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Methods in
Molecular Biology 2410
Sunil Thomas Editor
Vaccine Design
Methods and Protocols,
Volume 1: Vaccines for Human Diseases
Second Edition
METHODS IN MOLECULAR BIOLOGY
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, UK
For further volumes:

http://www.springer.com/series/7651
For over 35 years, biological scientists have come to rely on the research protocols and
methodologies in the critically acclaimed Methods in Molecular Biology series. The series was
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constitute the key ingredient in each and every volume of the Methods in Molecular Biology
series. Tested and trusted, comprehensive and reliable, all protocols from the series are
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Vaccine Design
Methods and Protocols, Volume 1: Vaccines

for Human Diseases
Second Edition
Edited by
Sunil Thomas
Lankenau Institute for Medical Research, Wynnewood, PA, USA
Editor
Sunil Thomas
Lankenau Institute for Medical Research
Wynnewood, PA, USA
ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-0716-1883-7 ISBN 978-1-0716-1884-4 (eBook)
https://doi.org/10.1007/978-1-0716-1884-4
© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part
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Dedication
The healthcare and frontline workers who worked tirelessly taking care of COVID-19
patients.
Researchers who studied diligently the biology of SARS-CoV-2 and developed vaccines
to protect against COVID-19.
v
Preface
A healthy society should not have just one voice.—Li Wenliang (1986–2020)
(the first physician to recognize the outbreak of COVID-19 in Wuhan, China)
Vaccinations have greatly reduced the burden of infectious diseases. Aggressive vaccination
strategies have helped eradicate smallpox in humans and rinderpest, a serious disease of
cattle. Vaccination has greatly reduced many pediatric infectious diseases. Vaccines not only
protect the immunized but can also reduce disease among unimmunized individuals in the
community through “herd protection.” Vaccines have also led to increased production of
fish and farm animals, thereby improving food security.
The development of vaccines has improved our understanding of immunology and the
principles of immunity. This has led to the research and development of vaccines for cancer
and neurodegenerative diseases.
The world’s health and economy deteriorated since the first report of COVID-19 in
China in December 2019. The pandemic has resulted in a huge interest in the development
of vaccines. Even the skeptics were clamoring for early development of vaccines. Generally,
vaccines take around 10–15 years to reach the clinic. Advances in the knowledge of
molecular biology, immunology, and bioinformatics have led to the development of
mRNA and adenovirus vector vaccines that are more efficacious than conventional vaccines.
Collaboration at multiple levels led to the development and quick employment of COVID-
19 vaccines in the clinic within a year of the observation of the disease, making it the quickest
vaccines ever to be developed and deployed.
In 2016, we published the first edition of the book Vaccine Design: Methods and
Protocols. Volume 1: Vaccines for Human Diseases and Volume 2: Vaccines for Veterinary
Diseases. The books were a tremendous success.
The Methods in Molecular Biology series Vaccine Design: Methods and Protocols, Second
Edition, contains 87 chapters in three volumes. Volume 1: Vaccines for Human Diseases, has
an introductory section on future challenges for vaccinologists, the immunological mecha-
nism of vaccines, and trends in vaccinology. The design of human vaccines for viral, bacterial,
fungal, and parasitic diseases as well as vaccines for tumors is also described in this volume.
Volume 2: Vaccines for Veterinary Diseases includes vaccines for farm animals and fishes.
Volume 3: Resources for Vaccine Development includes chapters on vaccine adjuvants, vaccine
vectors and production, vaccine delivery systems, vaccine bioinformatics, vaccine regulation,
and intellectual property.
It has been 225 years since Edward Jenner vaccinated his first patient in 1796 to protect
against smallpox. This book is a tribute to the pioneering effort of his work. The job of
publishing the second edition of the book Vaccine Design: Methods and Protocols was
assigned at a tough time. Most of the universities were closed due to COVID-19 immedi-
ately after I took up the assignment. Several of the authors, their collaborators, and families
were infected with the virus while contributing to the book. Nevertheless, the authors
completed their chapters within the stipulated time. I am extremely grateful to the authors
for completing the task in spite of the hardship faced while contributing to the books. My
sincere thanks to all the authors for contributing to Vaccine Design: Methods and Protocols
(Edition 2); Volume 1: Vaccines for Human Diseases; Volume 2: Vaccines for Veterinary
vii
viii Preface
Diseases; and Volume 3: Resources for Vaccine Development. I would also like to thank the
series editor of Methods in Molecular Biology™, Prof. John M. Walker, for giving me the
opportunity to edit this book. My profound thanks to my parents Thomas and Thresy, wife
Jyothi for the encouragement and support, and also our twins Teresa and Thomas for
patiently waiting for me while preparing the book. Working on the book was not an excuse
for staying away from the laboratory. I made sure that my children were told about new
exciting data generated in the laboratory and the advances in science published daily before
bedtime.
Wynnewood, PA, USA Sunil Thomas

Contents
Dedication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii
PART I VACCINES: INTRODUCTION
1 Challenges for Vaccinologists in the First Half of the

Twenty-First Century . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Sunil Thomas, Ann Abraham, Patrick J. Callaghan,
and Rino Rappuoli
2 Principles in Immunology for the Design and Development
of Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Claudius U. Meyer and Fred Zepp
3 Revisiting the Principles of Designing a Vaccine . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
Shubhranshu Zutshi, Sunil Kumar, Prashant Chauhan,
and Bhaskar Saha
4 Status of COVID-19 Pandemic Before the Administration
of Vaccine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
Sunil Thomas
PART II TRENDS IN VACCINOLOGY
5 mRNA Vaccines to Protect Against Diseases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111

Sunil Thomas and Ann Abraham
6 Artificial Intelligence in Vaccine and Drug Design . . . . . . . . . . . . . . . . . . . . . . . . . . 131
Sunil Thomas, Ann Abraham, Jeremy Baldwin, Sakshi Piplani,
and Nikolai Petrovsky
PART III VACCINES FOR HUMAN VIRAL DISEASES
7 Vaccines Targeting Numerous Coronavirus Antigens, Ensuring

Broader Global Population Coverage: Multi-epitope and
Multi-patch Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
Sukrit Srivastava, Spyros D. Chatziefthymiou, and Michael Kolbe
8 Use of Micro-Computed Tomography to Visualize and Quantify
COVID-19 Vaccine Efficiency in Free-Breathing Hamsters . . . . . . . . . . . . . . . . . . 177
Laura Seldeslachts, Christopher Cawthorne, Suzanne F. Kaptein,
Robbert Boudewijns, Hendrik Jan Thibaut, Lorena Sanchez Felipe,
Sapna Sharma, Georg Schramm, Birgit Weynand, Kai Dallmeier,
and Greetje Vande Velde
ix
x Contents
9 Design of Replication-Competent VSV- and Ervebo-Vectored

Vaccines Against SARS-CoV-2. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
Qixing Liu, Zhe Ding, Jiaming Lan, and Gary Wong
10 CRISPR Engineering of Bacteriophage T4 to Design Vaccines
Against SARS-CoV-2 and Emerging Pathogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
Jingen Zhu, Neeti Ananthaswamy, Swati Jain, Himanshu Batra,
Wei-Chun Tang, and Venigalla B. Rao
11 Techniques for Developing and Assessing Immune Responses
Induced by Synthetic DNA Vaccines for Emerging Infectious Diseases . . . . . . . . 229
Ziyang Xu, Michelle Ho, Devivasha Bordoloi, Sagar Kudchodkar,
Makan Khoshnejad, Leila Giron, Faraz Zaidi, Moonsup Jeong,
Christine C. Roberts, Young K. Park, Joel Maslow,
Mohamed Abdel-Mohsen, and Kar Muthumani
12 Towards Determining the Epitopes of the Structural Proteins
of SARS-CoV-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265
Sunil Thomas
13 Development, Production, and Characterization of Hepatitis B
Subviral Envelope Particles as a Third-Generation Vaccine . . . . . . . . . . . . . . . . . . . 273
Juan Manuel Battagliotti, Diego Fontana, Marina Etcheverrigaray,
Ricardo Kratje, and Claudio Prieto
14 Generation of CpG-Recoded Zika Virus Vaccine Candidates . . . . . . . . . . . . . . . . . 289
Ivan Trus, Daniel Udenze, and Uladzimir Karniychuk
PART IV VACCINES FOR HUMAN BACTERIAL DISEASES
15 Salmonella Uptake into Gut-Associated Lymphoid Tissues:

Implications for Targeted Mucosal Vaccine Design and Delivery. . . . . . . . . . . . . . 305
Angelene F. Richards, Fernando J. Torres-Velez, and Nicholas J. Mantis
16 Development of Human Recombinant Leptospirosis Vaccines . . . . . . . . . . . . . . . . 325
Natasha Rodrigues de Oliveira, Thaı́s Larré Oliveira,
Sérgio Jorge, and Odir Antônio Dellagostin
17 Induction of T Cell Responses by Vaccination of a
Streptococcus pneumoniae Whole-Cell Vaccine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345
Emily M. Roy, Fan Zhang, Richard Malley, and Ying-Jie Lu
18 Development of a Bacterial Nanoparticle Vaccine Against
Escherichia coli. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357
Melibea Berzosa, Yadira Pastor, Carlos Gamazo,
and Juan Manuel Irache
19 Construction of Novel Live Genetically Modified BCG Vaccine
Candidates Using Recombineering Tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367
Mario Alberto Flores-Valdez and Michel de Jesús Aceves-Sánchez
Contents xi
20 An Update on Tuberculosis Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 387

Radha Gopalaswamy and Selvakumar Subbian
21 Structure-Based Design of Diagnostics and Vaccines for
Lyme Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 411
Sunil Thomas
22 Development of a SONIX Vaccine to Protect Against Ehrlichiosis . . . . . . . . . . . . 423
Sunil Thomas
PART V VACCINES FOR HUMAN PARASITIC DISEASES
23 Development of the Antileishmanial Vaccine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 433

Sunil Kumar, Shubhranshu Zutshi, Mukesh Kumar Jha, Prashant
Chauhan, and Bhaskar Saha
24 In Silico Design of Recombinant Chimera T Cell Peptide Epitope
Vaccines for Visceral Leishmaniasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 463
Amanda Sanchez Machado, Vivian Tamietti Martins,
Maria Victoria Humbert, Myron Christodoulides,
and Eduardo Antonio Ferraz Coelho
25 Preclinical Assessment of the Immunogenicity of Experimental
Leishmania Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 481
Vivian Tamietti Martins, Amanda Sanchez Machado,
Maria Victoria Humbert, Myron Christodoulides,
and Eduardo Antonio Ferraz Coelho
26 Production of Oral Vaccines Based on Virus-Like Particles
Pseudotyped with Protozoan-Surface Proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 503
Lucı́a Lara Rupil, Marianela del Carmen Serradell,
and Hugo Daniel Luján
27 A Fast-Track Phenotypic Characterization of Plasmodium falciparum
Vaccine Antigens through Lyse-Reseal Erythrocytes
Mediated Delivery (LyRED) of RNA Interference for Targeted
Translational Repression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 539
Malabika Chakrabarti, Swati Garg, Akshay Munjal,
Sweta Karan, Soumya Pati, Lalit C. Garg, and Shailja Singh
28 Plasmodium falciparum Antigen Expression in Leishmania Parasite:
A Way Forward for Live Attenuated Vaccine Development . . . . . . . . . . . . . . . . . . . 555
Akriti Srivastava, Swati Garg, Sweta Karan, Shikha Kaushik,
Anand Ranganathan, Soumya Pati, Lalit C. Garg,
and Shailja Singh
29 Molecular Characterization of a Vector-Based Candidate Antigen
Using the 30 -RACE and Genome Walking Methods and
In Silico Analysis of the Correspondent Protein for Vaccine
Design and Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 567
Abbasali Raz, Mahdieh Manafi, and Mahdokht Ilbeigi Khamseh Nejad
30 In Vitro Culture of Plasmodium falciparum for the Production of
Mature Gametocytes for Performing Standard Membrane Feeding
Assay and Infection of Anopheles spp.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 581
Abbasali Raz and Mahdokht Ilbeigi Khamseh Nejad
xii Contents
31 Plasmodium berghei Infection in BALB/c Mice Model as an

Animal Model for Malaria Disease Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 589
Abbasali Raz
32 Standard Membrane Feeding Assay for Malaria Transmission Studies. . . . . . . . . . 597
Abbasali Raz, Jafar J. Sani, and Hemn Yousefi
PART VI DEVELOPMENT OF CANCER VACCINES
33 Generation of Tumor Targeted Dendritic Cell Vaccines with

Improved Immunogenic and Migratory Phenotype . . . . . . . . . . . . . . . . . . . . . . . . . 609
Adam M. Swartz, Kelly M. Hotchkiss, Smita K. Nair,
John H. Sampson, and Kristen A. Batich
34 Monocytes as a Cellular Vaccine Platform to Induce
Antitumor Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 627
Min-Nung Huang, Vincent M. D’Anniballe,
and Michael D. Gunn
35 Beyond Sequencing: Prioritizing and Delivering Neoantigens
for Cancer Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 649
Alexander S. Roesler and Karen S. Anderson
PART VII VACCINES FOR ALLERGY
36 Proteomics for Development of Food Allergy Vaccines . . . . . . . . . . . . . . . . . . . . . . 673

Monica Carrera and Susana Magadán
PART VIII VACCINES FOR TOXINS
37 Estimating Vaccine Potency Using Antibody-Based

Competition Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 693
Jennifer Doering, Greta Van Slyke, Oreola Donini,
and Nicholas J. Mantis
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 707
Contributors
MOHAMED ABDEL-MOHSEN • Vaccine & Immunotherapy Center, The Wistar Institute,

