2018 NIAID 

STRATEGIC PLAN 

FOR 

RESEARCH 

ON

VACCINE 

ADJUVANTS

NIAID

National Institute of Allergy and Infectious Diseases
2 

TABLE OF CONTENTS

1. INTRODUCTION ................................................................................................................... 3

1.1. BRIEF OVERVIEW OF ADJUVANTS IN LICENSED VACCINES ........................... 3

1.2. THE NEED FOR NEW VACCINE ADJUVANTS

1.3. NIAID’S MISSION AND EXTRAMURAL FOCUS ON VACCINE ADJUVANT 

 ........................................................ 4

RESEARCH ............................................................................................................................ 4

1.4. NIAID’S STRATEGIC PLAN FOR RESEARCH ON VACCINE ADJUVANTS ....... 5

2. FUNDAMENTAL IMMUNOLOGY AND ADJUVANT DISCOVERY ............................. 8

2.1. RECENT PROGRESS IN NIAID-SUPPORTED ACTIVITIES.................................... 8

2.2. PROGRAM GOALS AND RESEARCH CHALLENGES ........................................... 12

2.3. NIAID’S STRATEGY TO ADVANCE ADJUVANT DISCOVERY .......................... 13

3. ADJUVANT DEVELOPMENT AND PRECLINICAL EVALUATION ........................... 16

3.1. RECENT PROGRESS IN NIAID-SUPPORTED ACTIVITIES.................................. 16

3.2. PROGRAM GOALS AND RESEARCH CHALLENGES ......................................... .. 19

3.3. NIAID’S APPROACH TO ADVANCE ADJUVANT DEVELOPMENT AND 

PRECLINICAL EVALUATION .......................................................................................... 21

4. CLINICAL EVALUATION OF ADJUVANTED VACCINES ........................................... 23

4.1. RECENT PROGRESS IN NIAID-SUPPORTED ACTIVITIES ............................ 23

4.2. PROGRAM GOALS AND RESEARCH CHALLENGES ........................................... 25

4.3. NIAID’S APPROACH TO ADVANCE THE CLINICAL EVALUATION OF 

ADJUVANTED VACCINES ............................................................................................... 27

5. SUMMARY .......................................................................................................................... 28

6. APPENDICES ....................................................................................................................... 29

6.1. APPENDIX A: ABBREVIATIONS.............................................................................. 29

6.2. APPENDIX B: BLUE RIBBON PANEL MEMBERS ................................................. 31

6.3. APPENDIX C: NIAID-SPONSORED CLINICAL TRIALS FOR NON-HIV 

ADJUVANTED VACCINES, RECENTLY COMPLETED OR ONGOING (AS OF APRIL 

2018) ...................................................................................................................................... 33

6.4. APPENDIX D: NIAID-SPONSORED CLINICAL TRIALS FOR HIV ADJUVANTED 

VACCINES, COMPLETED OR ONGOING (AS OF APRIL 2018) .................................. 34
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1. INTRODUCTION 

1.1. BRIEF OVERVIEW OF ADJUVANTS IN LICENSED VACCINES 

The use of adjuvants began in 1920 when the French veterinarian Gaston Ramon 

discovered that co-administration of inactivated diphtheria toxoid with starch, 

breadcrumbs, or other substances led to an increase in antitoxin responses to diphtheria. In 

1926, this finding was followed by Alexander Glenny’s discovery of the adjuvanticity of 

aluminum salts and their clinical utility in diphtheria immunization. 

Until 2009, aluminum-based adjuvants, collectively termed “alum,” were the only 

adjuvants in vaccines licensed for use in the United States. That year, the U.S. Food and 

Drug Administration (FDA) licensed the first of five vaccines containing novel adjuvants, 

which are described below and summarized in Table 1: 

• Cervarix®, a human papillomavirus (HPV) vaccine containing AS04, a combination of 

alum and 3-O-desacyl-4'-monophosphoryl lipid A (MPL), an immune-stimulating lipid

developed by GlaxoSmithKline (GSK). In the United States, Cervarix® is approved for 

use in females 10 through 25 years of age to prevent infection from HPV types 16 and 

18, which cause about 70% of cervical cancer cases. 

• Q-Pan H5N1, a pandemic H5N1 influenza vaccine containing AS03, an oil-in-water 

emulsion combination adjuvant developed by GSK. This vaccine is part of the U.S. 

vaccine stockpile but is not commercially available. It is intended for use in people 18 

years of age and older. 

• FLUAD®, an influenza vaccine containing MF59®, a squalene-based oil-in-water 

emulsion adjuvant developed by Novartis, for use in people 65 years of age and older. 

FLUAD® was first licensed in Italy in 1997 and is now used as a pediatric and adult 

influenza vaccine in more than 30 countries. 

• Shingrix®, a shingles (herpes zoster) vaccine containing AS01, a liposome-based 

combination adjuvant containing MPL and the saponin QS-21, developed by GSK. 

Shingrix® is for use in adults 50 years of age and older. 

• Heplisav-B®, a hepatitis B vaccine containing CpG-oligodeoxynucleotide (ODN), a 

short, single-stranded DNA molecule that mimics unmethylated bacterial DNA and 

triggers innate immune responses through activation of Toll-like receptor 9 (TLR9), 

developed by Dynavax. This vaccine is for use in people 18 years of age and older. 

Table 1. FDA-approved vaccines containing novel adjuvants (2009-2018) 

Year Adjuvant Vaccine Name

2009 AS04 Cervarix®

2013 AS03 Q-Pan H5N1 

2015 MF59® FLUAD®

2017 AS01B Shingrix®

2017 CpG-oligodeoxynucleotide Heplisav-B®

 
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1.2. THE NEED FOR NEW VACCINE ADJUVANTS

Live-attenuated vaccines and some vaccines based on inactivated or killed agents do not 

require exogenous adjuvants, because the vaccines contain their own natural/endogenous 

adjuvants that can stimulate both the innate and adaptive arms of the immune system. 

However, subunit vaccines, which contain specific purified molecular entities, generally 

lack immune stimulators found in whole organism–based vaccines, leading to a need for 

exogenous adjuvants to induce protective immunity and desirable vaccine efficacy. 

Unfortunately, not all adjuvanted vaccines induce the types of immune responses required 

for durable protective immunity. New or improved adjuvants may enhance the safety and 

efficacy of current vaccines, and they also are needed to improve vaccine efficacy in at-risk

populations such as neonates, young children, pregnant women, the immunocompromised, 

and the elderly. Finally, vaccines do not exist for many known and newly emerging 

infectious agents, and the availability of new adjuvants, along with an understanding of 

their mode of action, may facilitate development of such vaccines.

1.3. NIAID’S MISSION AND EXTRAMURAL FOCUS ON VACCINE ADJUVANT 

RESEARCH

The overall mission of the National Institute of Allergy and Infectious Diseases (NIAID), 

part of the National Institutes of Health (NIH), is to improve human health by supporting

research to understand, treat, and prevent infectious and immune-mediated diseases. 

Critical components of NIAID’s research agenda in support of this mission are the 

discovery and development of adjuvants, traditionally defined as vaccine components that 

augment and target immune responses. 

NIAID’s three extramural divisions—the Division of Allergy, Immunology, and 

Transplantation (DAIT); the Division of Microbiology and Infectious Diseases (DMID); 

and the Division of AIDS (DAIDS)—all support aspects of adjuvant research that are

described in detail in Sections 2, 3, and 4. Briefly, the mission of each division and its 

adjuvant research focus is described below. 

