in several vaccines including those against Plasmodium yoelii and Mycobacterium tuberculosis. Machine learning techniques like black-box optimization, which included active learning, reinforcement learning, and Bayesian optimization, are used in high-throughput adjuvant screening to predict specific characteristics of adjuvants. These properties could include efficacy, safety, and interactions with immune cells. Technological advances, including next-generation sequencing, omics techniques, and ML, enable comprehensive data collection for understanding and evaluating adjuvant efficacy. High-content imaging uncovers hidden patterns, facilitating in silico binding simulations and streamlining adjuvant development at all design stages [5].
Databases and in silico tools work together in vaccine design. Databases store verified vaccines and components, setting standards for prediction programs, whereas in silico tools provide computational methods for designing new vaccines. VIOLIN, a comprehensive web-based vaccine database, includes Vaxign, a genome-based design program [6, 7]. A web-based database, Växjö, collects and analyzes information on vaccine adjuvants, including names, components, structure, and usage details. It connects these with 380 vaccines targeting 81 conditions and over 100 selected adjuvants [8].
2.2 In Silico Techniques for Adjuvant Discovery
In silico techniques for adjuvant discovery utilize computational methods to accelerate the identification and design of potential adjuvants, enhancing the efficiency and precision of vaccine development. Main features include the following:
Structure retrieval and preparation: All bound ligands are removed from the target protein during pre-processing by using the Protein Data Bank (PDB) database. Eliminating them leads to a simpler computing process and an optimal posture search [
9
].
Preparation of monoclonal antibody: The PDB database is used to obtain antibodies. In order to identify compounds with comparable structural orientation and possible actions, it is used as a ligand in the virtual screening of natural chemicals database [
9
].
Pharmacophore-based virtual screening: A limited number of hits are identified through virtual screening when 3D structures are uploaded to the PDB due to certain stereochemical features.
Druglikeness test: This is used to assess a compound’s suitability for use as a medication, helping identify compounds with favorable therapeutic properties [
9
].
Molecular dynamics simulation (MDS): Using MDS, docking results are verified, and the behavior of final candidate adjuvants within the protein binding pocket are investigated.
In vitro
validation of candidates:
In vitro
validation has become a standard approach in design and discovery processes following MDS [
9
].
2.2.1 Immunoinformatics Tools for Epitope Prediction
Immunoinformatics combines immunology with computational biology to analyze and predict immune system interactions, aiding in vaccine design, immunotherapy development, and understanding immune responses at a molecular level. High-throughput methods can provide crucial insights into immune cell-peptide interactions [10]. Immunoinformatics use bioinformatics to analyze immune system characteristics and predict B- and T-cell epitopes [11, 12]. This approach identifies antigenic regions of proteins [13] and offers a cost-effective and time-efficient alternative to laboratory based methods [14, 15].
2.2.1.1 Identification of B-Cell and T-Cell Epitopes
Antigenic peptides are presented by major histocompatibility complex [MHC class I (MHC-I) and MHC class II (MHC-II)] molecules, commonly referred to as human leukocyte antigen (HLA) in humans, for T-cell stimulation. CD8+ T cells recognize peptide-MHC-I complex and CD4+ T cells recognize peptide-MHC-II complex [16]. Using computational tools, immunoinformatics can rapidly predict peptide epitopes presented on MHC molecules, aiding in vaccine design. In contrast, B cells recognize peptides via their cell surface receptor (B-cell receptor), a membrane-bound immunoglobulin. B-cell epitopes can either be continuous (linear) or discontinuous (conformational) types. Computational tools and algorithms are important for accurate epitope prediction [17]. Linear epitopes are consecutive amino acids and do not require the protein to be folded in a 3D conformation, whereas conformational epitopes are amino acids, which are brought together in a 3D conformation in a protein, making their prediction challenging, and, as such, nuclear magnetic resonance (NMR) spectroscopy and/or crystal structures are required. The most antigenic epitopes, however, are conformational epitopes as they mimic the native structure more closely and bind to B-cell receptors with a higher affinity [18].