Philadelphia, PA, USA
ANN ABRAHAM • Lankenau Institute for Medical Research, Wynnewood, PA, USA
MICHEL DE JESÚS ACEVES-SÁNCHEZ • Centro de Investigacion y Asistencia en Tecnologı́a y
diseño del Estado de Jalisco, A.C. Biotecnologı́a Médica y Farmacéutica. Av. Normalistas
800, Col. Colinas de la Normal, Guadalajara, Jalisco, Mexico
NEETI ANANTHASWAMY • Department of Biology, The Catholic University of America,
Washington, DC, USA
KAREN S. ANDERSON • Mayo Clinic, Scottsdale, AZ, USA; Center for Personalized
Diagnostics, Biodesign Institute, Arizona State University, Tempe, AZ, USA
JEREMY BALDWIN • Vaxine Pty Ltd, Adelaide, SA, Australia
KRISTEN A. BATICH • Department of Neurosurgery, Duke University Medical Center,
Durham, NC, USA; The Preston Robert Tisch Brain Tumor Center, Duke University
Medical Center, Durham, NC, USA
HIMANSHU BATRA • Department of Biology, The Catholic University of America, Washington,
DC, USA
JUAN MANUEL BATTAGLIOTTI • UNL, CONICET, FBCB (School of Biochemistry and
Biological Sciences), CBL (Biotechnological Center of Litoral), Cell Culture Laboratory,
Ciudad Universitaria, Santa Fe, Argentina
MELIBEA BERZOSA • Department of Microbiology and Parasitology, Institute of Tropical
Health, University of Navarra, Pamplona, Spain
DEVIVASHA BORDOLOI • Vaccine & Immunotherapy Center, The Wistar Institute,
ROBBERT BOUDEWIJNS • Virology and Chemotherapy, Molecular Vaccinology & Vaccine
Discovery, KU Leuven Department of Microbiology, Immunology and Transplantation,
Rega Institute, Leuven, Belgium
PATRICK J. CALLAGHAN • Lankenau Institute for Medical Research, Wynnewood, PA, USA
MÓNICA CARRERA • Department of Food Technology, Spanish National Research Council
(CSIC), Institute of Marine Research (IIM), Pontevedra, Spain
CHRISTOPHER CAWTHORNE • KU Leuven Department of Imaging and Pathology, Nuclear
Medicine and Molecular Imaging, Leuven, Belgium
MALABIKA CHAKRABARTI • Special Centre for Molecular Medicine, Jawaharlal Nehru
University, New Delhi, India
SPYROS D. CHATZIEFTHYMIOU • Deutsches Elektronen-Synchrotron (DESY), Hamburg,
Germany; Department of Structural Infection Biology, Center for Structural Systems
Biology (CSSB), Helmholtz-Center for Infection Research (HZI), Hamburg, Germany
PRASHANT CHAUHAN • National Centre for Cell Science, Ganeshkhind, Pune, Maharashtra,
India
MYRON CHRISTODOULIDES • Neisseria Research Group, Molecular Microbiology, School of
Clinical and Experimental Sciences, University of Southampton Faculty of Medicine,
Southampton General Hospital, Southampton, England, UK
xiii
xiv Contributors
EDUARDO ANTONIO FERRAZ COELHO • Programa de Pos-Graduação em Ciências da Saúde:

Infectologia e Medicina Tropical, Faculdade de Medicina, Universidade Federal de Minas
Gerais, Belo Horizonte, Minas Gerais, Brazil
VINCENT M. D’ANNIBALLE • Department of Medicine, Duke University Medical Center,
Durham, NC, USA
KAI DALLMEIER • Virology and Chemotherapy, Molecular Vaccinology & Vaccine Discovery,
KU Leuven Department of Microbiology, Immunology and Transplantation, Rega
Institute, Leuven, Belgium
NATASHA RODRIGUES DE OLIVEIRA • Universidade Federal de Pelotas, Centro de
Desenvolvimento Tecnologico, Campus Universitário s/n, Pelotas, RS, Brazil
ODIR ANTÔNIO DELLAGOSTIN • Universidade Federal de Pelotas, Centro de Desenvolvimento
Tecnologico, Campus Universitário s/n, Pelotas, RS, Brazil
ZHE DING • Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China
JENNIFER DOERING • Division of Infectious Diseases, Wadsworth Center, New York State
Department of Health, Albany, NY, USA
OREOLA DONINI • Soligenix, Inc, Princeton, NJ, USA
MARINA ETCHEVERRIGARAY • UNL, CONICET, FBCB (School of Biochemistry and Biological
Sciences), CBL (Biotechnological Center of Litoral), Cell Culture Laboratory, Ciudad
Universitaria, Santa Fe, Argentina
MARIO ALBERTO FLORES-VALDEZ • Centro de Investigacion y Asistencia en Tecnologı́a y diseño
del Estado de Jalisco, A.C. Biotecnologı́a Médica y Farmacéutica. Av. Normalistas 800, Col.
Colinas de la Normal, Guadalajara, Jalisco, Mexico
DIEGO FONTANA • UNL, CONICET, FBCB (School of Biochemistry and Biological Sciences),
CBL (Biotechnological Center of Litoral), Cell Culture Laboratory, Ciudad Universitaria,
Santa Fe, Argentina; UNL, FBCB (School of Biochemistry and Biological Sciences), CBL
(Biotechnological Center of Litoral), Biotechnological Development Laboratory, Ciudad
CARLOS GAMAZO • Department of Microbiology and Parasitology, Institute of Tropical
Health, University of Navarra, Pamplona, Spain
LALIT C. GARG • Gene Regulation Laboratory, National Institute of Immunology, New
Delhi, India
SWATI GARG • Special Centre for Molecular Medicine, Jawaharlal Nehru University, New
Delhi, India
LEILA GIRON • Vaccine & Immunotherapy Center, The Wistar Institute, Philadelphia, PA,
USA
RADHA GOPALASWAMY • Department of Bacteriology, ICMR-National Institute for Research
in Tuberculosis, Chennai, Tamil Nadu, India
MICHAEL D. GUNN • Department of Medicine, Duke University Medical Center, Durham,
NC, USA
MICHELLE HO • Vaccine & Immunotherapy Center, The Wistar Institute, Philadelphia, PA,
USA
KELLY M. HOTCHKISS • Department of Neurosurgery, Duke University Medical Center,
Durham, NC, USA
MIN-NUNG HUANG • Department of Medicine, Duke University Medical Center, Durham,
NC, USA
MARIA VICTORIA HUMBERT • Neisseria Research Group, Molecular Microbiology, School of
Clinical and Experimental Sciences, University of Southampton Faculty of Medicine,
Southampton General Hospital, Southampton, England, UK
Contributors xv
MAHDOKHT ILBEIGI KHAMSEH NEJAD • Malaria and Vector Research Group (MVRG),
Biotechnology Research Center (BRC), Pasteur Institute of Iran, Tehran, Iran
JUAN MANUEL IRACHE • Department of Technology and Pharmaceutical Chemistry,
University of Navarra, Pamplona, Spain
SWATI JAIN • Department of Biology, The Catholic University of America, Washington, DC,
USA
MOONSUP JEONG • GeneOne Life Science Inc., Seoul, South Korea
MUKESH KUMAR JHA • National Centre for Cell Science, Ganeshkhind, Pune, Maharashtra,
India; Department of Microbiology and Immunology, Columbia University, New York,
NY, USA
SÉRGIO JORGE • Universidade Federal de Pelotas, Faculdade de Medicina Veterinária,
Campus Universitário s/n, Pelotas, RS, Brazil
SUZANNE F. KAPTEIN • Virology and Chemotherapy, Molecular Vaccinology & Vaccine
SWETA KARAN • Gene Regulation Laboratory, National Institute of Immunology, New Delhi,
India
ULADZIMIR KARNIYCHUK • Vaccine and Infectious Disease Organization-International
Vaccine Centre (VIDO-InterVac), University of Saskatchewan, Saskatoon, SK, Canada
SHIKHA KAUSHIK • Special Centre for Molecular Medicine, Jawaharlal Nehru University,
New Delhi, India
MAKAN KHOSHNEJAD • Vaccine & Immunotherapy Center, The Wistar Institute,
MICHAEL KOLBE • Department of Structural Infection Biology, Center for Structural Systems
Biology (CSSB), Helmholtz-Center for Infection Research (HZI), Hamburg, Germany;
MIN-Faculty University Hamburg, Hamburg, Germany
RICARDO KRATJE • UNL, CONICET, FBCB (School of Biochemistry and Biological Sciences),
CBL (Biotechnological Center of Litoral), Cell Culture Laboratory, Ciudad Universitaria,
Santa Fe, Argentina
SAGAR KUDCHODKAR • GeneOne Life Science Inc., Seoul, South Korea
SUNIL KUMAR • National Centre for Cell Science, Ganeshkhind, Pune, Maharashtra, India
JIAMING LAN • Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China
QIXING LIU • Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China
YING-JIE LU • Division of Infectious Diseases, Boston Children’s Hospital, Harvard Medical
School, Boston, MA, USA
HUGO DANIEL LUJÁN • Centro de Investigacion y Desarrollo en Inmunologı́a y Enfermedades
Infecciosas (CIDIE), Consejo Nacional de Investigaciones Cientı́ficas y Técnicas
(CONICET)/Universidad Catolica de Cordoba (UCC), Cordoba, Argentina; Facultad
de Ciencias de la Salud, Universidad Catolica de Cordoba (UCC), Cordoba, Argentina
AMANDA SANCHEZ MACHADO • Programa de Pos-Graduação em Ciências da Saúde:
SUSANA MAGADÁN • Biomedical Research Center (CINBIO), Universidade de Vigo,
Immunology, Pontevedra, Spain
RICHARD MALLEY • Division of Infectious Diseases, Boston Children’s Hospital, Harvard
Medical School, Boston, MA, USA
MAHDIEH MANAFI • Malaria and Vector Research Group (MVRG), Biotechnology Research
Center (BRC), Pasteur Institute of Iran, Tehran, Iran
xvi Contributors
NICHOLAS J. MANTIS • Department of Biomedical Sciences, University at Albany School of

Public Health, Albany, NY, USA; Division of Infectious Diseases, Wadsworth Center, New
York State Department of Health, Albany, NY, USA
VIVIAN TAMIETTI MARTINS • Programa de Pos-Graduação em Ciências da Saúde:
JOEL MASLOW • GeneOne Life Science Inc., Seoul, South Korea
CLAUDIUS U. MEYER • Department of Pediatrics, University Medical Center Mainz, Mainz,
Germany
AKSHAY MUNJAL • Special Centre for Molecular Medicine, Jawaharlal Nehru University,
New Delhi, India
KAR MUTHUMANI • Vaccine & Immunotherapy Center, The Wistar Institute, Philadelphia,
PA, USA; GeneOne Life Science Inc., Seoul, South Korea
SMITA K. NAIR • Division of Surgical Sciences, Department of Surgery, Duke University
Medical Center, Durham, NC, USA; The Preston Robert Tisch Brain Tumor Center, Duke
University Medical Center, Durham, NC, USA
THAÍS LARRÉ OLIVEIRA • Universidade Federal de Pelotas, Centro de Desenvolvimento
Tecnologico, Campus Universitário s/n, Pelotas, RS, Brazil
YOUNG K. PARK • GeneOne Life Science Inc., Seoul, South Korea
YADIRA PASTOR • Department of Microbiology and Parasitology, Institute of Tropical Health,
University of Navarra, Pamplona, Spain
SOUMYA PATI • Department of Life Science, School of Natural Sciences, Shiv Nadar
University, Gautam Buddh Nagar, Uttar Pradesh, India
NIKOLAI PETROVSKY • Vaxine Pty Ltd, Adelaide, SA, Australia; College of Medicine and
Public Health, Flinders University, Adelaide, SA, Australia
SAKSHI PIPLANI • Vaxine Pty Ltd, Adelaide, SA, Australia; College of Medicine and Public
Health, Flinders University, Adelaide, SA, Australia
CLAUDIO PRIETO • UNL, FBCB (School of Biochemistry and Biological Sciences), CBL
(Biotechnological Center of Litoral), Biotechnological Development Laboratory, Ciudad
ANAND RANGANATHAN • Special Centre for Molecular Medicine, Jawaharlal Nehru
University, New Delhi, India
VENIGALLA B. RAO • Department of Biology, The Catholic University of America,
Washington, DC, USA
RINO RAPPUOLI • GSK Vaccines, Siena, Italy
ABBASALI RAZ • Malaria and Vector Research Group (MVRG), Biotechnology Research
ANGELENE F. RICHARDS • Department of Biomedical Sciences, University at Albany School of
Public Health, Albany, NY, USA; Division of Infectious Diseases, Wadsworth Center, New
York State Department of Health, Albany, NY, USA
CHRISTINE C. ROBERTS • GeneOne Life Science Inc., Seoul, South Korea
ALEXANDER S. ROESLER • School of Medicine, Duke University, Durham, NC, USA; Mayo
Clinic, Scottsdale, AZ, USA
EMILY M. ROY • Division of Infectious Diseases, Boston Children’s Hospital, Harvard
Medical School, Boston, MA, USA
LUCÍA LARA RUPIL • Centro de Investigacion y Desarrollo en Inmunologı́a y Enfermedades
Infecciosas (CIDIE), Consejo Nacional de Investigaciones Cientı́ficas y Técnicas
Contributors xvii
(CONICET)/Universidad Catolica de Cordoba (UCC), Cordoba, Argentina; Facultad

de Ciencias de la Salud, Universidad Catolica de Cordoba (UCC), Cordoba, Argentina
BHASKAR SAHA • National Centre for Cell Science, Ganeshkhind, Pune, Maharashtra, India;
Trident Academy of Creative Technology, Bhubaneswar, Odisha, India
JOHN H. SAMPSON • Department of Neurosurgery, Duke University Medical Center,
Durham, NC, USA; The Preston Robert Tisch Brain Tumor Center, Duke University
LORENA SANCHEZ FELIPE • Virology and Chemotherapy, Molecular Vaccinology & Vaccine
JAFAR J. SANI • Malaria and Vector Research Group (MVRG), Biotechnology Research Center
(BRC), Pasteur Institute of Iran, Tehran, Iran
GEORG SCHRAMM • KU Leuven Department of Imaging and Pathology, Nuclear Medicine
and Molecular Imaging, Leuven, Belgium
LAURA SELDESLACHTS • KU Leuven Department of Imaging and Pathology, Biomedical
MRI/MoSAIC, Leuven, Belgium
MARIANELA DEL CARMEN SERRADELL • Centro de Investigacion y Desarrollo en Inmunologı́a y
Enfermedades Infecciosas (CIDIE), Consejo Nacional de Investigaciones Cientı́ficas y Té
cnicas (CONICET)/Universidad Catolica de Cordoba (UCC), Cordoba, Argentina;
Laboratorio de Parasitologı́a y Micologı́a, Departamento de Bioquı́mica Clı́nica, Facultad
de Ciencias Quı́micas, Universidad Nacional de Cordoba (UNC). Haya de la Torre y
Medina Allende, Ciudad Universitaria, Cordoba, Argentina
SAPNA SHARMA • Virology and Chemotherapy, Molecular Vaccinology & Vaccine Discovery,
KU Leuven Department of Microbiology, Immunology and Transplantation, Rega
Institute, Leuven, Belgium
SHAILJA SINGH • Special Centre for Molecular Medicine, Jawaharlal Nehru University, New
Delhi, India
AKRITI SRIVASTAVA • Department of Life Science, School of Natural Sciences, Shiv Nadar
University, Dadri, UP, India
SUKRIT SRIVASTAVA • Infection Biology Group, Indian Foundation for Fundamental
Research, Raebareli, Uttar Pradesh, India
SELVAKUMAR SUBBIAN • The Public Health Research Institute Center at New Jersey Medical
School, Rutgers University, Newark, NJ, USA
ADAM M. SWARTZ • Division of Surgical Sciences, Department of Surgery, Duke University
WEI-CHUN TANG • Department of Biology, The Catholic University of America, Washington,
DC, USA
HENDRIK JAN THIBAUT • Virology and Chemotherapy, Molecular Vaccinology & Vaccine
Rega Institute, Leuven, Belgium; Translational Platform Virology and Chemotherapy
(TPVC), KU Leuven Department of Microbiology, Immunology and Transplantation,
SUNIL THOMAS • Lankenau Institute for Medical Research, Wynnewood, PA, USA
FERNANDO J. TORRES-VELEZ • Division of Infectious Diseases, Wadsworth Center, New York
State Department of Health, Albany, NY, USA
IVAN TRUS • Vaccine and Infectious Disease Organization-International Vaccine Centre
(VIDO-InterVac), University of Saskatchewan, Saskatoon, SK, Canada
xviii Contributors
DANIEL UDENZE • Vaccine and Infectious Disease Organization-International Vaccine