DAIT supports basic and clinical research to understand immune system development; 

define the mechanisms governing immune system function across the lifespan, providing 

knowledge that can be applied to develop and improve treatment and prevention strategies 

for inducing immunity against pathogenic infections; and develop better diagnostic, treatment, 

and prevention strategies for immune-mediated diseases and the rejection of transplanted

organs. DAIT adjuvant research focuses on fundamental immunology, adjuvant discovery, and 

early-stage development. The latter includes compound screening, determination of adjuvant 

mechanisms of action, lead compound optimization and formulation, efficacy testing in 

animals, and investigational new drug (IND)–enabling studies. Adjuvant science programs 

supported by DAIT began in 2004 and include the Adjuvant Discovery Program, the 

Adjuvant Development Program, and the Molecular Mechanisms of Combination 

Adjuvants Program. Through these programs, more than 2 million compounds have been 

screened, and numerous adjuvant candidates, encompassing eight different adjuvant types 
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or classes, are being further developed. Other programs invested in characterizing immune

responses to adjuvants include the NIAID Systems Approach to Immunity and 

Inflammation Program and the Human Immunology Project Consortium (HIPC). 

DMID supports basic and applied research to control and prevent diseases caused by 

virtually all human infectious agents except HIV. The DMID portfolio includes a broad 

range of research from early discovery through product development for vaccines, 

therapeutics, and diagnostics, including novel vaccines (adjuvanted and unadjuvanted) and 

platform technologies. Currently, DMID is testing more than 100 different adjuvant 

candidates/formulations encompassing all stages of vaccine development. DMID also 

offers a comprehensive suite of preclinical services for the development and evaluation of 

adjuvanted and unadjuvanted vaccines. 

DAIDS supports a comprehensive research portfolio to advance biological knowledge of 

HIV/AIDS and its related coinfections and comorbidities. The ultimate goal is to halt the 

spread of HIV through the development and implementation of an effective vaccine and 

biomedical prevention strategies that are safe and desirable. The adjuvants used in HIV 

vaccine candidates, as of April 2018, are listed in Appendix D. 

1.4. NIAID’S STRATEGIC PLAN FOR RESEARCH ON VACCINE ADJUVANTS 

As technologic and scientific advances increased our understanding of the pathways 

regulating innate and adaptive immune responses, NIAID recognized opportunities to 

develop novel adjuvants or modify existing adjuvants to safely trigger long-term protective 

immunity and durable vaccine efficacy. In 2010, NIAID prepared its first Strategic Plan for

Research on Vaccine Adjuvants and convened an Adjuvant Blue Ribbon Panel (BRP) to 

review the plan. 

The 2010 BRP made the following recommendations: 

• Support additional research to understand how new and existing adjuvants and 

combinations of adjuvants enhance vaccine-specific immune protection

• Foster discovery and advancement of promising adjuvant candidates through 

optimization and preclinical testing stages

• Develop/improve and standardize animal models to evaluate adjuvant safety and 

efficacy, including development/use of animal models that more faithfully reflect 

immune responses of neonates/young children and the elderly 

• Expand/standardize reagents for adjuvant discovery and development, including 

pathogen-specific reagents, and immunologic reagents for animals beyond traditional 

mouse models 

• Identify more accurate predictors of adjuvant efficacy and/or reactogenicity

• Determine the effects of formulation on adjuvant mechanisms of action and safety and 

effectiveness of adjuvanted vaccines

Between 2010 and 2018, NIAID’s extramural adjuvant discovery and development 

research programs have implemented these recommendations. Because of significant
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advances in adjuvant science, many of which were driven by NIAID-supported programs, 

NIAID has updated its 2010 adjuvant research agenda, culminating in the 2018 Strategic 

Plan, which focuses on three areas: 

• Fundamental immunology and adjuvant discovery 

• Adjuvant development and preclinical evaluation

• Clinical evaluation of adjuvanted vaccines 

NIAID convened a second BRP on April 23-24, 2018, to assess this Plan and provide 

insights and recommendations, which are included in appropriate sections of the Plan and 

summarized in Table 2. 

Table 2. 2018 Blue Ribbon Panel Recommendations 

Fundamental Immunology and Adjuvant Discovery

Fundamental Immunology 

• Expand fundamental mechanistic studies to: 

o Identify innate correlates of adaptive immunity, including correlates of both protection 

and persistent immunity, for healthy and at-risk populations 

o Determine how antigen selection, including antigen processing/presentation 

mechanisms, impacts adjuvant functionality 

Adjuvant Discovery 

• Expand discovery efforts beyond TLRs, to include adjuvants that trigger 

inflammasome components, target specific immune cells, or mimic immunologic 

outcomes of natural infections that induce long-term protective immunity 

• Foster precision adjuvant discovery by first determining clinical/immunologic needs, 

followed by designing a discovery process to identify compounds that meet those needs 

• Support development of functional screening methods that better mimic in vivo responses, 

taking into account human variability and at-risk populations 

• Compare/optimize adjuvants for specific cell targeting and delivery routes 

• Encourage development of standard operating procedures and best practices that maximize 

screening efforts and data sharing for cross-study analyses and benchmarking to licensed 

products 

• Support central core services to further improve program integration: formulation 

assistance, production of standardized reference antigens, and animal testing (multiple 

models) for head-to-head adjuvant comparisons 

• Emphasize importance of considering formulation at the early stages of adjuvant discovery 
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Adjuvant Development and Preclinical Evaluation

• Explore iterative testing of candidate adjuvants with a broad panel of antigens to identify 

the most promising combinations for further development 

• Establish a public database/portal that describes the characteristics, functionality, and 

safety profiles of novel compounds developed in NIAID’s adjuvant programs, including 

links to publications and clinical trials information, and procedures for requesting access to 

these adjuvants 

• Support studies that develop methods to extrapolate adjuvant outcomes seen with one 

antigen to additional antigens 

• Expand support of current Good Manufacturing Practice (cGMP) capabilities to support 

small-scale cGMP production of promising adjuvants for clinical trials

Clinical Evaluation of Adjuvanted Vaccines

• Support clinical trials of novel adjuvants and expand clinical comparisons of different 

adjuvants formulated with the same antigen 

• Support clinical trials of adjuvanted vaccines in at-risk populations (e.g., elderly, children) 

• Support comprehensive immunological analyses of samples from clinical trials to generate 

detailed immune profiles of adjuvant functionality 

• Increase use of human challenge models, where applicable, to better assess the efficacy of 

adjuvanted vaccines 

• Ensure at least a 12-month post-vaccination assessment for safety and immune durability
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2. FUNDAMENTAL IMMUNOLOGY AND ADJUVANT DISCOVERY

2.1. RECENT PROGRESS IN NIAID-SUPPORTED ACTIVITIES

Fundamental Immunology

Fundamental immunology research supported by NIAID’s portfolio of investigatorinitiated grants and solicited research programs has identified many novel innate immune 

molecules and increased our understanding of innate and adaptive immunity. For example, 

investigator-initiated studies have led to the discovery of pathogen-associated molecular 

patterns (PAMPs), which trigger innate immune responses and are the basis of many 

adjuvants (e.g., they mimic PAMP-induced immune function). More recently, NIAIDsupported investigators have determined that live/viable bacteria contain a unique class of 

PAMPs, termed vita-PAMPs, which are bacterial messenger RNAs (mRNAs) and cyclic

di-adenosine monophosphate (c-di-AMP) that serve as signatures of viability and trigger 

specific innate immune responses. Similarly, investigators supported by one of NIAID’s 

solicited research programs, Systems Approach to Immunity and Inflammation, have: 

• Identified novel functions for more than 260 genes, many of which provide insights 

into signaling pathways triggered by innate immune receptors and will aid in 

mechanism-of-action studies of novel candidate adjuvants 

• Developed a suite of ENU-mutant mice (https://mutagenetix.utsouthwestern.edu/) and 

Collaborative Cross (CC) mice (http://csbio.unc.edu/CCstatus/index.py) that can be 

used to confirm or determine an adjuvant’s mechanism of action. Mice from both 

resources can be used in various ways to identify immune receptors or signaling 

pathways responsible for adjuvant function. 

Novel animal models also have been developed through investigator-initiated research 

projects. For example, researchers at the University of Minnesota have developed the “dirty 

mouse” model by determining that the microbiome of pet store mice dramatically impacts 

immune system function. Dirty mice exhibit immune functionality more reflective of adult 

human responses, while immune function in laboratory mice is more similar to that of 

human infants. Co-housing the pet store mice with laboratory mice increases the 

microbiome diversity of the laboratory mice and changes their immune functionality to that 

of the pet store mice (e.g., more similar to adult human responses). 