2.2.1.2 Tools Used for MHC and B-Cell Receptor Binding Prediction Tools
Bioinformatics tools play a crucial role in predicting MHC-I and MHC-II binding epitopes, which are important for understanding CD8+ T-cell immune responses. These tools, including lEDB, NetCTL, MHCPred, NetMHC, nHLAPred, CTLPred, SVMHC, RANKPEP, BIMAS, MAPPP, ProPred, SYFPEITHI, PREDEP, and MHCPEP (for MHC-I), as well as lEDB, NetMHC-II, MHCpred-II, MetaMHC-II, MetaSVMP, Propred-II, RANKPEP, PREDIVAC, SYFPEITH, BIMAS, CTL-Pred, EpiTOP, MHCPEP, EpiVax, PREDEPP, TEPITOPE, EPIPREDICT, EpiDOCK, Concensus, and EpiMatrix (for MHC-II), are instrumental in analyzing peptides’ binding potential to MHC molecules. These tools aid in predicting epitopes from various types of antigens, including those from bacteria, viruses, and tumors. Depending on the antigen type, the performance of each program in predicting MHC-II-bound epitopes can vary, highlighting the importance of choosing the appropriate tool for specific antigen analyses [19, 20].
Bepipred, BCpred, ABCpred, Pcipep, etc., are the tools available for predicting B-cell epitopes. The software Bepipred is commonly used to predict B-cell epitopes of viruses, and BCpred is commonly used for bacteria and ABCpred for tumor epitopes [21]. Discotope, Ellipro, CBTope, Epitope, etc., are tools used to predict conformational B-cell epitopes. Each software tool used to predict conformational epitopes of B cells is based on antigen types evaluated across three categories: viral, bacterial, and tumor-specific antigens [22]. All the above tools for both T- and B-cell epitope prediction assists in understanding immune responses mediated by T and B cells and facilitating the design of peptide-based vaccines.
2.2.2 Molecular Docking Simulation
The technique known as “molecular docking” evaluates how molecules align and conform into the binding site of a macromolecular target. Possibilities are generated by searching algorithms and ranked using scoring formulas. Over the past few decades, several softwares have been developed; some of the more well-known ones include AutoDock, AutoDock Vina, DockThor, GOLD, FlexX, and Molegro Virtual Docker. Obtaining the target structure, typically a large biological molecule (protein, DNA, or RNA) is the first step in conducting a docking calculation [23]. These macromolecular structures are readily accessed from the PDB, which offers access to three-dimensional atomic coordinates derived by experimental techniques, such as x-ray crystal structures [24]. Table 2.1 highlights the docking software and its scoring function.
Table 2.1 A list of the docking software and scoring function.
Software
Scoring
Posing
Reference
Molegro/MolDock
Semi-empirical
Alternatively Simplex Evolution and Iterated Simplex
[
25
]
AutoDock4
Semi-empirical
Algorithm, Genetic Algorithm, or Simulated Annealing
[
26
]
AutoDock Vina
Empirical/ knowledgebased
Iterated Local Search + BFGS Local Optimizer
[
26
,
27
]
SMINA
Empirical
Monte Carlo stochastic sampling + local optimization
[
27
]
ICM
Physics-based
Biased Probability Monte Carlo + Local Optimization
[
26
]
FRED
Empirical (defaults to Chemgauss3)
Conformer generation + Systematic rigid-body search
[
28
]
2.2.2.1 Application of Molecular Docking in Adjuvant Design
Molecular docking plays an important role in adjuvant design by determining the interactions between potential adjuvant molecules and target receptors. This computational approach aids in identifying and optimizing adjuvants that can enhance immune responses, thereby improving vaccine efficacy and design. Some key features include the following:
Hit Identification/Virtual Screening: By forecasting the binding affinity of small compounds to a protein or receptor of interest, it aids in the identification of possible therapeutic candidates [
27
].
Lead Optimization: Molecular docking can be utilized to refine the structure of the lead molecule to increase its binding affinity and selectivity after a hit compound has been identified [
29
].
Bioremediation: In bioremediation, molecular docking is used to determine the molecule’s binding affinity to enzymes that break down environmental contaminants [
30
].
ADMET Prediction: The absorption, distribution, metabolism, excretion, and toxicity (ADMET) characteristics of small compounds can also be predicted
via
docking [
31
].
Molecular Dynamics Simulation: Molecular docking and molecular dynamic simulations can be used together to determine the dynamic properties of protein-ligand complexes [
32
].
Structure Elucidation: Proteins with unclear structures can also have their structures clarified through the use of molecular docking [
32
].
2.2.3 Artificial Intelligence and Machine Learning for Adjuvant Design
Several fields of science, including medicine, are fast changing as a result of artificial intelligence (AI) and machine learning (ML). Whereas ML is a subset of AI, ML is the process of using data to create predictions or classifications, either with or without human supervision. AI is the construction of robots or systems that can replicate human thought and conduct. The advent of high performance computers in recent years has expedited the growth in these sectors. Digitalized medical fields like imaging are well-suited to be early users of AI and ML. Digital space facilitates the operation of the imaging pipeline, which includes picture acquisition, reconstruction, interpretation, reporting, and result sharing. This enables the efficient capture of data for AI and ML [33].