Centre (VIDO-InterVac), University of Saskatchewan, Saskatoon, SK, Canada
GRETA VAN SLYKE • Division of Infectious Diseases, Wadsworth Center, New York State
Department of Health, Albany, NY, USA
GREETJE VANDE VELDE • KU Leuven Department of Imaging and Pathology, Biomedical
MRI/MoSAIC, Leuven, Belgium
BIRGIT WEYNAND • KU Leuven Department of Imaging and Pathology, Division of
Translational Cell and Tissue Research, Leuven, Belgium
GARY WONG • Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China;
Département de microbiologie-infectiologie et d’immunologie, Université Laval, Québec,
QC, Canada
ZIYANG XU • Vaccine & Immunotherapy Center, The Wistar Institute, Philadelphia, PA,
USA
HEMN YOUSEFI • Malaria and Vector Research Group (MVRG), Biotechnology Research
FARAZ ZAIDI • Vaccine & Immunotherapy Center, The Wistar Institute, Philadelphia, PA,
USA
FRED ZEPP • Department of Pediatrics, University Medical Center Mainz, Mainz, Germany
FAN ZHANG • Division of Infectious Diseases, Boston Children’s Hospital, Harvard Medical
School, Boston, MA, USA
JINGEN ZHU • Department of Biology, The Catholic University of America, Washington, DC,
USA
SHUBHRANSHU ZUTSHI • National Centre for Cell Science, Ganeshkhind, Pune,
Maharashtra, India
Part I
Vaccines: Introduction
Chapter 1
Challenges for Vaccinologists in the First Half

of the Twenty-First Century
Sunil Thomas, Ann Abraham, Patrick J. Callaghan, and Rino Rappuoli
Abstract
The COVID-19 pandemic of 2020–2021 has highlighted the importance of vaccines and vaccination in
human health. The pandemic has resulted in social distancing, travel restrictions, decreased trade, high
unemployment, commodity price decline, and financial stress that has impacted the global economy. Since
December 2020, a massive vaccination campaign is undergoing in every country on the planet to protect
against SARS-CoV-2. Vaccination is the cheapest health-care interventions that can save more lives than any
other drugs or therapies. Some of the common diseases of the twentieth century including smallpox and
polio are seldom reported due to intense vaccination programs that eradicated it. Smallpox is completely
eradicated globally; whereas, polio is confined to only a couple of countries. Vaccination has not only
improved the health of man but also improved food security by preventing diseases in farm animals and
aquacultured fish. Awareness of the principles of immunology and novel vaccines has led to effective
vaccination strategies. Climate change could lead to generation of new strains of infectious microorganisms
that would require development of novel vaccines. Recent years have seen the increase in incidence of brain-
eating amoeba and flesh-eating bacteria (necrotizing fasciitis). There are no vaccines for these diseases.
Though vaccination programs have eradicated several diseases and increased the quality of life, there are
several diseases that have no effective vaccines. Currently there are no vaccines for cancer, neurodegenera-
tive diseases, autoimmune diseases, as well as infectious diseases like tuberculosis, AIDS, and parasitic
diseases including malaria. Spontaneous evolution of pathogenic microorganisms may lead to pandemics
that impact the health of not only humanity but also other animals. Hence, the challenge to vaccinologists is
the development of novel vaccines and vaccination strategies within limited time period and using mini-
mum resources. In addition, the vaccine developed should be administered globally within a short duration
so as to prevent generation of pathogenic variants more lethal than the parent strain.
Key words Climate change, Infectious disease, Vaccine, COVID-19, Vaccine challenge
1 Introduction
Infectious diseases are disorders caused by microorganisms includ-

ing bacteria, viruses, fungi, or parasites that are passed, directly or
indirectly, between people or due to exposure from infected ani-
mals. Infectious diseases are a leading cause of death worldwide,
particularly in low-income countries, especially in young children.
Sunil Thomas (ed.), Vaccine Design: Methods and Protocols, Volume 1: Vaccines for Human Diseases,
Methods in Molecular Biology, vol. 2410, https://doi.org/10.1007/978-1-0716-1884-4_1,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022
3
4 Sunil Thomas et al.
The current pandemic COVID-19 caused by SARS-CoV-2 (started

in December 2019) has led to millions of infections and deaths
within 1 year of its occurrence. The incidence of death due to
COVID-19 was more in developed countries compared to devel-
oping countries. Millions of deaths, travel restrictions, decreased
trade, high unemployment, and financial stress have impacted the
global economy; having an effective vaccine earlier would have
resulted in a different scenario. Vaccinologists raced against time
to develop and test COVID-19 vaccines; by December 2020, they
were ready to be administered globally.
The importance of vaccine in the health and well-being of the
society was confirmed during the COVID-19 pandemic. Vaccines
have revolutionized healthcare since the mid-twentieth century.
Though less than 1% of microbial species are human pathogens,
infectious diseases are still major cause of human morbidity and
mortality, accounting for tens of millions of deaths each year
[1, 2]. The number of deaths inflicted by microorganisms and the
number of individuals maimed from infectious disease would actu-
ally be much higher in a world without vaccinations [3, 4]. Millions
of deaths are prevented annually due to the reduced incidence of
infectious diseases such as measles, polio, diphtheria, pertussis,
tetanus, and whooping cough [4–8]. Undoubtedly, vaccines are
among the most important scientific and public health achieve-
ments since their discovery over 200 years ago [3]. Despite the
usage and popularity of vaccines in developed countries, one-third
of all deaths in several developing countries occur in children, and
infectious diseases are the leading cause of these deaths [9]. Hence,
there is a need for mass vaccination programs benefitting children
of these countries to prevent curable infectious disease.
The COVID-19 pandemic caused by SARS-CoV-2 has opened
new challenges to the vaccinologists. There is a need to quickly
develop novel vaccines against new and emerging diseases with
limited resources. Several diseases including AIDS, malaria, that
are yet to have vaccines require new strategies for vaccine develop-
ment. This chapter states the challenges that will be faced by
vaccinologists in the first half of the twenty-first century.
1.1 Change and Climate change will be the greatest threat to humanity in the
Emerging Infectious twenty-first century. Climate change is associated with increase in
Diseases temperature, changes in weather patterns, increase in sea level,
frequency of hurricanes and cyclones, increase or decrease in rain
fall patterns depending on the region, forest fires, expansion of
desertification, salinization of cultivable lands, decrease in snow,
and destruction of glaciers and ice shelves.
Climate change will impact food security, availability of drink-
ing water, flooding of coastal areas, air pollution, and may also lead
Challenges for Vaccinologists 5
to mass migration. Lack of fresh water would lead to use of con-

taminated water that may lead to waterborne diseases.
Changes in the weather and temperature have an impact on
insect vectors. Increase in temperature leads to increase in mosqui-
tos thereby increasing the range of diseases like malaria, dengue,
and Zika virus. The population of ticks is controlled during winter
season; however, a warm winter season would not regulate the tick
population [10], thereby increasing incidence of Lyme disease,
rickettsiosis, and ehrlichiosis. Changes in rain fall patterns especially
in the desert regions that are breeding grounds for locust could lead
to increase in locust population that threatens the global food
security. The recent locust swarms (year 2020) extended from
East Africa to India [11] and devastated crops in the region. Hun-
ger and malnutrition weaken the immune system; people subjected
to starvation or on low nutrient diet are more prone to infectious
diseases [12, 13].
1.2 Development of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is

Vaccines to Protect responsible for the disease COVID-19 that has decimated the
against COVID-19 health and economy of our planet. The virus causes the disease
not only in people but also in companion and wild animals. People
with diabetes are at risk of the disease. As yet we do not know why
the virus has been highly successful in causing the pandemic within
3 months of its first report [14, 15]. The disease was first reported
in Wuhan, China, in December 2019 [16]. As of March 2021,
116 million people are infected with COVID-19 with 2.5 million
deaths. The unofficial figures may be many times higher.
COVID-19 is regarded as a respiratory disease that manifests
with fever, cough, shortness of breath or difficulty breathing, chills,
muscle pain, headache, sore throat, and loss of taste and smell.
Other symptoms include diarrhea, nausea, and vomiting. Many
patients with the COVID-19 are asymptomatic but are able to
transmit the virus to others [14, 15]. The prolonged pandemic
has resulted in social distancing, travel restrictions, decreased
trade, high unemployment, commodity price decline, and financial
stress that has impacted the global economy. COVID-19 disease is
caused by the severe acute respiratory syndrome coronavirus
2 (SARS-CoV-2), a member of the betacoronavirus genus. The
disease has resulted in a mortality of 0.5–8.0%. Several factors
influenced the death rate in people with COVID-19. Age, health,
and behavior of the population impacted the death rate due to
COVID-19. Old people and people with underlying diseases such
as diabetes, lung diseases (due to smoking), liver disease, cardiovas-
cular disease, and obesity are more prone to death due to COVID-
19. As yet, there are no effective drugs available for treatment of the
disease [14].
The major structural proteins of SARS-CoV-2 are spike (S),
membrane (M), envelope (E), and the nucleocapsid (N) proteins
[14]. The spike protein of SARS-CoV-2 uses the host angiotensin-
converting enzyme 2 (ACE2) as the entry receptor. Hence, the

research community was interested in studying the S protein for
vaccine development. There are several variants of COVID-19
vaccines. The vaccines developed by companies are based on
mRNA, adenovirus vector, or live attenuated. The efficiency of
the vaccines ranges from 50% to 95%. The mRNA vaccines were
found to be more efficient than conventional vaccines. Vaccines
were administered beginning December 2020.
Most countries did not strictly enforce social distancing. This
has resulted in quick spread of the virus and its mutation. Viruses
generally acquire mutations over time, giving rise to new variants.
Several variants of the SARS-COV-2 are reported recently. Lineage
B.1.1.7 is also known as the UK variant. B.1.1.7 variant may be
associated with an increased risk of death compared with other
variants. This variant has a mutation in the receptor binding
domain (RBD) of the spike protein at position 501, where the
amino acid asparagine (N) has been replaced with tyrosine (Y).
The 501Y.V2 variant (lineage B.1.351) is the South African strain.
This variant has multiple mutations in the spike protein, including
K417N, E484K, and N501Y. Some of the vaccines may not be
protected by this variant. P.1 is a close relative of the B.1.351
lineage and is the Brazil variant. This variant has 17 unique muta-
tions, including three in the receptor binding domain of the spike
protein. Lineage B.1.429 is the California variant (CAL.20C). The
S protein L452R mutation is within a known receptor binding
domain that has been found to be resistant to certain S protein
monoclonal antibodies [17]. The vaccines currently developed do
not protect against all the variants. Hence, the challenge is to
develop vaccines that could protect against all the variants of
SARS-CoV-2.
The COVID-19 vaccine was developed during lockdown con-
ditions with minimum support. The Universities and laboratories
were under lockdown during the vaccine development. Companies
had to use their resources to achieve their goals. Pfizer BioNTech
and Moderna had to use a never tested vaccine technology—
mRNA—to develop an effective COVID-19 vaccine. The mRNA
vaccine was successful because there was prior knowledge on the
use of the strategy for overexpression of proteins and use of tech-
nology to protect against diseases [18–21].
Companies and research organizations engaged in basic and
translational research, collaborate effectively and have a team that
could perform tasks with minimum support and at minimum time
has a cutting edge in developing new and innovative vaccines.
1.3 Development of Acquired immunodeficiency syndrome (AIDS) caused by human

Vaccines to Protect immunodeficiency virus (HIV) has caused millions of infections
against HIV and deaths since early 1980s. Despite the countless efforts of
research, there is yet to develop a vaccine or drug that can
successfully prevent or eradicate the disease. It is a continual raging

pandemic that is affecting millions of people globally. Approxi-
mately, 38 million people are living with HIV around the globe
[22]. Tiredness and fatigue are common problems among people
with HIV. Other symptoms of HIV include lack of energy, sleep
disturbance, anxiety, and depression [23]. AIDS costs billions of
dollars in lost earnings, largely as a result of the deaths of hundreds
of thousands of workers that could have been prevented with
effective treatment. Lack of energy and fatigue in people with
AIDS contribute to loss of income.
The structure of the HIV virus is uniquely complex due to its
mutability and genetic diversity, which in turn adds to the challenge
of inducing an immune response in the host. HIV-1, is an RNA
virus, has been estimated to have the highest mutation rate of any
biological product [24, 25]. This introduction of antigenic varia-
tion limits recognition of the virus by the immune system, and this
issue is compounded by antibodies having minimal accessibility and
specificity for native envelope proteins on virus’ surface [26]. Fur-
ther, infected cells that express HIV have reduced expression of
MHC class I on their surface, causing these infected cells to be
resistance to cytolysis, effectively providing a hideout for the virus
[26]. As yet there are no vaccines to protect against HIV. There is
an urgent need to develop a vaccine against the life-threatening
disease.
2 Development of Vaccines for Flaviviruses
The Flaviviridae family consists of 70 different viruses that are

arthropod-borne viruses and transmitted by mosquitoes or ticks.
The mosquitoes-transmitted viruses include yellow fever, dengue
fever, Japanese encephalitis, West Nile virus, and Zika virus. The
flaviviruses transmitted by ticks are responsible for encephalitis and
hemorrhagic diseases and include tick-borne encephalitis (TBE),
Kyasanur Forest disease (KFD) and Alkhurma disease, and Omsk
hemorrhagic fever [27].
The major flaviviruses of economic importance include the
West Nile virus (WNV), dengue, and Zika virus, and none of
them have vaccines for humans. The West Nile virus is known to
infect mosquitoes, birds, horses, and humans. It is a member of the
Flavivirus family and is spread through infected mosquitoes
[28]. Despite the significant health risk, including three million
infections in the United States, there are no vaccines for the disease.
Approximately 80% of cases are asymptomatic, which makes it
difficult to diagnose patients who are infected [29]. Several vaccine
candidates are in various stages of clinical development; however,
demonstrating efficacy and the unpredictability of WNV outbreaks
pose a challenge for securing an effective vaccine. ChimeriVAX-
WN02 is a promising candidate with 96% seroconversion rates