 

Adjuvant Discovery 

NIAID began its first solicited program in adjuvant science in 2003 with the Adjuvant 

Discovery Program. This contract program supported the identification and 

characterization of novel, effective, and safe vaccine adjuvants. The scientific scope of this 

program included: 

• Use of high-throughput screening methods for adjuvant identification 
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• Validation of adjuvant activity in human cells 

• Optimization of lead adjuvant candidates through formulation and medicinal chemistry 

guided by structure–activity relationship (SAR) studies 

• Mechanism of action studies 

• Verification of adjuvanticity in animal models 

The program was renewed in 2009. Building on knowledge gained from the 2003 and 2009 

programs and to address recommendations made by the 2010 Adjuvant BRP, the 2014 

program renewal emphasized discovery of adjuvants (1) for use in at-risk populations and 

(2) that induced mucosal immune responses. Recognizing the limitations of a program that 

renews only every five years, and following the recommendations of the 2010 Adjuvant 

Blue Ribbon Panel to increase the investment in adjuvant research, in 2017, NIAID began 

using the annual Small Business Innovation Research (SBIR) contract solicitation to 

support additional adjuvant discovery activities. This program, while new, already has 

expanded the investigator pool and increased the diversity of adjuvant candidates for the 

development pipeline. 

Since its inception, researchers funded by NIAID’s Adjuvant Discovery Program have 

screened more than 2 million compounds in vitro and more than 1024 in silico and 

identified a wide variety of promising adjuvant candidates, including: 

• Synthetic TLR2, 4, 5, 7, 8, 7/8, and 9 agonists; nanoemulsions (for mucosal 

delivery)

• C-type lectin receptor (CLR) agonists 

• CD1d ligands (natural killer T [NKT] cell inducers) 

• Synthetic RIG-I agonists 

• Carbohydrate-based adjuvants 

Figure 1 shows representative immune receptors and pathways targeted by compounds and 

formulations that have resulted from the NIAID Adjuvant Discovery Program.
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Fig. 1

Figure 1: Vaccine adjuvants from NIAID’s adjuvant discovery and adjuvant 

development programs target a variety of cellular receptors and signaling pathways. 

Some of these adjuvants (solid arrows) engage cell-surface and intracellular RIG-I-like 

receptors and C-type lectin receptors. In addition, adjuvants have been developed that 

directly act on signaling molecules of these receptors, act by rearranging lipid rafts and 

associated receptors, or provide adjuvanticity through yet undefined mechanisms 

(dashed-line arrows).

Specific accomplishments of these programs include identification of the following 

molecules with adjuvant activity, including: 

• Novel CpG-ODNs that exhibit higher adjuvanticity than previously published CpGODN and are not species specific (e.g., able to trigger both mouse and human TLR9).

One of these novel CpG-ODNs, originally identified through in silico screening, is now

part of an inulin-CpG combination adjuvant that has been used in two clinical trials of

influenza vaccines.

• Co-adjuvants in the form of small molecules that enhance the activity of an adjuvant

but do not exhibit intrinsic adjuvant activity themselves. Such co-adjuvants have been

shown to extend NF-KB signaling in antigen-presenting cells (APCs) and thus enhance

lymphocyte activation.

• Novel oxoadenine-based TLR7/8 agonists with high potency and low reactogenicity
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• Amphotericin B as a novel TLR2/TLR4 agonist with adjuvant activity and low

reactogenicity

• Adjuvant formulations that target adjuvant activity to specific APCs or lymph nodes,

reducing reactogenicity of potent imidazoquinoline (IMDQ)-based TLR7 agonists, such

as:

o Lysophospholipid conjugates of IMDQ

o IMDQ-ligated nanogel

o Hyaluronic-acid conjugated IMDQ

• First small-molecule TLR4 agonist, which is currently being developed as a

combination adjuvant with TLR7 agonists for influenza vaccines

• Novel RIG-I agonists, as follows:

o A small molecule compound called KIN, currently being developed for vaccines

against emerging RNA viruses, including pandemic influenza and Zika

o Synthetic, Sendai-virus-derived double-strand RNA (dsRNA) adjuvant for

intranasal immunization against influenza

• Nanoemulsions (NEs) for intranasal or intramuscular injection, which act as delivery

platform and adjuvant. NEs inactivate viruses and bacteria and can be used to produce

killed, whole organism adjuvanted vaccines. Intranasal formulations can elicit strong

Th1/Th17 responses upon boost with NE-adjuvanted vaccine, overcoming preexisting

antigen-specific Th2 dominance. This approach is being developed for a recombinant

influenza vaccine and a B. pertussis vaccine.

• Selective TLR8-agonist (methylpyrimidine-diamine) without TLR7 activity and ultralow inflammatory profile

• Dendrimeric IMDQ construct with pure TLR7 activity, which has low reactogenicity

and enhanced adjuvanticity (compared to monomeric IMDQ) and promotes antibody

affinity maturation and epitope spreading

• First chemokine receptor CCR1 agonist adjuvant (bis-quinoline)

• NOD-1 agonist adjuvant (glutamyldiaminopimelic acid-derivative)

• Novel water-soluble TLR2 agonist

• Synthetic CRX compounds (MPL-derivatives), which are TLR4 agonists with high

safety profiles. Several of these compounds were developed as adjuvants for

intramuscular delivery and sublingual formulation (combination adjuvant with

methylglycol chitosan) for influenza vaccines. They also are being developed as

combination adjuvants with novel TLR7/8 agonists.

• Protein cage nanoparticles (PCN) for mucosal delivery to induce tertiary lymphatic

tissue

The Adjuvant Discovery Program has also identified molecules with valuable, nonadjuvant properties that include: 

• Novel antimicrobials

o Antivirals against Zika and dengue viruses

o Antibiotics such as a modified TLR7/8 agonist without TLR activity, which acts as 

a potent inhibitor of Gram-positive bacteria, including methicillin-resistant

Staphylococcus aureus (MRSA)

o Pure TLR7 agonists currently being evaluated as HIV latency-reversal agents
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• Large libraries of TLR7, TLR8, and TLR7/8 agonists that modulate immune responses

and can be used as tools to study TLR7 and TLR8 signaling pathways

2.2. PROGRAM GOALS AND RESEARCH CHALLENGES

Goal 1: Strengthen Immunology Research 

The most significant advances in NIAID’s Adjuvant Discovery Programs have come from 

increased knowledge of innate immune receptors and their mechanisms of action (Figure 

1). 

The first innate immune receptor agonists developed as adjuvants targeted TLR4 and 

TLR9. NIAID-supported researchers have expanded screening to include molecules that 

bind to NOD-like receptors and C-type lectin receptors, as well as molecules involved in 

signal transduction from innate immune receptors, such as various kinases and interferon 

regulatory factors. Furthermore, an increased understanding of how innate and adaptive 

immune cell subsets contribute to vaccine-induced immunity is leading to the discovery of 

adjuvants that target cells other than macrophages and dendritic cells. These cells include B 

cells, mast cells, and NKT cells. 

While considerable progress has been made in discovery of innate immune receptors and 

their mechanisms of action, several challenges remain. These include: 

• Limitations of existing methods to evaluate potential adjuvant function in vivo.

Most screening approaches are based on a limited number of parameters, such as NFKB activation or the induction of select cytokines from either cell lines or primary cells.

In vitro induction of those factors may not translate into adjuvanticity in vivo. Better

methods to evaluate adjuvant function, based on increased knowledge of immunologic

mechanisms, will ensure that promising candidates are not being overlooked.

• Selection of appropriate animal models for adjuvant screening. Host species

differences in immune receptor specificity and cellular distribution, immunogenetics,

and pathogen susceptibility can complicate the discovery of adjuvants for human use.

One example is TLR8, which was thought to be nonfunctional in mice until the recent

discovery of the mouse TLR8 ligand and the realization that it differs significantly from

the human TLR8 ligand. The selection of appropriate animal models for adjuvant

screening is critically important.

• Adjuvants for at-risk populations, especially the very young and frail elderly.

The recent successes of the AS01-adjuvanted Shingrix® vaccine and MF59®-

adjuvanted influenza vaccine in the elderly and the AS03- and MF59®-adjuvanted

influenza vaccines licensed outside the U.S. for young children clearly demonstrate that

adjuvants can overcome age-associated immune deficits. However, additional adjuvants

are still needed for use in the very young and the frail elderly.
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Goal 2: Support Technology Development for Adjuvant Discovery 

Adjuvant discovery methods have improved in recent years for two main reasons: 

• Technologic advances have allowed for more efficient in vitro screening of compounds

with progressively higher throughput rates.

• Advances in medicinal chemistry have enabled rapid and systematic modifications of

molecules to alter their immune stimulatory properties.

In addition, the focus of adjuvant discovery has shifted from natural compounds and/or 

derivatives of known innate immune receptor agonists to small molecules that bear no 

structural similarities to natural compounds. These molecules may bind to sites on an 

immune molecule that may or may not overlap with the binding site for the natural ligand. 

These types of adjuvants are identified by high-throughput screening of chemical libraries. 

Recently, in silico approaches have been employed (1) to pre-screen compound libraries to 

reduce the number of compounds that need to be screened in vitro or (2) to explore the 

potential utility of computer-generated structures before synthesis and in vitro testing.

While in silico screening has the potential to revolutionize adjuvant science, this approach 

still faces challenges, which include the needs to: 

• Determine structure–activity relationship (SAR) to improve the accuracy of in

silico screening methods. SAR analysis defines the relationship between a

compound’s chemical or three-dimensional structure and its biological activity. Insights

gained through SAR analysis can be used to rationally alter structure and hence the

potency or functionality of a compound. Application of SAR to in silico screening of

adjuvants requires knowledge of the structure of the immune receptor being targeted.

• Define binding site requirements. Recent identification of small-molecule compounds

that can bind to innate immune receptors but lack structural similarity to the natural

receptor ligands highlights gaps in our understanding of the requirements for receptor

activation. In addition, ligand binding is not necessarily associated with receptor

activation. Thus, in silico modeling alone can be misleading and needs to be coupled

with laboratory validation to improve overall accuracy.

• Provide access to supercomputing facilities. The most frequently used algorithms for

in silico screening simulate static docking of putative ligands to their receptors, rather

than the dynamic interactions that occur naturally between receptor and ligands.

Modeling such dynamic interactions requires supercomputing capacity that is not

readily available to many researchers.

2.3. NIAID’S STRATEGY TO ADVANCE ADJUVANT DISCOVERY

Based on NIAID’s goals and recommendations from the BRP, NIAID’s 2018 Strategic 

Plan for Research on Vaccine Adjuvants focuses on two major areas: strengthen 
14 

immunology research and support technology development.

• Strengthen immunology research, to specifically improve evaluation of adjuvant 

function, provide new animal models for adjuvant screening, and facilitate the 

discovery of adjuvants for use in at-risk populations. To meet this goal, NIAID will 

continue to support and expand current programs, and develop new programs, as 

follows: 

o Current programs

 The Systems Approach to Immunity and Inflammation Program, which is

identifying novel immune response genes through development and analysis of 

novel mouse models. The data and novel mouse models generated through this 

program will enhance adjuvant discovery efforts. 

 The Immunity in Neonates and Infants program and the Immunity in the Elderly 

program, which are characterizing immune functionality and facilitating 

identification of possible adjuvant targets in these at-risk populations. 

 The Adjuvant Discovery Program, which continues screening, preclinical 

testing, and early-stage development of new adjuvant candidates and will pursue 

discovery of immune modulating or tolerogenic adjuvants. 

 The Molecular Mechanisms of Combination Adjuvants (MMCA) program, 

which is providing novel insights into the mechanisms of combination adjuvant 

activity and the basis for synergistic adjuvanticity.

o FY 2019 new NIAID initiatives

 Maintaining Immunity After Immunization, which will increase understanding 

of how durable immunity is induced and maintained in response to infection or 

vaccination and will identify novel targets for adjuvant discovery 

 Collaborative Cross (CC) Mouse Model Generation and Discovery of 

Immunoregulatory Mechanisms, which will support the use of CC mouse lines 

to increase understanding of the host genetics involved in immune regulation 

and function 

o NIAID will continue to use the SBIR contract mechanism to fund additional 

adjuvant discovery by small business entrepreneurs and the development of 

immunologic reagents for underrepresented animal models that may be better 

models of human immune responses (e.g., ferrets for influenza research). 

o In addition to its solicited adjuvant portfolio, NIAID will maintain and expand its 

portfolio of unsolicited adjuvant grants using a variety of mechanisms, such as 

SBIR, R01, R03, R21, and T grants. 

 

• Support technology development for adjuvant discovery. For example:

o NIAID will expand support of structural immunology studies to enhance SAR 

analyses and improve the accuracy of in silico adjuvant discovery methods. 

Currently, most of the structural immunology research supported by NIAID is 

through investigator-initiated grants. While these studies have provided valuable 

insights into the structure–function relationships of many important immune 

molecules, technologic advances in this area would increase the number of 
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available structures for use by the adjuvant discovery community. NIAID will 

consider using the SBIR contract funding mechanism and other approaches to grow 

this important research area.

o NIAID’s Office of Cyber Infrastructure and Computational Biology (OCICB) 

provides sophisticated biocomputing resources to NIAID’s intramural researchers.

NIAID extramural staff will work with OCICB staff to make these supercomputing 

capabilities available to adjuvant researchers in the extramural community. 
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3. ADJUVANT DEVELOPMENT AND PRECLINICAL EVALUATION

3.1. RECENT PROGRESS IN NIAID-SUPPORTED ACTIVITIES

Adjuvant Development Program

NIAID expanded its solicited program in adjuvant science in 2008 with the Adjuvant 

Development Program. This contract program supports: 

• Preclinical development of novel adjuvants coupled with specific vaccines, including 

mechanistic analyses of the novel adjuvant/antigen combinations 

• Formulation and dosing studies 

• Optimization of immunization regimens and delivery routes 

• cGMP production 

This program was recompeted in 2013 and 2018 with the goal of ensuring further 

development of promising adjuvant candidates through preclinical testing and INDenabling studies, thus expanding the number of adjuvants available for clinical evaluation. 

Several of the compounds evaluated through the Adjuvant Development Program

originated from NIAID’s Adjuvant Discovery Program, and some have been or are 

currently being tested in more than 20 clinical trials. In 2016, NIAID began using the 

annual SBIR contract solicitation to support additional adjuvant development activities. 

The adjuvant/antigen or adjuvant/vaccine combinations assessed through the Adjuvant 

Development program and SBIR contract program include: 

• Microcrystalline inulin with seasonal, pandemic, and universal influenza vaccine 

candidates

• Nanoemulsions with a plant-derived, recombinant H5 influenza vaccine or HIV 

envelope virus-like particle (VLP) vaccine

• Fully synthetic TLR4 agonists plus plant-derived saponin (QS-21), coupled with a 

recombinant E protein from West Nile virus 

• A small-molecule RIG-I agonist combined with split H5N1 influenza virus, UVinactivated West Nile virus, or a VLP-based vaccine for Zika virus 

• The protein-based mucosal double-mutant heat-labile toxin (dmLT) adjuvant, with 

BCG and the recombinant fusion protein, 5fu, from Mycobacterium tuberculosis and a 

recombinant IpaB-IpaD fusion protein from Shigella sonnei

• A novel semi-synthetic analog of QS-21 (TiterQuil-1055TM) with Fluzone®, Fluarix®, 

and Flublok® influenza vaccines

Adjuvants in HIV Preclinical Vaccine Development

NIAID also supports adjuvant development within vaccine-focused research programs. 