observed during phase II of clinical trials. Another phase II clinical
trial observed a 92% seroconversion rates in all tested doses for
50 year olds and older. Other phase I clinical trials include
WNV-DENV chimeric vaccine, a naked DNA vaccine, and
WN-80E, which have been completed. These candidates have
shown promising immunogenic properties in humans; however, a
major hurdle is the cost-effectiveness of developing a vaccine for
WNV. Due to the low incidence rates and inconsistent activity, it
would not be beneficial for pharmaceutical companies to introduce
WNV routine immunization [30]. The West Nile virus is increasing
in vulnerable populations; therefore, it is essential for vaccinologists
to reconsider the cost-effectiveness and determine the importance
of immunizing humans.
Zika virus emerged slowly yet made an outstanding impact in
the lives of many. The virus was first found in a Rhesus monkey and
in mosquitoes in the Zika forest (Uganda) in 1947 and 1948. In
1952, the first human case was reported [28]. It is primarily trans-
mitted by mosquitoes. The Zika virus caught the world’s attention
in 2007 when it caused an epidemic on Yap Island, Micronesia.
Later in 2013–2014, large epidemics arose in French Polynesia and
other regions of the South Pacific. In May 2015, the World Health
Organization reported the first local transmission of Zika virus in
the Region of the Americas (Americas), with autochthonous cases
identified in Brazil. In December, the Ministry of Health estimated
that 440,000–1,300,000 suspected cases of Zika virus (ZIKV)
disease had occurred in Brazil in 2015. By January 20, 2016, locally
transmitted cases had been reported to the Pan American Health
Organization from Puerto Rico and 19 other countries or terri-
tories in the Americas [31].
ZIKV infection is linked with activation of Guillian-Barré syn-
drome (GBS) in adults infected with the virus and microcephaly in
infants following maternal infection [32]. Association between
ZIKV infection and microcephaly via vertical transmission of virus
from pregnant mother to her fetus was illustrated based on an
increased incidence of microcephaly possibly pertaining to ZIKV
outbreak, presence of ZIKV in microcephalic fetal brain as well as
the presence of viral RNA in the amniotic fluid of pregnant women
[33–35]. Zika-related birth defects are also linked to pregnancy
losses, which include miscarriages and abortions (CDC)
[32]. Once infected, the disease symptoms include fever, rash,
headache, joint pain, red eyes, and muscle pain. The World Health
Organization declared the Zika virus (ZIKV) as a Public Health
Emergency of International Concern in 2016 [36]. The virus has
infected approximately 1.5 million people. ZIKV stems from two
genetic lineages (African and Asian) with <5% amino acid variation
among the various strains. It is understood that ZIKV can be
characterized with a single serotype. As of 2018, vaccine candidates
have advanced to clinical phase I/II trials. Candidates for the

vaccine have been tested using inactivated vaccines, subunit vac-
cines, and live-attenuated vaccines. Inactivated vaccines and DNA
subunit vaccines have demonstrated safety and immunogenicity.
Live-attenuated vaccines may be desirable due to the advantage of
administering a single dose, immediate immunity, protection, and
low cost. Since ZIKV is mostly native to developing countries, a
single-dose vaccine is practical and beneficial. Despite the progress,
vaccine development for ZIKV has faced obstacles: animal models
that can represent human GBS are not available, the epitopes
responsible for virus-specific neutralizing antibodies need to be
determined, the decline of ZIKV human cases limits the testing
needed in both nonpregnant and pregnant women, 80% of infected
individuals are asymptomatic, a diagnostic assay is necessary to
support clinical trials, and better understanding the biology of
ZIKV in humans, especially nonpregnant and pregnant
women [37].
Dengue fever is a very prominent anthropod-borne disease
globally. Dengue fever (DF) and dengue hemorrhagic fever
(DHF) are caused by DENV serotypes. It is estimated that
50–100 million infections and 50,0000–500,0000 DHF cases
with more than 20,000 deaths occur annually [30]. The virus is
transmitted by the yellow fever mosquito, which has adapted to
urban conditions, and commonly concentrated in tropical and
subtropical areas. Currently, mosquito control is the only way to
prevent an increase in cases. The pathogenic implications of DHF
development are not fully grasped by vaccinologists. It is under-
stood that both viral and host factors may be involved. The viru-
lence of each DENV strain and the genetic information of the
infected individuals may be significant factors in DENV infection.
Intensive research for the past 50 years has led to the development
of the dengue vaccine Dengvaxia in 2015.
Dengvaxia is a live-attenuated dengue vaccine consisting of a
mixture of four yellow fever–dengue virus chimeras developed by
Sanofi Pasteur, completed phase IIb, III efficacy trials in >35,000
children, ages 2–16 years in 10 dengue-endemic countries in Asia
and the Americas [38]. Dengvaxia is efficacious in seropositive
individuals, but increases the risk for severe dengue in seronegative
persons about 2 years after administration of the first dose. In a
phase IIb trial in Thailand, 2.8% of children vaccinated at ages
2–5 years and 1.4% of children vaccinated at age 9 or above were
hospitalized for breakthrough dengue infections [39]. For
countries considering the introduction of Dengvaxia, WHO
recommends a prevaccination screening strategy whereby only per-
sons with evidence of a past dengue infection would be
vaccinated [40].
Dengvaxia, lacking DENV nonstructural protein antigens,
does not protect seronegatives because it failed to raise a competent
T-cell response and/or antibodies to NS1 [39]. Dengvaxia was

used in the Philippines to vaccinate 9–10-year-old school children,
living in areas highly endemic for dengue. After about 830,000 had
received at least 1–3 recommended doses, risks of enhanced disease
in dengue-naı̈ve vaccinees were reported [41].
Several types of dengue vaccines are in the developing stages,
and some are under preclinical and clinical evaluation [30]. Takeda’s
tetravalent dengue vaccine candidate (TAK-003) is based on a live-
attenuated DENV-2 virus that provides the genetic backbone for all
four of the vaccine viruses. TAK-003 demonstrated continued
benefit independent of baseline serostatus in reducing dengue
with some decline in efficacy during the second year [42]. Biswal
et al. (2020) [43] demonstrated that TAK-003 has an acceptable
safety profile in healthy children aged 4–16 years and is efficacious
in the prevention of symptomatic dengue disease in both indivi-
duals who are dengue-naive and those previously exposed. Efficacy
varied against individual serotypes, with an overall efficacy of 66% in
individuals who were dengue-naive and 76% in those who were
preexposed. In addition, TAK-003 reduced the number of dengue
cases that were hospitalized by 90% along with an 86% reduction in
dengue hemorrhagic fever. These data represent a major step for-
ward in the development of an effective and safe dengue vaccine for
use in people of all ages, irrespective of previous dengue exposure at
the time of vaccination. The immunogenicity and protective effi-
cacy of a candidate tetravalent dengue virus purified inactivated
vaccine (TDENV PIV) formulated with alum or an Adjuvant Sys-
tem (AS01, AS03, or AS04) was evaluated in a 0, 1-month vaccina-
tion schedule in rhesus macaques. When tested in a nonhuman
primate infection model, adjuvanted TDENV PIV vaccine formu-
lations showed an acceptable safety profile and were highly immu-
nogenic, with all formulations inducing robust and persistent
neutralizing antibody responses against each of the four DENV
serotypes [44]. However, currently the only available
WHO-approved dengue vaccine is Dengvaxia.
3 Development of Vaccines for Norovirus
Norovirus, also known as Norwalk virus, is responsible for gastro-

intestinal distress or commonly known as the “stomach flu.” The
first recorded outbreak of the norovirus was in Norwalk, Ohio in
1968, where 50% of the elementary students were infected. Symp-
toms of the virus are vomiting, diarrhea, and a low fever [45]. Nor-
oviruses are the leading cause for gastroenteritis spanning all age
groups. Outbreaks of noroviruses are likely to occur in close contact
communities, such as schools, prisons, hospitals, hotels, or cruise
ships [28].
Norovirus is the leading cause of acute gastroenteritis (AGE)

worldwide, and in the United States, norovirus is estimated to
cause 19–21 million illnesses, 1.7–1.9 million outpatient visits,
56,000–71,000 hospitalizations, and 570–800 deaths annually
[46]. The main mode of transmission is the fecal–oral route
which can be through food, water, and environmental contamina-
tion. The direct contamination of food during production and
contamination of food during preparation have been responsible
for foodborne transmission of the virus. Raw fruits, vegetables, and
shellfish are main risks of infection. Waterborne transmission of the
virus may be due to municipal water systems, commercial ice con-
sumption, water sources at camps, and recreational water exposure.
The general population is prone to acquire the viral infection, but
the majority of deaths occurs at extreme ages, such as infants and
the elderly. Although prevention (good hygiene, washing fruits and
vegetables thoroughly, and cooking seafood properly) is the only
approach to limit infection, a vaccine is ideal to encourage an
immune response in humans. The main obstacles facing vaccine
development for noroviruses are the lack of an animal model that
resembles the human disease, the viral and human host diversity,
and virus evolution [45]. Another major barrier to studying the
pathogenesis, virus–host interactions, and effect of control mea-
sures to prevent and treat norovirus gastroenteritis has been the
lack of a robust and reproducible cell culture system [47].
Vaxart, a US biotechnology company, is developing an oral
vaccine platform to protect against norovirus. This tablet vaccine
comprises a nonreplicating adenovirus-based vector expressing the
VP1 gene from the GI.1 norovirus strain and a double-stranded
RNA adjuvant. Sixty-six adult subjects meeting inclusion/exclu-
sion criteria were randomized 2:1 to receive a single vaccine dose or
placebo, respectively. This oral norovirus vaccine was well-tolerated
and generated substantial immune responses, including systemic
and mucosal antibodies as well as memory IgA/IgG. These results
are a major step forward for the development of a safe and immu-
nogenic oral norovirus vaccine [48]. Treanor et al. (2020) [49]
investigated the safety and immunogenicity of bivalent virus-like
particle (VLP) vaccine candidate formulations with and without
monophosphoryl lipid A (adjuvant MPL). Adults over 60 years of
age displayed no safety concerns and had similar immune responses
to the norovirus VLP vaccine candidate as younger adults, unaf-
fected by increasing age, a second dose, or inclusion of MPL.
4 Development of Vaccines for Influenza
Influenza is an infectious respiratory disease; in humans, it is caused

by influenza A (genus influenzavirus A) and influenza B (genus
influenzavirus B) viruses. Symptoms associated with influenza virus
infection vary from a mild respiratory disease confined to the upper

respiratory tract and characterized by fever, sore throat, runny nose,
cough, headache, muscle pain, and fatigue to severe and in some
cases lethal pneumonia owing to influenza virus or to secondary
bacterial infection of the lower respiratory tract. Influenza A viruses
cause flu pandemics. A pandemic can occur when a new and very
different influenza A virus emerges that both infects people and has
the ability to spread efficiently between people [50].
The WHO estimates that annual epidemics of influenza result
in ~1 billion infections, 3–5 million cases of severe illness and
300,000–500,000 deaths. The severity of pandemic influenza
depends on multiple factors, including the virulence of the pan-
demic virus strain and the level of preexisting immunity. The most
severe influenza pandemic, in 1918, resulted in >40 million deaths
worldwide. Influenza vaccines are formulated every year to match
the circulating strains, as they evolve antigenically owing to anti-
genic drift. Nevertheless, vaccine efficacy is not optimal and is
dramatically low in the case of an antigenic mismatch between the
vaccine and the circulating virus strain [50].
Influenza viruses are members of the Orthomyxoviridae family
and are enveloped negative-sense single-strand RNA viruses with a
segmented genome. Influenza A and influenza B viruses contain
eight RNA segments, which encode RNA polymerase subunits,
viral glycoproteins (namely, hemagglutinin (HA), with its distinct
globular “head” and “stalk” structures, which facilitate viral entry,
and neuraminidase (NA), which facilitates viral release), viral nucle-
oprotein (NP), matrix protein (M1) and membrane protein (M2),
the nonstructural protein (NS1), and nuclear export protein
(NEP). The HA and NA viral proteins are the most antigenically
variable, and in the case of influenza A virus, they are classified into
antigenically diverse subtypes. These two viral glycoproteins are
located at the surface of the virus particle and are the main targets
for protective antibodies induced by influenza virus infection and
vaccination [50]. There are 18 different hemagglutinin subtypes
and 11 different neuraminidase subtypes (H1 through H18 and N1
through N11, respectively). While there are potentially 198 differ-
ent influenza A subtype combinations, only 131 subtypes have been
detected in nature. Current subtypes of influenza A viruses that
routinely circulate in people include A(H1N1) and A(H3N2) [51].
The challenges in developing an influenza vaccine include the
dependence on embryonated eggs for vaccine production, the
lengthy timeline for vaccine production, the need for annual vacci-
nation, the emergence of antigenically novel viruses, the need for
improved immunogenicity in the elderly, and the need for an
improved correlate of protection. The ultimate goal of a universal
influenza vaccine is to protect against all influenza A viruses, obviat-
ing the need for annual revaccination. Influenza vaccines must
protect all age groups, particularly those most vulnerable to
complications of severe influenza. Ideally, new vaccines should

increase the breadth of the immune response to include antigeni-
cally distinct viruses within the same subtype and viruses of other
subtypes, should not be manufactured in eggs, and should require
less time to manufacture than currently licensed technologies [52].
Strategies employed to improve vaccine efficacy involve using
structure-based design and nanoparticle display to optimize the
antigenicity and immunogenicity of target antigens; increasing the
antigen dose; using novel adjuvants; stimulating cellular immunity;
and targeting other viral proteins, including neuraminidase, matrix
protein 2, or nucleoprotein [53].
5 Development of Vaccines for Sepsis
Sepsis (septicemia) is a medical emergency that describes the body’s

systemic immunological response to an infectious process that can
lead to end-stage organ dysfunction and death [54].
The global epidemiological burden of sepsis is, however, diffi-
cult to ascertain. It is estimated that more than 30 million people
are affected by sepsis every year worldwide, resulting in six million
deaths annually. Mortality rates from sepsis are approximately 41%
in Europe versus approximately 28.3% in the United States [55].
There are currently no effective pharmacological treatments for
sepsis, making early recognition, resuscitation, and immediate
treatment with appropriate antibiotics the key to reducing the
burden of resulting disease. The majority of sepsis, around
70–80%, is community acquired making emergency departments
and primary care key targets to improve recognition and early
management. Case fatality rates for sepsis are decreasing in many
countries with the reduction attributed to national or regional
screening and quality improvement programs focused on early
identification and immediate treatment.
Sepsis, now defined as life-threatening organ dysfunction due
to a dysregulated host response to infection, was recently recog-
nized by the World Health Organization as a global health priority.
Sepsis causes or contributes to up to half of all in-hospital deaths in
the USA [56].
Sepsis may be due to bacterial, viral, mycoplasma, or fungal
pathogens. The common species causing sepsis includes Pneumo-
coccus, Staphylococcus, Mycoplasma, Legionella, Escherichia coli,
Klebsiella, Enterococcus, Candida, Streptococcus,
methicillin-sensitive Staphylococcus aureus and Neisseria
sp. [56]. GBS septicemia is caused by the bacterium Streptococcus
agalactiae, which is commonly called group B strep, or GBS. GBS
causes sepsis in newborns. GBS can also cause serious infections in
adults that include bloodstream infections, pneumonia, skin and
soft-tissue infections, and bone and joint infections [57].
Secretion of extracellular vesicles (EVs), a process common to

eukaryotes, archaea, and bacteria, represents a secretory pathway
that allows cell-free intercellular communication. EVs purified from
a S. aureus mutant that is genetically engineered to express detox-
ified cytolysins are immunogenic in mice, elicit cytolysin-
neutralizing antibodies, and protect the animals in a lethal sepsis
model [58].
Gram-negative bacterial lipopolysaccharide (LPS, endotoxin) is
an initiator of sepsis. Vaccines directed against core LPS structures
that are widely conserved among Gram-negative bacteria could be
used in the prevention of sepsis. Killed whole bacterial vaccines
(E. coli O111:B4, J5 [Rc chemotype] mutant and S. minnesota,
Re chemotype) protected mice against experimental sepsis [59].
6 Development of Vaccines for Tuberculosis
Tuberculosis (TB) is a detrimental disease that has affected millions

and is the leading cause of death due to an infectious agent. The
infectious agent Mycobacterium tuberculosis was discovered in
1882. The only vaccine effective for TB is the Bacillus Calmette-
Guerin (BCG) vaccine; however, it is only effective in infants and
children [60]. Therefore, there is a dire need to develop a vaccine
that is universally effective. It is suggested that a 60% effective
vaccine which will provide protection for 10 years could prevent
17 million cases of TB between the years 2040 and 2050. Despite
intense efforts, designing a vaccine for TB is difficult. The immu-
nological correlates of protection have not been clarified, the pre-
clinical animal models pose uncertainty, and M. tuberculosis has the
ability to evade host immunity. In 2019, many vaccine candidates
have been analyzed in clinical trials. The recombinant strains of
BCG and the attenuated strains of M. tuberculosis have been tested
to replace BCG vaccination in infancy. Protein and adjuvant com-
binations and recombinant viral vector subunit vaccines have been
designed to increase the efficacy of neonatal BCG vaccinations
[61]. TB vaccine development has been hindered by the lack of
animal models accurately predicting the effects in humans and the
lack of knowledge of the M. tuberculosis epitopes.
A phase 2b trial of the M72/AS01E tuberculosis vaccine con-
ducted in different countries of Africa. Human immunodeficiency
virus (HIV)-negative adults 18–50 years of age with latent M. tuber-
culosis infection (by interferon-γ release assay) were randomly
assigned (in a 1:1 ratio) to receive two doses of either
M72/AS01E or placebo intramuscularly 1 month apart. Most
participants had previously received the Bacillus Calmette-Guérin
vaccine. The authors assessed the safety of M72/AS01E and its
efficacy against progression to bacteriologically confirmed active
pulmonary tuberculosis disease. Clinical suspicion of tuberculosis
was confirmed with sputum by means of a polymerase chain reac-

tion test, mycobacterial culture, or both. M72/AS01E provided
54.0% protection for M. tuberculosis-infected adults against active
pulmonary tuberculosis disease, without evident safety concerns
[62]. A further study was conducted among adults infected with
M. tuberculosis. Vaccination with M72/AS01E elicited an immune
response and provided protection against progression to pulmo-
nary tuberculosis disease for at least 3 years [63].
H4:IC31 (AERAS-404) is a field-reconstituted vaccine with
H4 antigen (Sanofi Pasteur) and IC31® proprietary adjuvant (Val-
neva, formerly Intercell) supplied in different vials. H56:IC31 is a
vaccine with the H56 antigen (Statens Serum Institut; SSI) formu-
lated in IC31® adjuvant. H4:IC31 has an acceptable safety profile,
immunogenic, and capable of triggering multifunctional CD4+ T
cell responses in previously BCG-vaccinated healthy individuals
[64]. A trial of BCG revaccination and vaccination with H4:IC31,
in South African adolescents, showed efficacy in preventing Myco-
bacterium tuberculosis infection. BCG revaccination administered
as a single-dose ID and both H4:IC31 and H56:IC31 administered
as 2 doses IM had acceptable safety profiles in healthy,
QFT-negative, previously BCG-vaccinated adolescents [65].
7 Development of Vaccines for Tick-Borne Diseases
Increases in tick-borne disease prevalence and transmission are

important public health issues [66]. Lyme disease, caused by Bor-
relia burgdorferi, is a major tick-borne disease. Borrelia mayonii are
a type of bacteria recently found in North America that can cause
Lyme disease [67]. There is currently no vaccine available to pre-
vent Lyme borreliosis in humans. Borrelia outer membrane pro-
teins have been investigated as vaccine candidates but are not
successful yet.
The other tick-borne diseases include ehrlichiosis (caused by
Ehrlichia sp.), babesiosis (caused by the parasite Babesia sp.), Rocky
Mountain spotted fever (caused by Rickettsia rickettsia), tularemia
(caused by Francisella tularensis), and anaplasmosis (cause by Ana-
plasma sp.). These diseases are not diagnosed properly and if
untreated may be fatal. The diseases caused by these pathogens
significantly lower health quality status, may lead to impairment
in their ability to work, increased utilization of healthcare services,
and greater out of pocket medical costs. Development of vaccines
could provide protection against these diseases.
8 Development of Vaccines for Flesh-Eating Bacteria
Flesh-eating bacteria, also known as necrotizing fasciitis (NF), is a

life-threatening infection which infects the subcutaneous tissue,
fascia, and muscles. It is responsible for approximately 500,000
deaths annually. Inflammation is the initial sign of NF and then it
progresses to skin necrosis that causes deep tissue damage. Escher-
ichia coli, Group A streptococcal infections, methicillin-resistant
Staphylococcus aureus, methicillin-sensitive Staphylococcus aureus,
and fungal infections are the major bacterial organisms responsible
for NF. Those who are infected by flesh-eating bacteria experience
systemic toxicity with multiorgan failure, which may result in death.
Currently, treatment for flesh-eating bacteria is surgery, antibi-
otic therapy, skin grafting, reconstructive surgery, negative pressure
wound therapy, and even amputation of limbs. The severity of flesh-
eating bacteria on patients is immensely painful and calls for an
urgent need to design a vaccine for patients. A vaccine will provide
an effective and direct route of elimination without the need for
expensive and time-consuming procedures [68].
9 Development of Vaccines for Parasites
The parasites include ectoparasites, like ticks, mosquitoes, fleas, and

itch mite, and endoparasites including Plasmodium, Entamoeba,
Leishmania, Trypanosoma, Babesia, Toxoplasma, Wuchereria, Bru-
gia, Giardia, Ascaris, tapeworm, hookworm, pinworm, whip-
worm, Onchocerca, Fasciola, and Schistosoma. Most of the diseases
are classified under neglected tropical diseases and are the major
causes of fatality in poverty-stricken regions of the developing
world. Though the diseases caused by these parasites affect millions
of people in the developing world, in the long term (due to climate
change, movement of refugees, etc.) they pose a risk to people all
over the world. As yet there are no vaccines against these parasites.
Hence there is an urgent need to develop vaccines against these
parasites causing misery to millions of people [8].
10 Development of Vaccines for Malaria
Malaria has affected millions of people over the centuries and has
caused 410,000 deaths in 2019 [69]. The protozoan parasite caus-
ing malaria stems from the Plasmodium species, which is native to
subtropical and tropical regions. Severe and fatal malaria are known
to be caused by P. falciparum. An infected female Anopheles mos-
quito is the causative agent that initiates infection. When the mos-
quito bites into the human, it injects sporozoite into the skin,
where it travels through the blood stream to the liver. The sporo-
zoite travels by multiple cells and then resides in a hepatocyte. The
sporozoite grows into 40,000 merozoites that invade red blood
cells and erythrocytes, which eventually leads to erythrocyte inva-
sion and growth in the blood stream [70]. The epidemiology of
transmission varies in different regions, villages, and even person-
to-person. Therefore, it is challenging to develop a vaccine that
caters to the severity of each individual case. Another major chal-
lenge facing vaccine development is that many infected people are
asymptomatic, who acts as carriers for transmission of malaria
parasites. During pregnancy, malaria can cause miscarriages, death
of the fetus, decreased birth weight, and premature delivery. Preg-
nant women are at risk for anemia and sometimes even death.
The malaria parasite has an extraordinary ability to evade the
immune system, which may explain the failure of malaria vaccines to
date [71]. Due to the impact of the disease in the developing world,
there is an urgent need to develop vaccines to protect against
malaria. The circumsporozoite protein (CSP), the major protein
expressed on the surface of the infecting sporozoite, is essential for
mediating liver infection. A truncated form of CSP is linked to
hepatitis B surface antigen (HBsAg) to produce RTS. RTS is
co-expressed in yeast cells with HBsAg to produce RTS,S
[72, 73]. Despite the advancements in malaria vaccine develop-
ment, we do not have an efficient vaccine that protects against
young and old adults.
RTS,S is the first malaria vaccine to provide protection against
malaria in children [74]. The RTS,S/AS01 vaccine against malaria
infection completed phase III trials in 2014 and demonstrated
efficacy against clinical malaria of approximately 36% over 4 years
for a four-dose schedule in children aged 5–17 months [75].
Saponins, particularly those obtained from Quillaja saponaria
Molina, are known potent adjuvants and Quillaja saponins
(QS) have for long been used in animal vaccines. Saponin-based
adjuvants can be formulated in different ways; in free form, with
aluminum hydroxide, in ISCOMs (immunostimulating complex)
or in ISCOM-Matrix/Matrix structures. The ISCOM, a potent
adjuvant formulation, consists of stable complexes composed of
saponin, cholesterol, phospholipid, and incorporated antigen(s).
The hallmarks of the ISCOM technology are the dose-sparing
potential, induction of high and long-lasting antibody titers and
potent T cell responses. However, later it was shown that antigen
incorporation is not critical for these immune properties. Antigen
and empty ISCOMs, i.e., ISCOM-Matrix/Matrix could simply be
mixed with sustained vaccine efficacy. A novel adjuvant formulation
based on two different Matrix particles made from two separate
purified fractions of saponins, yielding Matrix-A™ and Matrix-
C™. These Matrix particles, approximately 40 nm large, are
subsequently mixed at defined ratios to get the Matrix-M™

adjuvant [76].
Collins et al. (2017) [77] developed a more immunogenic
CSP-based RTS,S-like vaccine called R21. The major improvement
is that in contrast to RTS,S, R21 particles are formed from a single
CSP-hepatitis B surface antigen (HBsAg) fusion protein, and this
leads to a vaccine composed of a much higher proportion of CSP
than in RTS,S. In animal studies, R21 is immunogenic at very low
doses and when administered with the adjuvants Matrix-M, it elicits
sterile protection against transgenic sporozoite challenge. In a
phase I study, R21 adjuvanted with Matrix-M adjuvant was consid-
ered safe and had good immunogenicity even when administered at
a fivefold lower 10 μg dose in UK and African populations [78]. A
large-scale malaria vaccine implementation program coordinated by
the World Health Organization to investigate RTS,S/AS01 efficacy
is now ongoing in Malawi, Ghana, and Kenya. The program aims to
vaccinate about 360,000 children per year from 2019 to 2023 and
will examine safety, compliance with the booster dose, and reduc-
tion in mortality [79]. Serum Institute of India (SII) and the
US-based biotechnology company, Novavax, Inc. have announced
an agreement for a commercial license for the use of Novavax’s
proprietary Matrix-M vaccine adjuvant with SII’s malaria vaccine
candidate R21 [80].
11 Development of Vaccines for Cancer, Neurodegenerative Diseases, Substance

Abuse, and Autoimmune Diseases
Cancer is the leading cause of death in the world. Though there are
vaccines for some cancers induced by virus (e.g., human papilloma-
virus (HPV)–related cervical or oropharyngeal cancer; Merkel cell
polyomavirus (MCPyV)–related Merkel cell carcinoma (MCC) and
Epstein-Barr virus (EBV)–related head and neck cancers), any epi-
topes derived from open reading frames (ORFs) in the viral genome
could contribute to the potential source of antigens [81]. However,
there are no vaccines against the majority of cancers including
cancer of the lung, breast, colon, pancreas, skin, brain, blood, etc.
Development of cancer vaccines should be a priority as it could
reduce the incidence of the disease, thereby reducing emotional
and economic hardship to millions of people.
Tumor neoantigen, or tumor-specific antigen (TSA), is the
repertoire of peptides that displays on the tumor cell surface and
could be specifically recognized by neoantigen-specific T cell recep-
tors (TCRs) in the context of major histocompatibility complexes
(MHCs) molecules. Neoantigens could play a critical role in
tumor-specific T cell-mediated antitumor immune response and
successful cancer vaccines [81].
As people live longer, they are more prone to neurodegenera-

tive diseases including Alzheimer’s and Parkinson’s diseases. As yet
there are no cures for these diseases. A vaccine to prevent this
disease will decrease the enormous burden on society.
The currently available medications for the treatment of drug
abuse have had only limited success. Antiaddiction vaccines, aimed
at eliciting antibodies that block the pharmacological effects of
drugs, have great potential for treating drug abuse [82].
As yet there are no vaccines for arthritis, type I diabetes, allergy,
multiple sclerosis, and other autoimmune diseases. A vaccine for
these diseases could improve the quality of life of people suffering
from these debilitating diseases [8].
12 Antibody-Dependent Enhancement
Vaccines have been one of the most effective public health initia-
tives in human history. However, not every attempt at introducing
immunity is safe. Paradoxically, antibodies produced in response to
a vaccine have actually increased susceptibility to illness from infec-
tious agents and dangerous unforeseen consequences have been
reported in response to vaccination.
Antibodies neutralize pathogens when they block its attach-
ment and/or entry into a cell, thereby reducing infectivity of the
pathogen. Paradoxically, nonneutralizing antibodies can actually
increase infectivity of pathogens in a process coined antibody-
dependent enhancement (ADE) or viral infection [83]. One theory
for how this could occur involves antibodies produced in a prior
infection or vaccination having affinity for epitopes on a related—
but serologically distinct—pathogen and facilitating FcR-mediated
endocytosis of this related pathogen into hose immune cells, sup-
pressing antiviral pathways, and ultimately triggering a proinflam-
matory cytokine storm to exacerbate severity of the infection
[83]. This paradox has contributed to futile vaccine development
attempts for various flaviviruses, coronaviruses, and immunodefi-
ciency viruses.
Dengue virus (DENV) represents perhaps the most infamous
example of ADE among flaviviruses. Young infants and children
with antibodies for one DENV serotype (produced during a prior,
subclinical infection, or passively introduced from their mother)
were observed to suffer severe illness upon infection with a different
DENV serotype [84, 85]. This phenomenon also showed up in
vaccine trials. For example, in the CYD14 phase 3 clinical trial of a
DENV vaccine, participants aged 2–5 that received the vaccine
were found to be 7.45 times more likely to be hospitalized for
virologically confirmed dengue compared to control after
3 years [86].
Antibodies against related flaviviruses such as dengue virus