NIAID’s HIV preclinical vaccine development activities focus predominately on variations 

of HIV Env immunogens in combination with an array of adjuvants and/or delivery 
17 

systems to enhance potency and durability. These studies are conducted in both small 

animals and nonhuman primates. An extensive variety of adjuvants and adjuvant 

combinations have been tested, with the most common adjuvants being Alhydrogel and 

other aluminum salts formulations, MF59®, AS0 series, Adjuplex, ISCOMATRIX®, pIL12, and multiple TLR agonists alone or in combination (3M-052, CpG, poly ICLC, R848, 

GLA-SE). 

Adjuvants in Non-HIV Preclinical Vaccine Development

NIAID’s vaccine research and development activities for non-HIV infectious diseases 

include three different types of vaccines: live attenuated vaccines; inactivated vaccines; and 

subunit vaccines composed of purified protein, recombinant protein, or carbohydrate. These 

preclinical research and development efforts focus on early discovery of adjuvanted vaccine 

targets and IND-enabling activities, including providing biological resources, animal 

testing, process development, Good Laboratory Practice (GLP) toxicity assessment, and 

cGMP production of vaccines or vaccine components. Early-stage research on adjuvanted 

vaccines includes pairing of antigens with adjuvants, formulation studies, and the design 

and construction of novel vaccine candidates in which an adjuvant is built into the vaccine 

construct. Examples of this latter approach include co-display of the adjuvant and antigen 

on the surface of nonpathogenic engineered microbes and nanoparticle molding 

technologies for co-delivery of adjuvant and antigen. NIAID also supports development of 

animal models that are relevant for or mimic natural infection or diseases of public health 

importance to evaluate vaccine formulation. These models are especially important for 

vaccine research for biodefense or emerging infectious diseases, since the data generated 

may be utilized for vaccine emergency use authorization, accelerated approval, or pivotal 

efficacy studies under the Animal Rule. 

NIAID also supports the development of adjuvanted vaccines in some high-priority 

research areas through investigator-initiated research grants and various program 

initiatives, such as SBIR contracts, Centers of Excellence for Translational Research, 

Advanced Development of Vaccine Candidates for Biodefense and Emerging Infectious 

Diseases, and Research to Advance Vaccine Safety. Selected examples of current activities

include evaluation of: 

• Candidate tuberculosis (TB) vaccines containing traditional and novel adjuvants or 

adjuvant systems, as follows: 

o The cyclic dinucleotide (CDN) class of molecules (a vita-PAMP), which includes cdi-AMP, as regulators of M. tuberculosis physiology and the host immune response 

to infection and vaccination. Research is underway to determine the adjuvant 

potential of c-di-AMP in a preventative tuberculosis vaccine. 

o The cationic liposomal adjuvant CAF09, which induces both CD4+ and CD8+ T 

cells

o An adjuvant platform to produce micelles and vesicles incorporating mycolic acid, 

CpG, and MPL for intranasal immunization 

o A wide range of adjuvants, including alum, GLA-SE, GLA-AF, QS-21, CpG-SE, 

and liposomes, which are being evaluated independently and in combination with 
18 

either anionic, neutral, or PEG liposomes to identify the optimal adjuvant pairing 

with the M. tuberculosis antigen ID93

• Candidate malaria vaccines containing: 

o The novel glycolipid adjuvant 7DW8-5 combined with a live attenuated sporozoite 

vaccine

o Traditional adjuvants such as Alhydrogel, MPL/Alhydrogel, ISA 51 VG, ISA

720 VG, GLA-SE, GLA-LSQ, AddaVaxTM, and CpG 7909, in malaria subunit-based 

vaccines

o Nano/microparticle technology platforms for a malaria transmission-blocking 

vaccine

Programs in Support of Adjuvanted Vaccine Development 

Several of NIAID’s basic immunology programs facilitate selection of appropriate antigens 

and evaluation of antigen-specific immunity generated by candidate adjuvanted vaccines, 

including: 

• The Immune Epitope Database and Analysis Resource (IEDB, www.iedb.org), which 

provides public access to B and T cell immune epitope information curated from the 

scientific literature or through direct submission by the research community, and

sophisticated epitope prediction and analysis bioinformatics tools

• The B-Cell Epitope Discovery and Mechanisms of Antibody Protection program and 

the Large-Scale T-Cell Epitope Discovery program 

• The NIH Tetramer Core Facility (http://tetramer.yerkes.emory.edu/) 

• The Human Immunology Project Consortium (HIPC) program, which uses novel 

approaches, including metabolomics, transcriptomics, and proteomics, to conduct 

comprehensive systems immunologic analyses of human immune responses to 

infections, adjuvants, and/or vaccines

• The Cooperative Centers on Human Immunology (CCHI) program, which is 

developing novel technologies and bioinformatics tools to conduct mechanistic 

analyses of human immune responses to infections and vaccines (adjuvanted and 

unadjuvanted) 

• The Modeling Immunity for Biodefense program, which includes the development of 

computational tools that analyze antibody-antigen interactions and facilitate design of 

more immunogenic or cross-protective antigens that can be paired with adjuvants

Preclinical Development Services and Support

NIAID also provides a comprehensive suite of preclinical development services that fill 

particular knowledge and experimental gaps critical to move adjuvanted vaccine candidates 

along the product development pathway. These resources include: 

• BEI Resources, a central repository that supplies organisms and reagents, including 

those relevant for vaccine or adjuvant research to the research community

• The Preclinical Models of Infectious Diseases program, which provides services to 

researchers to study the full range of pathogens, including bacteria, viruses, parasites,
19 

fungi, and other agents such as toxins and prion proteins. The program’s main focus is 

to help researchers who have developed a product such as a vaccine that needs to be 

tested in an animal model, but who lack either the resources or expertise needed to 

perform that testing, and to support the development and refinement of animal models 

for infectious diseases of importance to humans. 

• NIAID’s Vaccine Development Services, which are intended for use in the 

investigation, control, prevention, and treatment of a wide range of infectious agents 

and to support the development of vaccines; vaccine components, including adjuvants; 

vaccine delivery systems; and challenge material. Vaccine testing services include 

assay development, immunogenicity and efficacy studies, clinical and nonclinical 

sample testing, and safety and toxicity testing. Vaccine manufacturing services include 

writing product development plans, product optimization, non-cGMP and cGMP 

manufacturing of antigens and adjuvants, assay development, and regulatory activities, 

including audits. 

NIAID will continue to support new program initiatives to address the development and 

evaluation of important vaccines and vaccine technologies, including adjuvants. Examples 

of some existing programs are: 

• Centers of Excellence for Translational Research (for emerging/reemerging vaccine 

technologies) 

• Novel Vaccine Technologies and Strategies to Promote Sustained Vaccine Efficacy (for 

malaria and pertussis) 

• Advanced Development of Vaccine Candidates for Biodefense and Emerging 

Infectious Disease (for vaccines, including influenza and TB; and technologies, 

including adjuvants) 

• Research to Advance Vaccine Safety

These services and programs support a wide range of preclinical development needs for 

vaccines, including adjuvanted vaccine construction; in vitro and in vivo assessment of 

candidate products; generation of data regarding optimization, synthesis, and formulation 

of candidates; manufacturing using cGMP standards; and safety assessments such as GLP

toxicity studies. 

3.2. PROGRAM GOALS AND RESEARCH CHALLENGES

Goal 1: Expand Availability of Novel Adjuvants for Preclinical Vaccine Development

and Clinical Evaluation

Vaccine developers tend to look for adjuvants with well-established safety records, 

preferably in humans, when selecting an adjuvant for their candidate vaccines. NIAID’s 

adjuvant development activities include mechanism-of-action studies to gain insights into 

immune pathways triggered by adjuvants and ensure that off-target effects are identified 

and mitigated early in the development process. These efforts will be crucial in assembling 

a pool of well-characterized, potent adjuvants with proven safety records that can be used 

to accelerate development of effective vaccines. 
20 

The major challenge associated with early stage development and preclinical assessment of 

novel adjuvants and adjuvant vaccines is: 

• The ability to correlate safety and efficacy observed in animal models to human 

responses. Animal models, while valuable, are not able to emulate many of the 

factors affecting responses in humans. These factors include:

o Pre-existing conditions and medications; recent infection history and chronic 

infection status (e.g., cytomegalovirus [CMV], Epstein-Barr virus [EBV]) 

o Genetics/epigenetics (e.g., species-specific TLR expression patterns and ligand 

binding) 

o Microbiome composition 

o Behavior, including exercise and recreational drug use

o Nutrition and diet 

o Environmental exposures 

In addition, current animal models may not identify the most effective vaccine regimens or 

predict adjuvant-vaccine reactogenicity in humans. Although certain cytokines, other 

soluble molecules, and transcriptional networks have been associated with reactogenicity, 

well-defined signatures that reliably predict reactogenicity have not been identified.