(DENV) and West Nile virus (WNV) can cross-react with Zika
virus (ZIKV) and could thereby increase disease severity. ADE
may explain the severe disease manifestations associated with
ZIKV outbreaks and highlights the need to exert caution when
designing flavivirus vaccines [87]. Bardina et al. (2017) [87]
showed that low titers of DENV and WNV antibodies enhanced
ZIKV viremia.
Similarly, immunodeficiency viruses have demonstrated ADE in
multiple species. Antibodies produced in cats in response to FIV
vaccine have been shown to enhance infection in subsequent infec-
tions [88]. ADE has been observed in macaques infected with SIV
as well [89]. HIV-1 has increased infectivity in vitro in the presence
of HIV-1 antibodies [90]. Demonstration of ADE in vaccine clini-
cal trials for HIV further complicates this paradox. For example,
attenuated recombinant adenovirus-5 (Ad5) expressing HIV genes
(gag, pol, and nef) increased HIV-1 infection rate among partici-
pants with high Ad5 antibodies prior to the vaccination [91]. It is
unclear how these Ad5 antibodies could contribute to HIV-1
infectivity but results like these highlight the complex challenges
faced by vaccinologists in ensuring their products are not only
effective in stimulating neutralizing antibodies but also safe.
13 Future Challenges
The COVID-19 pandemic showed the need for developing and

manufacturing vaccines with minimum resources and within a short
time frame. The pandemic led to the development of mRNA vac-
cines that were never developed commercially before. The mRNA
vaccines developed were highly efficient than the conventional
vaccines. The novel vaccines developed showed the importance of
investing in basic and translational research as well as the need for
collaboration between academia and industries. With climate
change and the increase in temperature and the movement of
people, there may be pandemics in the future. The scientific and
management knowledge of the current pandemic may be helpful in
tackling future pandemics.
There is a need to develop new adjuvants to induce the immune
system and make the vaccine highly potent. There is also a need to
develop new classes of vaccines (DNA, RNA, VLPs, AAV vector,
structure-based vaccines) that are more efficient in inducing immu-
nity. Use of artificial intelligence (AI), deep learning, and bioinfor-
matic software will accelerate the design of efficient vaccine
candidates by predicting better immunogenic regions that may
require fewer tests before clinical trials.
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Chapter 2
Principles in Immunology for the Design and Development

of Vaccines
Claudius U. Meyer and Fred Zepp
Abstract
Vaccinology has come a long way from early, empirically developed vaccines to modern vaccines rationally
designed and produced. Vaccines are meant to cooperate with the human immune system, the later largely
unknown in the early years of vaccine development. In the recent years, a tremendous depth of knowledge
has been accumulated in the field of immunology that has provided an opportunity to understand the
mechanisms of action of the vaccine components. In parallel, our knowledge in microbiology, molecular
biology, infectiology, epidemiology, and furthermore in bioinformatics has fostered our understanding of
the interaction of microorganisms with the human immune system. Strategies engaged by pathogens
strongly determine the targets of a vaccine, which should be formulated to stimulate potent and efficiently
protective immune responses. The improved knowledge of immune response mechanisms has facilitated the
development of new vaccines with the capacity to selectively address the key pathogenic mechanisms. The
primary goal of a vaccine design might no longer be to mimic the pathogen but to identify the relevant
processes of the pathogenic mechanisms to be effectively interrupted by a highly specific immune response,
eventually surpassing natural limitations. Vaccines have become complex sets of components meant to
orchestrate the fine-tuning of the immune processes leading to a lasting and specific immune memory. In
addition to antigenic materials, which are comprised of the most critical immunogenic epitopes, adjuvant
components are frequently added to induce a favorable immunological activation. Furthermore, for reasons
of production and product stability preservatives, stabilizers, inactivators, antibiotics, or diluents could be
present, but need to be evaluated. While on the one hand vaccine effectiveness is a primary goal, on the
other hand side effects need to be excluded due to safety and tolerability. Further challenges in vaccinology
include variability of the vaccinees, the variability of the pathogen, the population-based settings of vaccine
application, and the process technology in vaccine production. Vaccine design has become more tailored
and in turn has opened up the potential of extending its application to hitherto not accessible complex
microbial pathogens plus providing new immunotherapies to tackle diseases such as cancer, Alzheimer’s
disease, and autoimmune disease. This chapter gives an overview of the key considerations and processes
involved in vaccine design and development. It also describes the basic principles of normal immune
responses and in their function in defense of infectious agents by vaccination.
Key words Vaccine, Vaccination, Immune memory, Pathogen, T cell, B cell, Infectious disease,
Adjuvant
Sunil Thomas (ed.), Vaccine Design: Methods and Protocols, Volume 1: Vaccines for Human Diseases,
Methods in Molecular Biology, vol. 2410, https://doi.org/10.1007/978-1-0716-1884-4_2,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022
27
28 Claudius U. Meyer and Fred Zepp
1 Introduction
Vaccination has been proven to be one of the most effective medical

interventions to reduce morbidity and mortality of infectious dis-
eases. The outstanding theme of vaccination is to induce a protec-
tive immune memory using principles identified for the natural
interaction of an infectious pathogen (bacteria, viruses, etc.) with
the (human) immune system (Fig. 1). In contrast to the natural
infectious pathogens, vaccines ideally achieve their protective
effects without clinical symptoms of disease or side effects.
Vaccine design in principle builds on the structure and
biological properties of an infectious agent. It is of utmost impor-
tance for vaccine developers to understand the physiology of the
pathogen, its underlying genetics, the epidemiology, the pathogen-
esis, and immunobiology. As pathogens tend to generate variants as
a strategy to escape previous immune memory, an ideal vaccine
should also have the capacity to induce cross-protective immune
responses against those potential variants of the pathogen.
The common understanding is that a vaccine should stimulate
all those steps naturally initiated by a pathogen contact leading to
immune activation and promoting an adequate effector mecha-
nism, involving mediators and cellular responses, which are tailored
to address the specific pathogen.
At all times in varying degrees, the development of vaccines was
started in alignment with observed natural phenomena [1]. Early
vaccinologists including Chinese pediatricians and Edward Jenner
(in Europe) [2] deducted their concepts from the observation that
under certain conditions individuals were spared from highly con-
tagious diseases. Over the last two centuries, vaccination was
strongly endorsed by the progress in biological sciences, the emer-
gence of biochemical techniques, and the discoveries in immunol-
ogy and genetics. The techniques available in the late twentieth
century further facilitated the development of new vaccine concepts
such as subunit vaccines (purified or recombinant protein or poly-
saccharide), DNA- or mRNA-vaccines, and the application of
reverse vaccinology resulting in rationally designed, genetically
engineered antigenic vaccine components [3]. The first two dec-
ades of the twenty-first century have seen an ever-growing choice of
new vaccine designs, including viral vectors, RNA- or DNA-vac-
cines, and virus-like protein particles, additionally stimulated by the
advent of systems vaccinology, including data-driven approaches
based on bioinformatic methodology [4, 5].
The advantages of modern vaccine concepts have often been
associated with specific drawbacks, including the fact that highly
purified vaccine antigens often provide less potent immunogens.
Moreover, there are challenging diseases such as malaria, tubercu-
losis, or HIV/AIDS that at least to date remain out of reach of
Principles of Vaccination 29
Pathogen Vaccine
Infecon Toxins Angens relevant for

protecon
Adjuvants: Control of
Disease immune response
Immune response Immune response
Cure and Protecon

protecon
Fig. 1 After infection with a pathogen a complex interaction between pathogen

and the host immune system includes illness and harmful processes. The
vaccine offers similar antigens like the pathogen, but the whole part of
disease is ommitted, thus directly leading to protection without a need for
healing
classical vaccine design. To overcome these impediments, adjuvant

formulations had been developed to stimulate innate immune
components as natural support for a vaccine-driven immunization
[6]. Following the recognition of the important role of the innate
immunity for the induction of an adaptive immune response, new
adjuvant mechanisms were developed that together with an ever-
growing armament from systems biology have generated new
insights and options to modulate the type of immune response,
specifically increasing the level of immune activity at least to those
levels typically seen with original live-attenuated or inactivated
vaccines. Thus, the hope is that modern vaccines may have the
potential not only to compensate for limitations of natural occur-
ring immune responses but even also to surpass natural limitations.
1.1 A Brief History Already in the ancient world it was common knowledge that for
of Vaccination some infectious diseases an individual was rarely infested twice. This
observation led to the practice of inoculation that has been docu-
mented in China more than 1000 years before Jenner’s remarkable
studies [1]. Even the term “immunity” was used in reference to
plague during the fourteenth century. Progress in natural sciences
and the development of experimental techniques during the eigh-
teenth century led to the systematic use of inoculation to fight
smallpox, one of the most serious threats during that time. In the
early eighteenth century variolation, the transmission of small,
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uskottomaksi hänestä, joka läksi luotani… Häntä minä katson
suurimmaksi kanssarikollisekseni kaikessa mitä olen rikkonut.
Thora Thammers oli hypähtänyt kiveltä, jolla hän oli istunut. Hänen
huulensa olivat valkoiset, kuin kuihtuneet. Hän oli huomannut että
jotain oli tulossa — oli huomannut sen äänestä, joka muuttui
muuntumistaan, kunnes hän ei sitä enää tuntenut.
Hän katsahti ylös.
Samuel Stern seisoi hänen edessään muuttuneena — — —

voimakkaana, mahtavana, tumma säihky katseessa — — —
Nyt vasta tunsi hän hänet samaksi, joka hän oli ollut muinoin. Oli
kuin hän olisi astunut hänen eteensä muuttumatonna pitkäin,
menneitten aikojen takaa — Thora katsoi häneen sanatonna
kauhusta.
Oliko se hänen syynsä! — — — Oliko hän syypää Samuel Sternin

rikokseen?
Samuel Stern nauroi.
— Ja entä te? Onko kehityksenne tuottanut teille iloa?… Eikö ollut

perin herttaista, että te valitsitte tulla tuollaisten lasten äidiksi?
Eiköhän olisi paikoillaan nimittää juuri sellaisia lapsia äpäröiksi?
Thora Thammers kävi äkkiä aivan tyyneksi ja levolliseksi. Hänestä

tuntui kuin hänen verensä jähmettyisi, ja hän tunsi sen helpotuksena.
Hän painoi päänsä alas ja seisoi hiljaa.
He olivat vaiti molemmat. Samuel Stern hengitti raskaasti, kuin

ruumiillisen ponnistuksen jälkeen.
— Minun täytyi sanoa se, sanoi hän vihdoin hiljaa, — se oli kerran
sanottava!
— Niin… niin, niin! sanoi Thora epävarmasti.
Sitten hän hymyili ja kohotti päänsä — kaula suoristui, veri pääsi

liikkeelle, ja hänen silmissänsä säteili jotakin.
— Ei se tee mitään! sanoi hän ja nauroi. — Muutoin olettekin aivan

oikeassa!
Hän käänsi kasvonsa Samuel Sterniä kohden. He katsoivat

hetkisen toisiinsa.
— Thora, sanoi Samuel surumielisesti, — saanko vielä kerran
sanoa sen nimen yhtä hiljaa ja suljetusti kuin te itse.
Thora hymyili katkerasti. — Mitä se hyödyttää?
Mitä te ajattelette?
— Jotain, jonka kerran olen lukenut koptilaisessa raamatussa,

sanoi Thora hitaasti: »Mutta Herra sanoi opetuslapsillensa: Ettekö
sitten tekään näe, että eläin vuotaa verta, ja ettekö kuule kuinka se
huutaa ja vaikeroi? Mutta opetuslapset vastasivat: Ei, Herra, emme
kuule sen huutavan ja vaikeroivan…» Sellaista elämä on!… me
emme kuule toisiamme!
— Puolustautukaa! pyysi Samuel. — Vastatkaa minulle! Sanokaa

jotain lisää.
— Sen minä teen — joskus — myöhemmin!
— Huomennako?
Thora ei vastannut. Hän läksi.
Samuel jäi katsomaan hänen jälkeensä. Hän näki tuon solakan,

hiukan kumarassa kulkevan olennon kuvastuvan valoisaa ilmaa
vastaan — ja ikäänkuin häipyvän nummelle.
Hän ei voinut enää nähdä Thoraa — vaan hän kuuli kaikkialla

hänen viime sanansa: nummi oli omaksunut sen — se täytti koko
nummen… Huomennako? Tulisiko hän huomenna?
Mutta varhain seuraavana aamuna oli rouva Thammers

matkustanut pois.
Sanottiin että hän oli saanut sähkösanoman mieheltänsä.
Itse asiassa alettiin jo vähitellen väsyä täälläoloon ja matkustaa

kotiin.
Ja ikäänkuin kesävieraat olisivat kaikessa hiljaisuudessa sopineet

keskenään, etteivät enää jaksaneet sietää toisiansa, kävi pysyväksi
keskusteluaineeksi, ken ensi kerralla läksisi tiehensä.
Ei enää välitetty niistä, jotka tulivat. Kaikkien huomio oli kiintynyt

niihin, jotka läksivät.
Vanhemmat punnitsivat kysymystä pitäisikö mennä saattamaan

vai ei — nuoremmat sitä, kuinka monta tavanmukaista
jäähyväiskyyneltä asianomainen oli ansainnut.
*****
Oli myöskin muutamia, joiden mielestä oli kaikkein hauskinta nyt

kesäkauden lopulla, kun ei enää ollut niin paljon ihmisiä.
Näihin kuului Marit Hennerud, parantolan nuori emäntä. Nyt oli

hänellä enemmän aikaa olla mukana sisällä. Juuri näin kesäkauden
lopulla hänellä tavallisesti oli pienet rakkausjuttunsa. Varhain kesällä
oli siellä ollut eräs nuori mies, joka oli ollut oikein onneton hänen
tähtensä — mutta eihän hän voinut siihen mitään, kun hänellä oli niin
paljon tehtävää.
Nyt hän sensijaan voi olla mukana sekä tunturiretkillä että

varsinkin illalla, kun mentiin soutelemaan järvelle.
Jos sitten sattui että sen, johon hänen sydämensä oli kiintynyt,
täytyi matkustaa liian pian, voi hän itkeä vuolaita kyyneliä — oli kuin
kuolema olisi ollut lähellä.
Mutta muutaman päivän perästä häntä pyrki naurattamaan sitä

muistellessa.
Olihan jonkun kerran tapahtunut, että asia alkoi näyttää

vakavammalta. Silloin oli hän aina ajatellut että nyt teki oikea
rakkaus tuloansa, ja hänen oli täytynyt uskoa kaikki vanhalle
tädillensä.
Ja vanhus kehotti häntä joka kerran olemaan uskollinen ja

kestäväinen, niin että asiasta voisi tulla jotain valmista. Sillä perheen
mielipahaksi oli Marit Hennerudilla päähänpisto antaa aina rukkaset.
Ei hän voinut siihen mitään että rakkaus katosi, kun ehdittiin niin
pitkälle. Hänelle soveltuivat parhaiten vähäiset kesähakkailut.
Ja päivät menivät menojansa — kunnes viimeinen tuli, ja

viimeinen vieras matkusti tiehensä kuullen jälkeensä ystävällisen
kehotuksen: — Tervetuloa toistekin! — — — ja sitten vielä aivan
viimeisen hilpeän huudon, joka kajahteli mäkiä alas: — Sillä tuleehan
toistekin kesä!
Sitten parantola suljettiin ja telkittiin kiinni.
Ja Maritin täytyi palata kylään, missä hylättyjen kosijain tuli kuuma

olla, joka kerran kun kohtasivat hänet.
Se oli hänen silmiensä syy. Ne katsoivat liian lempeästi ja

haaveellisesti heitä kaikkia. Mutta hän ei itse tiennyt siitä mitään, niin
sitä ei käynyt hevillä korjaaminen.
II.
Metsäruusun kukkiessa.
8.
Niin oli siis kukin jälleen saapunut kotiin.
Tuli talvi.
Monesta kävi aika pitkäksi. Tuli kylmä, päivät kävivät lyhyiksi.
Ulkona metsissä ja kedoilla oli niin autiota. Tyhjää oli kaikkialla ja

tiet ummessa, kuin ei ketään olisi kotona.
Puut nukkuivat tajuttomina, valkoisiin kaapuihin verhottuina. Kaikki

vaikenivat, paitsi tuuli, joka kävi saalistamassa.
Lumi kimmelsi ja hohti, kokosi kultaa, hopeata ja timantteja ja

rakenteli muhkeita taloja. Mutta kylmä henki vastaan noista
valkeista, hiljaisista saleista, joissa kuolon enkeli tuntui
käyskentelevän uneksivana.
Paljon oli sellaisia, jotka koko pitkän talven kestäessä vain

odottelivat että se läksisi tiehensä.
Vihdoin tuli sanoma, että kesä teki tuloa.
Kaikki muuttui. Ihmisiin ja eläimiin tuli levotonta eloa. Metsissä ja

lehdoissa kohosivat mehut maasta, kuohuvina kuin nuori viini.
Kullahtavana ja sinisenä pulpahti kevään kaiho esiin lumen alta
jokaisella mäenrinteellä.
Kun niityt alkoivat hohtaa vihreinä valkoisten jäätiköitten

alapuolella, silloin ei Marit Hennerud enää voinut viihtyä kylässä.
Ja kun tuulet kävivät lämpimiksi ja yöt valoisiksi, silloin oli