Concerns about adverse events have slowed the development and licensure of adjuvanted 

vaccines, especially for at-risk populations such as the very young, pregnant women, and 

the elderly.

Goal 2: Optimize Formulation, Antigen/Adjuvant Pairing, and Support cGMP 

Production

Development of novel adjuvanted vaccines involves activities that require specialized, 

multidisciplinary teams with strong formulation expertise. NIAID’s adjuvant and vaccine 

development programs support formulation studies, including the iterative process required 

to optimize antigen/adjuvant pairing, and cGMP production of lead adjuvant and adjuvantantigen formulations for further downstream preclinical development and clinical 

evaluation. Formulation studies are crucial during adjuvant development and vaccine 

construction. However, they also must be considered during the early stages of adjuvant 

discovery and adjuvant-antigen pairing, since inadequate or suboptimal formulation may 

prevent promising novel adjuvants or adjuvanted vaccines from moving forward in 

development. 

The major challenges associated with these activities include: 

• Formulation considerations. Subtle differences in formulations can significantly 

impact immunogenicity, stability, and safety of adjuvanted vaccines. In addition, due to 

significant advances in antigen discovery and development of protein expression 

platforms, most vaccines currently in development are subunit vaccines. Pairing the 

correct protein antigens with the appropriate adjuvants during the early development 

stage is critical for future success of a safe and efficacious vaccine. Currently, given the 
21 

lack of correlates or surrogates of protection for most pathogens, head-to-head 

comparisons of multiple adjuvants with specific vaccine antigens may be the most 

efficient process for identifying the optimal adjuvants for a given antigen. 

Despite the vital importance of formulation, including co-formulation of 

adjuvant/antigen pairing, adjuvant development efforts frequently lack adequate 

consideration of formulation, which can impact: 

o Proper adjuvant selection and retention of antigenicity/immunogenicity 

o Targeting of the adjuvant to the correct immune cells and lymphatic tissues

o Co-localization of antigen and adjuvant, or proper antigen conformation 

o Timed release of the adjuvant to prevent induction of excessive innate immune 

responses and the potential for reactogenicity 

o Selection of the optimal delivery system and vaccination strategy

• cGMP manufacturing. Bottlenecks for vaccine developers in academia and small 

businesses are (1) limited access to proprietary adjuvants; (2) development of scalable, 

robust manufacturing processes for promising novel adjuvants; and (3) lack of 

sufficient quantities of cGMP material for well-characterized adjuvants/adjuvanted 

vaccines required for early phase clinical trials.

3.3. NIAID’S APPROACH TO ADVANCE ADJUVANT DEVELOPMENT AND

PRECLINICAL EVALUATION

To address the issues raised above and to continue the development of promising 

adjuvanted vaccine candidates, NIAID will support the following activities: 

• Expand availability of novel adjuvants for preclinical vaccine development and clinical 

evaluation by improving the ability to correlate safety and efficacy profiles generated in 

animal models to human responses. 

To accomplish these efforts, NIAID will: 

o Continue NIAID’s Preclinical Models of Infectious Disease program, which 

provides a central resource for development and refinement of animal models, 

including traditional small laboratory animals, nonhuman primates, and 

nontraditional animals such as swine. This program also offers in vivo screening

and efficacy testing in appropriate animal models.

o Continue NIAID’s SBIR contract program for the development of immunologic 

reagents for underrepresented mammalian models, including ferrets, guinea pigs, 

and swine, to expand the types of animal models that can be used for detailed 

immunologic analyses of adjuvant function 

o Maintain and expand development of novel animal models that more accurately 

reflect human immune responses to adjuvanted vaccines across all age 

groups/populations through NIAID’s Systems Approach to Immunity and 

Inflammation program and through a new funding opportunity, starting in 2019, to 
22 

support the use of CC and CC-RIX mouse lines that more faithfully reproduce 

human immune responses 

o Increase support to HIPC and CCHI investigators to: 

 Accelerate further development and validation of fully human artificial 

“immune systems” and immune tissue explants 

 Conduct additional systems immunology analyses of head-to-head comparisons 

of novel adjuvant/antigen combinations 

• Optimize formulation and antigen/adjuvant pairing activities. 

To accomplish these efforts, NIAID will: 

o Renew the Adjuvant Development Program in 2018, which includes a stronger 

emphasis on formulation and head-to-head comparisons of adjuvant/antigen 

combinations. NIAID also will continue to award SBIR contracts to increase 

adjuvant development. Community access to adjuvants developed under these 

programs will be expanded through targeted NIAID supplements. 

o Expand development of novel adjuvanting technologies, including conjugation 

technologies or self-adjuvanting vaccine platforms, through the SBIR adjuvant 

development contracts and other funding mechanisms 

o Expand development of co-adjuvants to include checkpoint inhibitors that enhance 

APC or T-cell responses by extending immune activation or by blocking negative 

regulators

o Continue to support adjuvant process development to enable cGMP production 

capabilities for adjuvant production through NIAID’s Adjuvant Development 

program 

o Encourage and continue to support preclinical development of adjuvanted vaccines 

in the areas of product optimization, process development, formulation studies, 

cGMP production, and GLP safety assessment through NIAID’s Vaccine 

Development Services, Broad Agency Announcements (BAAs), partnership 

programs, and SBIR contract solicitations to accelerate the transition of adjuvanted 

vaccine candidates into clinical evaluation
23 

4. CLINICAL EVALUATION OF ADJUVANTED VACCINES

4.1. RECENT PROGRESS IN NIAID-SUPPORTED ACTIVITIES

Adjuvanted Vaccines for Non-HIV Infectious Diseases

Since 1998, NIAID has supported 133 vaccine clinical trials for non-HIV infectious 

diseases, primarily through the Vaccine and Treatment Evaluation Units (VTEUs). Fortyseven of these clinical trials have included adjuvanted vaccines, with adjuvants such as 

Alum (used in 13 of these trials), MF59®, AS03, glucopyranosyl lipid adjuvant (GLA, a 

synthetic TLR4 agonist), and GLA with QS-21 in liposomal formulation. Recently 

completed and ongoing trials of adjuvanted vaccines for non-HIV infectious diseases are 

summarized in Appendix C and described below: 

• Phase I and II clinical trials of inactivated influenza vaccine candidates with established 

adjuvants in multiple combinations, as part of its influenza pandemic preparedness 

program 

• Exploration of novel vaccination strategies, including heterologous prime-boost interval 

studies using H7 influenza vaccines 

• In collaboration with GSK, the direct comparison of two adjuvants, AS03 and MF59®, 

with seasonal influenza vaccines. The trial has been completed and results will be 

released to the public once data analyses are completed.

• A first-in-human Phase I clinical trial with a purified formalin-inactivated Zika virus 

vaccine candidates with alum adjuvant, in a rapid response to this emerging viral 

disease threat

• Two clinical trials exploring vaccines with novel adjuvants 

o TLR7/8 agonist, Imiquimod, to enhance the efficacy of a pre-pandemic H5

influenza vaccine

o Recombinant double-mutant heat-labile toxin (dmLT), which acts as both antigen 

and adjuvant in a vaccine against enterotoxigenic E. coli (ETEC). The dmLT 

adjuvant can induce strong mucosal immune responses after parenteral delivery.