Metsolan parantola jo valmiina ottamaan vastaan viimekesäiset
vieraat.
Vähitellen saapuivat ne kaikki tyyni.
Hienoisena ja punaposkisena, lempeästi hymyilevänä kuten

ennenkin, mutta vielä kaunopuheisempana käyskenteli siellä lehtori,
väenlaskua pitäen.
Kaikki olivat saapuneet — paitsi tukkukauppias Stern ja hänen

molemmat koiransa.
Lehtori ei kaivannut heitä. Mutta Marit Hennerud ja monet muut

olivat siitä huolissansa.
Rouva von Astenilta ei saatu mitään lohtua. Häntä huvitti
enemmän puhua nuoresta insinööristä, joka oli puuhassa keksiä
uuden hävitysvälineen.
Oli tullut useita uusiakin vieraita ja sitäpaitsi muutamia aviomiehiä,

jotka olivat saattaneet vaimonsa tänne ja aikoivat viipyä muutaman
päivän.
Kesä lupaili käydä oikein hauskaksi. Kaikki olivat hyvillä mielin. Oli
jo alettu maalata koskea.
Oli ehditty ensi päiviin, jolloin oli niin lämmin, että voi täyttä totta
istuskella ulkona.
Istuttiin suuressa kuistissa, keskustellen pienissä ryhmissä.
Rouva Thamar Gyllenskjold puheli miehensä kanssa ja omisti

hänelle kaiken huomionsa.
Rouva Thamarin mies oli komea katsella. Hänen avoimet tyynet

kasvonsa älykkäine ilmeineen muodostivat omituisen vastakohdan
hänen vaimonsa kasvoille. Tosinhan niilläkin kuvastui jonkinmoinen
tyyneys, mutta ne kertoelivat myöskin salaisista teistä, missä
ajatukset hiiviskelivät, — siellä vilahti välistä esiin jotakin, joka katosi
samassa kuin se oli havaittu. — Ja silmiin voi tulla itsetietoinen
valtijanilme, joka jälleen häipyi hänen rauhalliseen, aurinkoiseen
hymyynsä.
Thamar rouva oli varsin viehättävä, kun hän nyt istui kysellen
mieheltänsä kaikenmoisia pikkuseikkoja. Hänellä oli erityinen kyky
keksiä tuontapaisia kysymyksiä — eikä kenenkään olisi tarvinnut
niitä arkailla, sillä hän ei koskaan tarkannut vastausta.
Mutta erästä seikkaa ei tirehtööri ollut koskaan oivaltanut. Hänelle
oli niin mieleen tuo, että hänen oli aina päätettävä kaikesta ja että
hänen vaimonsa oli niin tyytyväinen kaikkiin hänen järjestelyihinsä.
Hiukan kauempana istui rouva Liss, alias rouva Wanda Arescho,

oivan, erinomaisen miehensä kanssa.
Tämä kuului myöskin niihin onnellisiin aviomiehiin, jotka olivat

voineet saattaa pikku vaimonsa tänne ja tulla vakuutetuiksi että
hänen olisi täällä oikein hyvä olla.
Rouva Liss oli kukoistava kuten ennenkin. Hänen lempivärinsä

tänä kesänä oli hiedanväri. Se puki häntä oivallisesti, — yleensä puki
häntä melkein mikä tahansa.
Hän istui jutellen ja piti pienoisia sormiansa innokkaasti liikkeessä.
Aivan läheisyydessä istui Don Miguel, ulkoasu yhtä huolellisesti

hoidettuna kuin edellisenäkin kesänä ja viikset pyöräytettyinä, jos
mahdollista, vieläkin uljaammin ylöspäin.
Konttoripäällikkö Arescho ei saanut silmiänsä käännetyiksi pikku

vaimostansa. Sitä ei kukaan voinut paheksua, — pitihän hänen
matkustaa pois seuraavana päivänä.
Jotkut arvelivat että hän juuri olisi tarvinnut lepoa täällä

vuoristossa, niin rasittuneelta kuin hän näytti.
Rouva Liss myönsi niin olevan, mutta se ei käynyt päinsä.

Fredrikin täytyi tehdä työtä. Eikä hän voinut kuvaillakaan Fredrikiä
muutoin kuin täydessä työssä. Ja Fredrik oli niin tottunut siihen, että
rouva arveli ettei se häntä ollenkaan rasittanut. Mutta olihan ikävää
että hän alati näytti niin huonolta. Se oli hänestä oikein ikävää.
Mies ei sanonut mitään, — hymyili vain.
Niiden mielestä, jotka olivat tunteneet herra Areschon muinoin,

hän oli muuttunut paljon.
Naimattomana ollessaan oli hän asunut yhdessä äitinsä ja tätinsä

kanssa. Siihen aikaan ei hän ollut osoittanut tuollaista hienoa
kunnioitusta naista kohtaan, joka voi tehdä sentapaiset suhteet niin
kauniiksi. Rakkautta oli hän silti saanut osaksensa. Niin oli hänen
itsekkäisyytensä ja mukavuuden rakkautensa tehnyt hänet
välinpitämättömäksi ja opettanut hänet ottamaan vastaan,
vaivautumatta antamalla jälleen.
Tiesiväthän hänen omaisensa niin hyvin, että hän piti heistä.
Nyt oli kaikki toisin. Nyt oli hänen huolehdittava kaikesta, vaikkei
hän vielä ollut täysin ehtinyt sitä huomata, ollen tuollaisen rakkauden
pauloissa, joka häikäisee ja orjuuttaa, niin kauan kuin se kestää.
Joskus hän sentään tuli ajatelleeksi, kuinka välttämättömiltä hänestä
nyt tuntuivat kaikki nuo vähäiset huolenpidon ilmaukset, joita hän ei
ennen tullut koskaan osoittaneeksi, ja hänen mielessänsä heräsi
kuin hämmästelevä tunne, että hän oli laiminlyönyt jotain, jota ei
voinut enää koskaan hyvittää.
Nyt juuri oli hän tuollaisen mielentilan vallassa ja oli tullut

näyttäneeksi hiukan miettiväiseltä, eikä siis ollut ihme, jos rouva Liss
hetkiseksi unohti hänen läsnäolonsa, — varsinkin kun Don Miguel
alkoi kertoella kiehtovalla tenoriäänellänsä ilkeitä juttuja.
Kaikki muutkin läsnäolevat alkoivat vilkastua.

Rouva Sahm oli saanut nuoren, oppineen tohtorin suljetuksi
nurkkaan. Hän halusi nyt tietää, oliko rakkaus tunnettu jo
sekundäärikaudella ja oliko ihmistä katsottava lajin muunnokseksi
vai alkulajiksi.
Ärsyttääkseen lehtoria tarjoutui muuan nuori nainen pitämään

esitelmää aineesta, jota ei vielä ollut käsitelty tarpeeksi: miehen
heikkouksista.
Muutamat toiset nuoret olivat piirittäneet ylioppilas Adelssonin,

tummatukkaisen pohjan pojan, jolla oli mitä rohkeimmat sinisilmät.
Hänessä oli jotain yleiseurooppalaiselta vaikuttavaa. Hän oli ollut
pari vuotta matkoilla ja oli tullut tänne suoraa päätä Nizzasta. Hän
kiinnitti naisten mieltä. Hän kertoeli niin rohkeasti ja
häikäilemättömästi. Hän oli suorastaan vielä kauheampi kuin Omar
Pasha.
Hän kuohui vihasta ajatellessaan että Ranska voi rajojensa sisällä

sietää sellaista pelipankkia kuin Monte Carloa. Ranskan keisari oli
antanut suostumuksensa sen perustamiseen. Tasavallan tulisi
sulkea se. Sen olemassaolo oli rikos ihmiskuntaa kohtaan.— — —
Tästä johtui puhe vanhoihin ruhtinassukuihin, sellaisina kuin

historia ne meille esittää — rappeutuneina, mitä kauheimpaan
mädännäisyyteen vajonneina… Tuollaisten sukujen jäsenten näytti
olevan vaikea kehittyä tavallisiksi kunnon ihmisiksi, — vielä
vaikeampi kuin muiden saavuttaa todellista ihmisarvoa. —
Hänestä pyrittiin laskettelemaan sukkeluuksia, sanoen että hänen

pääparkansa ilmeisesti oli parantolahoidon tarpeessa.
Mutta hän ei ollut vielä ehtinyt loppuun, vaan jatkoi yhä.

— — Tuolla etelässä hän oli tutustunut uuteen lajiin ihmisiä. Ne
olivat hänestä köyhälistöä, kurjempaa kuin aineellista puutetta
kärsivä. Ne luulivat, että Kaitselmus oli suonut heille rahaa sitä
varten että he huvittelisivat — eivätkä ne pystyneet siihen. Voi hyvä
Jumala, kuinka niiden oli ikävä! Hän näki ne edessään
haukottelevina, kaiken maailman ihanuuden keskellä. —
Nuoret naiset halusivat kernaammin puhua jostakin muusta. Ne

pyysivät häntä kertomaan näkemistään hienon maailman puvuista.
Hän nauroi. Sehän kuului juuri samaan asiaan! Niin, hänen oli ollut
mahdoton olla niitä näkemättä — ja huomaamatta että nykyajan
naiset pyrkivät ratkaisemaan saman tehtävän kuin muinoin
kuningatar Kraaka, mutta vastakkaiseen suuntaan: olla puettu ja
kumminkin alaston. Oli ihmeellistä nähdä, kuinka kunnialliset naiset
kilvan jäljittelivät kuoseja, jotka olivat siveettömäin naisten keksintöjä
tai heitä varten keksittyjä. Erikoisen älykkäältä ei se hänestä
vaikuttanut, mutta kaiketi onkin älykkäisyys jo vanhentunut
ominaisuus… Ennenmuinoinhan poltettiin viisaita naisia!… Erääseen
toiseenkin seikkaan oli hänen huomionsa kiintynyt, nimittäin tuohon
erinomaisen kauhistuttavaan tapaan, millä he kantoivat helmojansa.
Minne olikaan joutunut sulottarien perintö?
Naiset menivät tiehensä. Joko oli tuo mies maankiertäjä tai

sosialisti, taikka myöskin yli-ihminen.
Lehtori seisoi juuri esittämässä rovasti Hembille, että pappien

tehtävä oli tutustuttaa ihmiset ylempään ilmapiiriin, mutta samalla
hän myöskin tarkkasi nuorison puheita.
Nyt hän tuli esiin ja huomautti nuorukaista että hänen käytöksensä

oli sopimatonta.
Tämä nauroi ja lupasi olla vaiti. Hän asettui oveen silmäilemään
Thamar rouvaa.
Lähtiessään oli hän niin silmittömästi rakastunut, että tämä seikka

jäi kahden viikon ajaksi suosituimmaksi keskusteluaineeksi.
Rovasti käytti hyväkseen vapauttaan hakeakseen käsiinsä

tukkukauppias Iversenin. Tämä oli viisas mies. Hänen kanssaan voi
puhua mistä tahansa, ja hyväksi aluksi ryhtyi rovasti nyt puhumaan
fosforihappoisista lannoitusaineista.
Iversen oli juuri aikeissa ostaa maatilan, jossa oli oivallista

suomaata. Mutta tuuma ei miellyttänyt Ninaa. — — —
— Jospa voisit mainita minulle syysi, Nina-muoriseni! — — —
Rouva Iversen istui miehensä rinnalla, kirjaillen hienolla

ruusunpunaisella silkillä, ja tuo työ vaati kaikki hänen ajatuksensa.
Mutta kun mies ei hellittänyt, harmistui hän lopuksi hiukan.
— Anna minun olla rauhassa, Iversen! Syyt — tuo on ilkeimpiä

sanoja mitä on olemassa. Mutta hyvä Jumala, tuollahan tulee rouva
Harder, tietysti mustassa puvussaan. —
— Niin, sanoi eräs naisista, — ihmiset sanovat että hän suree

aivan ujostelematta miestä, joka ei ollenkaan ollut hänen
miehensä… Jos vain tiedettäisiin, ken hän oli, mutta ikävintä on, että
sitä ei tiedetä. —
Kaikki vaikenivat. Oli niin harvinaista että rouva Harder ilmestyi

tänne.
Yksin Don Miguelkin kääntyi, vaikka oli juuri ehtinyt
kertomuksensa jännittävimpään kohtaan. Pyörähyttäen viiksiänsä jäi
hän katsomaan rouva Harderiin.
Tuossa ihmisessä oli jotakin… ei hienostunutta… jotain enemmän

— — — Hän oli kuin »syntynyt prinsessaksi», yksi niitä harvoja sekä
prinsessojen että muiden joukossa, jotka todella vastaavat sitä, mitä
tuolla puhetavalla tarkoitetaan. — — —
Hän seurasi rouva Harderia katseellaan, tämän tullessa portaita

ylös: Ei, hänessä oli jotain tuollaista, jota on totuttu nimittämään
kuninkaalliseksi, kuninkaallista viehättäväisyyttä.
Don Miguelilta ei myöskään jäänyt huomaamatta että rouva