• Evaluation of tuberculosis (TB) vaccine candidates in Phase I clinical trials using new 

combination adjuvants, GLA-LSQ and GLA-SE 

• Evaluation of new combination adjuvants such as GLA-LSQ for a malaria vaccine or 

Alhydrogel plus GLA in Aqueous Formulation (GLA-AF) for schistosomiasis

vaccines

• Use of the human challenge models to evaluate, compare, or narrow the field of choices

of different vaccine formulations for hookworm vaccines 

Adjuvanted Vaccines for HIV 

NIAID’s HIV vaccine program has been exploring multiple vaccine platforms to achieve 

protective immunity against HIV that include HIV DNA vaccines or viral vectors boosted 

or combined with adjuvanted recombinant Env protein(s). Two complementary strategies

are being used: an empirical approach that builds on partial clinical success to 

expeditiously move vaccine candidates into human testing and a theoretical approach that 
24 

selects vaccine candidates for further development based on an understanding of the 

immune response to HIV infection. Completed and ongoing trials of HIV adjuvanted 

vaccines are described below and summarized in Appendix D. 

The Empirical Approach

• This approach relies mostly on the landmark RV144 study in Thailand, the first and 

only (as of April 2018) large clinical trial to demonstrate modest efficacy for an 

investigational HIV vaccine. RV144 evaluated the safety and estimated the efficacy of 

a prime-boost combination of two vaccine components given sequentially: ALVACHIV, which uses a canarypox virus as a vector—or carrier—to deliver HIV genes; and 

the recombinant AIDSVAX B/E, HIV Env protein formulated with alum hydroxide gel 

(Rehydragel). At the end of the 3.5-year study period, investigators observed a 31% 

reduction in risk of HIV infection among vaccine recipients compared to those who 

received a placebo. 

Analysis of the immune responses induced by the vaccine suggests that a lower risk of 

infection is associated with: 

o Plasma immunoglobulin G (IgG) antibodies targeting the V1V2 region of HIV Env

o Low plasma levels of HIV-Env-specific IgA (which compete with protective IgG) 

o Polyfunctional CD4+ T cells (CD154, interleukin-2 [IL-2], IL-4, interferon-gamma 

[IFN-γ], and tumor necrosis factor alpha [TNF-α]) 

o IgG antibodies harboring Fc effector functions needed for antibody-dependent cellmediated cytotoxicity (ADCC), but only in vaccinees with low-plasma, HIV-Envspecific IgA

To follow up on this encouraging outcome, the P5 partnership (Pox-Protein-PublicPrivate-Partnership, http://www.vaccineenterprise.org/content/P5Partnership) was 

established in 2010 to substantiate and improve upon RV144 using a modified poxprotein HIV vaccine. Phase I studies include: 

o Side-by-side comparison of adjuvant formulations 

 Boost of HIV Env formulated with Rehydragel vs. MF59® after ALVAC 

prime (HVTN 107)

 Boost of HIV Env formulated with MF59® vs. AS01B after DNA prime (HVTN 

108) 

 Boost of HIV Env formulated with MF59® vs. AS01B after ALVAC prime 

(HVTN 120)

• Prior to completion of the RV144 trial, many adjuvants were tested in Phase I clinical 

trials in combination with HIV immunogens: Rehydragel, Alhydrogel, Adju-Phos, 

MF59®, AS01B, and GLA-SE. Two Phase IIb studies used similar recombinant Env 

subunit proteins only (without the ALVAC prime) as part of the regimen (VAX003, 

VAX004) that were formulated with Rehydragel, but these studies did not show 

efficacy.
25 

The Theoretical Approach 

• This approach targets the induction of broadly neutralizing antibodies (bNAbs) against 

HIV. Based on HIV natural infection studies and an exquisite characterization of the 

structure of the HIV Env spike, there is a better understanding of the requirements to 

induce bNAbs. The immunogens must first engage the B cell harboring the germline 

lineage of desired antibodies, and then, through multiple cycles of affinity maturation, 

the gene encoding immunoglobulins may acquire the mutations needed to provide 

broad and potent HIV neutralization activity. T follicular helper (Tfh) cells, the drivers 

of affinity maturation in the lymph node, also are sought to generate better bNAbs. In 

addition to high levels of somatic hypermutation, other unusual structural features of 

bNAbs include a long heavy chain complementarity-determining region 3 (HCDR3), 

capable of reaching through the glycan shield of the virus, and autoreactivity.

• Two additional clinical trials involving direct comparisons of adjuvants are under 

development. The first is supported by the HIV Vaccine Trials Network (HVTN) and 

will use a stabilized trimer BG505 SOSIP.664 immunogen, adjuvanted with 

3M052+Alum, CpG 1018, or GLA-LSQ. The second trial, a partnership between 

NIAID and the U.S. Military HIV Research Program, will test a DNA prime, gp145 

HIV Env protein boost, adjuvanted with several Army Liposome Formulations (ALFs), 

Alhydrogel®, and dmLT. 

4.2. PROGRAM GOALS AND RESEARCH CHALLENGES

Goal 1: Increase the Availability of Well-Characterized Novel Adjuvants for Use in 

Vaccine Clinical Trials

One of NIAID’s major goals is to develop vaccines against pathogens and their toxins. This 

goal includes clinical evaluation in both domestic and international populations, which 

often have distinct pathogen exposure histories that can impact host immunity. Therefore, 

adjuvants capable of inducing Th1/Th17, Tfh, CD8+ T cell, or mucosal immune responses 

or that are effective in at-risk populations are especially needed. 

Challenges associated with these activities include: 

• Difficulty in reconciling human clinical data and animal preclinical data to 

improve the design and further evaluation of adjuvanted vaccine candidates. The 

primary endpoints of early-phase clinical trials of adjuvanted vaccines are safety and 

immunogenicity. However, sample sizes are generally small and lack sufficient 

statistical power to dissect safety or identify correlates of protection, both of which are 

critical parameters in determining whether a vaccine candidate moves into later-phase 

clinical trials or needs further refinement. Preclinical animal efficacy studies may 

provide some insights regarding safety and efficacy, but species differences in 

immunity, pharmacogenetics, and reactogenicity need to be considered. 

• Limited number of adjuvants available from commercial sources. Although NIAID 

has supported many clinical trials of vaccine candidates against infectious diseases, 
26 

those requiring adjuvants continue to rely on a limited number of sources. Access to 

novel adjuvants already in clinical development is hampered by intellectual property 

(IP) restrictions. In addition, adjuvant IP holders may either oppose or not be interested 

in direct comparisons of their formulation with those they do not own. Bringing more 

potent adjuvants into the clinic represents a significant financial and administrative 

burden for any adjuvant developer. Many of these developers reside at academic

institutions or own small businesses and have neither experience with the process nor 

the funding needed to bring products into the clinic.

Goal 2: Maximize Evaluation of Data from Clinical Trials

Although many well-designed clinical trials generate valuable datasets and contain a wealth 

of information, the lack of correlates of protection for most pathogens complicates the 

rational selection of an adjuvant for the clinical evaluation of a novel vaccine. Other issues 

related to evaluation of data from clinical trials of adjuvanted vaccines include: 

• Formulations identified in Phase I clinical trials as safe and immunogenic that may fail 

in advanced clinical development or efficacy field trials

• Immunological analyses of clinical trial samples commonly focus on certain parameters 

known or assumed to be associated with protection against a specific disease. However, 

such assumptions limit the breadth of immunologic information produced and our 

ability to identify and validate immune correlates of protection associated with adjuvant 

functionality. 

• The lack of standardized assays to measure the immunogenicity of an adjuvant 

combined with different antigens. This data would establish immunological profiles of 

adjuvants and determine how much those profiles are affected by the antigen 

component of the vaccine. 

Sample-sparing techniques, such as those supported by NIAID’s Sample Sparing Program, 

have improved significantly in recent years allowing more parameters to be determined 

with small numbers of cells or volumes of whole blood or serum. Simultaneously, the cost 

of multiplex assays has decreased making the routine generation of high-density datasets 

from clinical trials samples highly feasible. 