Harderin olennossa ilmeni tuollaista hienoa naisellista viehkeyttä,
jonka vanhemmat naimattomat naiset tavallisesti säilyttävät, jos ovat
sitä konsanaan omistaneet, ja jonka naimisissa olevat, jos sitä
omistavat, tavallisesti menettävät, melkein heti.
Ja hän, joka muutoin aina tiesi kuinka naiset olivat i puetut, ei

kiinnittänyt huomiota yksinkertaiseen mustaan pukuun, joka oli
herättänyt niin paljon pahennusta. Hänestä tuo nainen vain oli
erilainen joka kerran kun hän näki hänet, ja niin erilainen kuin nuo
muut kunnon ihmiset, joihin väsyi niin pian.
Se oli todellakin ihmeellistä, sillä rouva Harder oli kalpea ja

riutunut, oli vailla kauneutta ja näytti vanhalta. Mutta — kauneutta
hänessä sittenkin oli! Sitä oli hänen päänsä asennossa ja tyynessä,
valoisassa katseessa, joka kuvasti pyhää haaveilua.
Ei ainoakaan nainen ollut tätä ennen saattanut Don Miguelia

tekemään tuollaisia havaintoja.
Monen muun valtasi samantapainen tunne, kun rouva Harder
tervehtäen hymyili heille.
Don Miguel nouti tuolin vierashuoneesta. Rouva Harder kiitti,

mutta jäi seisomaan, käsi laskettuna korkealle selkänojalle.
— Tahdoin vain tulla sanomaan jotain… Tuolla alhaalla kylässä

asuu köyhä papinleski. Hänellä on kuusi pientä lasta… Nyt on heillä
ollut tulipalo. Hän on menettänyt pienen talonsa ja kaiken muun
omaisuutensa. Se ei ollut vakuutettua… Ettekö kaikki haluaisi olla
mukana häntä auttamassa?
Toiset alkoivat tuumia että pantaisiin lista kiertämään.
— Kuinka kauhean surullista! sanoi rouva Liss äkkiä. Ja hänen

suuret siniset silmänsä kyyneltyivät melkein. — Mutta, lisäsi hän,
pannen pienet kätösensä ristiin, — lesket selviävät sentään aina
jollakin tavoin! Hyvä Jumala, senhän näkee niin usein, kuinka
uskomattoman hyvin ne selviävät — — — niin että kenties ei olisi
tarpeenkaan — arvelen melkein — — —
Hän jätti lauseensa kesken. Hän tuli ajatelleeksi jotakin, jonka oli
kuullut kerran rouva Thamarilta: että jos ei kenenkään olisi vaikea
olla, niin eihän tietäisikään kuinka hyviä päiviä itse viettää. — Ja
mistä runoilijat sitten kirjoittaisivat? Kaikki ihmiset menehtyisivät
ikävään. — — — Tuota sieti todellakin miettiä!… Ja kun nyt kerran
maailman meno oli järjestetty sillä tavoin… Tietysti oli Luoja
asettanut kaikki sillä tavoin kuin tahtoi sen olevan. —
Tuo ajatus näytti tuottavan hänelle helpotusta.

Mutta tuo listatuuma ei ainakaan häntä miellyttänyt Jos piti
johonkin ryhtyä, niin oli basaari sopivinta. Siitä pääsisi helpoimmalla
hinnalla, Hänen lahjoihinsa nähden oli rahallinen puoli sangen tärkeä
asia. Kun hän osti itsellensä, tuli kaikki niin kalliiksi, että hänen oli
pakko säästää, kun oli puhe muista. Ja nythän hän voisi päästä
asiasta jollakin turhanpäiväisellä romulla, kun se vain näyttäisi
hiukan sievältä.
Ja rouva Liss äänesti basaaria, vaikka hän todellakin oli ajatellut

pääsevänsä täällä kaikesta tuollaisesta… Sitäpaitsi olivat tuollaiset
ihmiset aina niin kiittämättömiä!
Adèle Harder oli seisonut ja kuunnellut ja katsonut häneen hieman

hymyillen.
Sitten hän lausui: — Useimmat ihmiset eivät osaa antaa oikealla

tavalla, — antaa varovaisesti, niin ettei riistä autettavalta enemmän
kuin hänelle antaa, — ei tee köyhyyden taakkaa yhä raskaammaksi
kantaa. Vahvistaahan tulisi ja suoda hoivaa!… Ja useimmat eivät
myöskään osaa ottaa vastaan oikealla tavalla! Heillä ei ole tarpeeksi
itsetuntoa. He antavat nöyryyttää itseään. Sitä ei tule tehdä. Pitää
ymmärtää, että jos on saanut elämän rasitukset monenkertaisina
kannettavaksensa, niin on soturin kaltainen, joka on asetettu
vaaralliselle paikalle. Olemme velkaa kunnioitusta niille, jotka
suoriutuvat tuosta kaikesta, — sekä niille, jotka tukevat heitä… Jos
voimme auttaa jotakin, niin on meillä auttajilla enin syytä
kiitollisuuteen! Antamisen ilo on suurin kaikesta… Niin, siksi tulee
sen, joka antaa, olla kiitollinen sille, joka ottaa vastaan. — Tämän
vain halusin sanoa.
Don Miguel oli ottanut vadin ja kierteli herrojen keskellä, jotka

kaikki osoittivat uhrautuvaisuutta.
Tuo harmitti rouva Lissiä. Sehän teki tyhjäksi hänen basaarinsa.
Don Miguelin palatessa Adèle Harderin tykö, katsoi tämä häntä

hämmästyneenä ja anteeksianovasti, ikäänkuin olisi tuntenut
arvostelleensa häntä väärin. Ja Don Miguel tuli katsoneeksi hänen
silmiinsä. Ne olivat kuin sammuneet. Ne olivat silmät, jotka olivat
vuodattaneet paljon kyyneleitä — muinoin.
Hetkellinen heikkous saattoi ne Don Miguelista tuntumaan

kauniimmilta kuin rouva Areschon silmät.
Adèle Harderin lähdettyä alettiin harmitella hänen ihmeellisiä

oppejansa sekä tuota, että oli tultu ryhtyneeksi johonkin.
Lehtorin posket olivat käyneet hiukan punaisemmiksi kuin

tavallisesti. Hän oli loukkaantunut. Hän piti itseänsä kaikessa
hiljaisuudessa erinomaisen jalona henkilönä. Väliin vain hänen
mielessänsä heräsi epäilyksiä, etteivät kaikki tienneet sitä. Niinpä
esimerkiksi nyt. Millä tavoin olikaan tuo nainen katsonut häneen!
Ja hän alkoi esittää omaa katsantokantaansa.
Mutta väliin voi käydä niin, että lehtori ei enää tiennyt puheensa
alkua eikä loppua, ja nytkään ei hän itse eivätkä naiset tienneet,
kuinka hän oli tullut puhuneeksi — analogia-todistuksista ja sitten
lopettaneeksi puheensa herttaisella myönnytyksellä, että henkisesti
voimakkaat naiset kohoavat ympäristönsä yläpuolelle.
Eikö jokin kirjailija ollut lausunut niin?
Hän kääntyi lähellä seisovan ylioppilas Adelssonin puoleen —

pitihän tälle osoittaa hiukan ystävällisyyttä.
Nuori mies sukaisi tukkaansa kädellään. Hän ei tiennyt, oliko joku
niin lausunut, mutta tuollainen väite oli kumottava, sillä sehän oli
aivan väärä. Kukin saavutti suurimman täydellisyytensä juuri
pysymällä omassa lajissansa, — lajin rajoja täytyi olla valmis
siirtämään.
Lehtorin vastustaminen tuotti hänelle todellista tyydytystä.
Noustiin paikoilta. Liikuskelu tuntui olevan tarpeen. Rouva Liss

kääntyi innokkaasti Don Miguelin puoleen.
— No totta tosiaan! Rouva Harder on todellakin aivan kuin… eikö

teistäkin?
— On kyllä.
Ensi kerran loi nyt Don Miguel rouva Lissiin katseen, joka ei ollut
ehdottomasti ihaileva.
Rouva Thammers oli noussut ja kulki heidän ohitsensa.
— Ja rouva Thammers, sanoi Don Miguel hymyillen, — eikö

hänkin ole aivan kuin — — —?
Rouva Liss katsoi hänen jälkeensä.
— Ollaksensa niin vanha ihminen, on hän suunnattoman norea,

sanoi hän, käyden oikein miettiväiseksi.
Juuri tuo suunnaton noreus häntä harmitti, ja hieman huoahtaen

hän loi katseen omaan täyteläiseen vartaloonsa. Hän oli päättänyt,
ettei tahtonut tulla lihavaksi. Hänen täytyi täyttä totta käydä käsiksi
tuohon asiaan. Voimistelullako? Hyvä Jumala, siihen tarvittiin niin
paljon tarmoa!
Useimmat vieraista jaloittelivat pihassa.
Kuinka sää olikaan äkkiä käynyt päivänpaisteiseksi ja hohtoisaksi!

Toinen kertoi sitä väsymättömästi toiselleen, samalla kuin myöskin
käsiteltiin basaarikysymystä.
Lehtorille kerrottiin siitä myöskin.
— Hyvät naiset, sanoi hän ystävällisesti, — ja mikä on syy?

Nähkääs, se on vain luonnonlakien seurauksia. Jos teillä, arvoisat
naiset, olisi tarpeelliset fyysismatemaattiset tiedot, niin olisitte
voineet etukäteen laskea että niin kävisi. Eikö totta, herra professori?
Professori Maurus kulki juuri ohitse vatsa ulospäin ja kädet

selässä.
Hän hymyili alentuvaisesti, mutta ei sanonut mitään.
Eihän tuollaista sopinut sanoa naisille, ei edes leikilläkään.
Mieliala kävi yhä hilpeämmäksi. Basaarikysymystä pohdittiin

innokkaasti.
Juuri rouva Thammersin ikkunan alla oli muutamia raikasmielisiä

nuoria leikkimässä:
»Rosvo, rosvo olit sä,

Kun varastit mun ystävän'!
Mutt' on mulla sulotoivo,
Että löydän ystävän!
Luulen, rallalaa, luulen, lallalaa!
Luulen rallala, ralla lallallaa!»
Rouva Thammers kuunteli heitä alakuloinen ilme kasvoillaan.
Hiukan kauempana kudottiin sarkaa.
Sitä oli sateesta huolimatta jatkunut koko aamupäivän, ja nyt olivat

he jälleen alkaneet väsymättömällä innolla.
»Näin vedämme verkaa, näin kudomme sarkaa.

Vedä verkaa, kudo sarkaa,
Anna kaiteitten käydä!
Näin vedämme verkaa — — —»
Ja he toistivat tuota samaa viheliäistä säveltä — uudelleen ja aina

uudelleen.
Thora Thammers istui heitä katsellen, kunnes hänestä alkoi tuntua

että hän itse liukui mukaan tuohon loppumattomaan sarankudontaan
— hiljaiseen, harmaaseen iankaikkisuuteen.
Mutta seurusteluhuoneesta kajahtivat Racoczy-marssin säveleet,

tummina, säkenöivinä, pehmoisina — — — houkuttelevan hurjina.
Tuo pohjolan poika siellä soitteli.
9.
Tuolla ylhäällä leviää nummi.

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Vaccine Design Methods and Protocols

Vaccines for Human Diseases Methods

Vaccine Design Methods and Protocols Volume 2 Vaccines

Rhodopsin Methods and Protocols Methods in Molecular

Ferroptosis Methods and Protocols Methods in Molecular

DNAzymes Methods and Protocols Methods in Molecular

Monoamine Oxidase Methods and Protocols Methods in

Enzyme Engineering: Methods and Protocols (Methods in

Chromosome Analysis Methods and Protocols Methods in

Candida auris Methods and Protocols Methods in

Sunil Thomas Editor

For further volumes:

Methods and Protocols, Volume 1: Vaccines

ISSN 1064-3745 ISSN 1940-6029 (electronic)

Wynnewood, PA, USA Sunil Thomas

PART I VACCINES: INTRODUCTION

1 Challenges for Vaccinologists in the First Half of the

PART II TRENDS IN VACCINOLOGY

5 mRNA Vaccines to Protect Against Diseases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111

PART III VACCINES FOR HUMAN VIRAL DISEASES

7 Vaccines Targeting Numerous Coronavirus Antigens, Ensuring

9 Design of Replication-Competent VSV- and Ervebo-Vectored

PART IV VACCINES FOR HUMAN BACTERIAL DISEASES

15 Salmonella Uptake into Gut-Associated Lymphoid Tissues:

20 An Update on Tuberculosis Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 387

PART V VACCINES FOR HUMAN PARASITIC DISEASES

23 Development of the Antileishmanial Vaccine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 433

31 Plasmodium berghei Infection in BALB/c Mice Model as an

PART VI DEVELOPMENT OF CANCER VACCINES

33 Generation of Tumor Targeted Dendritic Cell Vaccines with

PART VII VACCINES FOR ALLERGY

36 Proteomics for Development of Food Allergy Vaccines . . . . . . . . . . . . . . . . . . . . . . 673

PART VIII VACCINES FOR TOXINS

37 Estimating Vaccine Potency Using Antibody-Based

MOHAMED ABDEL-MOHSEN • Vaccine & Immunotherapy Center, The Wistar Institute,

EDUARDO ANTONIO FERRAZ COELHO • Programa de Pos-Graduação em Ciências da Saúde:

NICHOLAS J. MANTIS • Department of Biomedical Sciences, University at Albany School of

(CONICET)/Universidad Catolica de Cordoba (UCC), Cordoba, Argentina; Facultad

DANIEL UDENZE • Vaccine and Infectious Disease Organization-International Vaccine

Challenges for Vaccinologists in the First Half

Infectious diseases are disorders caused by microorganisms includ-

The current pandemic COVID-19 caused by SARS-CoV-2 (started

to mass migration. Lack of fresh water would lead to use of con-

1.2 Development of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is

converting enzyme 2 (ACE2) as the entry receptor. Hence, the

1.3 Development of Acquired immunodeficiency syndrome (AIDS) caused by human

successfully prevent or eradicate the disease. It is a continual raging

2 Development of Vaccines for Flaviviruses

The Flaviviridae family consists of 70 different viruses that are

WN02 is a promising candidate with  96% seroconversion rates

have advanced to clinical phase I/II trials. Candidates for the

T-cell response and/or antibodies to NS1 [39]. Dengvaxia was

3 Development of Vaccines for Norovirus

Norovirus, also known as Norwalk virus, is responsible for gastro-

Norovirus is the leading cause of acute gastroenteritis (AGE)

4 Development of Vaccines for Influenza

Influenza is an infectious respiratory disease; in humans, it is caused

infection vary from a mild respiratory disease confined to the upper

complications of severe influenza. Ideally, new vaccines should

5 Development of Vaccines for Sepsis

Sepsis (septicemia) is a medical emergency that describes the body’s

Secretion of extracellular vesicles (EVs), a process common to

6 Development of Vaccines for Tuberculosis

EDUARDO ANTONIO FERRAZ COELHO • Programa de Pos-Graduação em Ciências da Saúde:

(CONICET)/Universidad Catolica de Cordoba (UCC), Cordoba, Argentina; Facultad

WN02 is a promising candidate with 96% seroconversion rates