The major challenge associated with this goal is: 

• Access to data from adjuvant studies and adjuvanted vaccine and human 

challenge clinical trials. Many investigators conducting adjuvant research or clinical 

trials do not deposit their datasets into publicly accessible databases such as ImmPort, 

because they either are unaware of relevant databases or lack incentives or the expertise 

to comply with data submission requirements. These requirements may include the 

collection of specific meta-data parameters, data types, and use of controlled 

vocabularies. 
27 

4.3. NIAID’S APPROACH TO ADVANCE THE CLINICAL EVALUATION OF

ADJUVANTED VACCINES

To address the challenges raised above and to continue the evaluation of novel adjuvants in 

clinical trials, NIAID plans to foster/support the following activities: 

• Generation, collection, curation, and public availability of clinical data on the 

immunological activity of vaccine adjuvants to inform their use in subsequent trials 

• Expansion of NIAID-sponsored research using human challenge models to evaluate the 

efficacy of candidate adjuvanted vaccine formulations 

• Support of clinical trials to compare and down-select novel adjuvanted vaccine 

candidates

• Continued/expanded investment in the development of computational models that can 

reconcile data from human clinical trials and preclinical animal studies to improve the 

design and efficacy of adjuvanted vaccines 

• Continued investment in the development of sample sparing assays to enable evaluation 

of more immunological parameters in small clinical samples 

• Use of NIAID-sponsored programs such as HIPC, CCHI, and Systems Biology 

programs to: 

o Conduct research to identify biomarkers and correlates/surrogates of immune 

protection or safety/reactogenicity 

o Assist in the design of immunological analyses of clinical trials samples

o Enable collaborations between existing NIAID networks and clinical trials sites 

through targeted administrative supplements 

• Expansion of NIAID’s Bioinformatics resources, including ImmPort 

(www.immport.org), to: 

o Develop a public database of reference adjuvants and their immunologic activity 

o Provide access to immunologic and other datasets generated by NIAID-supported 

clinical trials
28 

5. SUMMARY

The goal of NIAID’s adjuvant discovery and development programs is to generate a diverse 

panel of well-defined, safe, and effective adjuvants that can be paired with candidate vaccines 

to induce the desired protective immune profiles in all relevant populations. 

To achieve this goal, NIAID will continue to: 

• Support and expand its fundamental immunology research portfolio to identify the 

requirements for inducing effective immune responses and long-lasting protection in all 

individuals 

• Advance adjuvant discovery and early stage development, specifically targeting the critical 

immunologic parameters described in the relevant sections above 

• Support the discovery of adjuvanted vaccines; animal testing and screening, including GLP 

safety assessment of adjuvanted vaccine formulations; process development; and 

production of clinical grade materials of vaccines and vaccine adjuvants

• Explore the feasibility of a NIAID-sponsored cGMP vaccine/adjuvant manufacturing 

facility for small-scale clinical trials

• Explore creative approaches to promote and accelerate clinical testing of promising vaccine

candidates formulated with novel adjuvants 
29 

6. APPENDICES

6.1. APPENDIX A: ABBREVIATIONS 

ADCC Antibody-Dependent Cell-Mediated Cytotoxicity

ALF Army Liposome Formulation

APC Antigen-Presenting Cell

BAA Broad Agency Announcement 

bNAb Broadly Neutralizing Antibody 

BRP Blue Ribbon Panel 

CC Collaborative Cross 

CCHI Cooperative Centers for Human Immunology

c-di-AMP Cyclic Di-Adenosine Monophosphate 

CDN Cyclic Dinucleotide

cGMP Current Good Manufacturing Practice 

CLR C-Type Lectin Receptor

CMV Cytomegalovirus 

DAIDS Division of AIDS 

DAIT Division of Allergy, Immunology, and Transplantation 

DMID Division of Microbiology and Infectious Diseases 

dmLT Double-Mutant Heat-Labile E. coli Toxin

dsRNA double-strand RNA 

EBV Epstein-Barr virus

ETEC Enterotoxigenic E. coli

FDA Food and Drug Administration 

GLA Glucopyranosyl Lipid Adjuvant 

GLP Good Laboratory Practice 

GSK GlaxoSmithKline

HCDR3 Heavy Chain Complementarity-Determining Region 3

HIPC Human Immunology Project Consortium 

HPV Human Papillomavirus 

HVTN HIV Vaccine Trials Network

IEDB Immune Epitope Database and Analysis Resource 

IgG immunoglobulin G 

IL-2 interleukin-2 

IFN-γ interferon-gamma 

MMCA Molecular Mechanisms of Combination Adjuvants 

MPL Monophosphoryl Lipid

MRSA Methicillin-Resistant Staphylococcus aureus

NE Nanoemulsion 

NIAID National Institute of Allergy and Infectious Diseases

NIH National Institutes of Health

NKT Natural Killer T 

OCICB Office of Cyber Infrastructure and Computational Biology 

ODN Oligodeoxynucleotide 

PAMP Pathogen-Associated Molecular Patterns
30 

PCN Protein Cage Nanoparticles

SAR Structure–Activity Relationship 

SBIR Small Business Innovation Research

TB Tuberculosis 

Tfh T Follicular Helper

TLR Toll-Like Receptor

TNF-α tumor necrosis factor alpha

VLP Virus-Like Particle

VTEU Vaccine and Treatment Evaluation Units
31 

6.2. APPENDIX B: BLUE RIBBON PANEL MEMBERS

Rafi Ahmed, Ph.D. 

Professor

Emory University School of Medicine 

James Baker, M.D.

Director, Michigan Nanotechnology Institute for Medicine and Biological Sciences

University of Michigan 

Mario Barro, Ph.D. 

Senior Director 

Head of Technology and External Networks 

Sanofi Pasteur

John Clements, Ph.D. 

Professor

Tulane University 

Robert Coffman, Ph.D. 

Senior Vice President and Chief Scientific Officer

Dynavax Technologies

Victor DeFilippis, Ph.D. 

Assistant Professor

Oregon Health and Sciences University/Vaccine and Gene Therapy Institute 

Jay Evans, Ph.D. 

Professor

Director, Center for Translational Medicine

University of Montana 

Michael Gale, Ph.D.

Professor

University of Washington 

Hana Golding, Ph.D. 

Chief, Laboratory of Retroviruses 

Center for Biologics Evaluation and Research 

FDA
32 

Nir Hacohen, Ph.D. 

Professor

Broad Institute and Massachusetts General Hospital

Ross Kedl, Ph.D. 

Professor

University of Colorado 

Marian Kohut, Ph.D. 

Professor

Iowa State University 

Ofer Levy, M.D., Ph.D. 

Director

Precision Vaccines Program

Boston Children’s Hospital 

Julie McElrath, M.D., Ph.D. 

Senior Vice President

Director, Vaccine and Infectious Disease Division

Fred Hutchinson Cancer Research Center

Nikolai Petrovsky, M.B.B.S., Ph.D. 

Professor

Vaxine/Flinders University 

Rino Rappuoli, Ph.D. 

Chief Scientist

GSK Vaccines

Steven Reed, Ph.D. 

President and CEO

Infectious Disease Research Institute

Amy Weiner, Ph.D. 

Senior Program Officer, Discovery & Translational Sciences

Bill & Melinda Gates Foundation
33 

6.3. APPENDIX C: NIAID-SPONSORED CLINICAL TRIALS FOR NON-HIV 

ADJUVANTED VACCINES, RECENTLY COMPLETED OR ONGOING (AS 

OF APRIL 2018) 

Adjuvant Pathogen Pathogen 

Vaccine Antigen/Approach

Phase

ClinicalTrials.gov 

Identifier

dmLT Enterotoxigenic

E. coli dmLT antigen Phase I

NCT02531685 

AS03 vs. 

MF59® 

Influenza 

A/H5N8

A/gyrfalcon/Washington/41088-

6/2014 

Phase I

NCT02624219 

AS03 vs. 

MF59® 

Influenza 

A/H5N8

A/gyrfalcon/Washington/41088-

6/2014 

Phase I

NCT03014310 

AS03

Influenza 

A/H7N9 Monovalent A/Hong 

Kong/125/2017 

Phase II

NCT03312231 

AS03

Influenza 

A/H7N9

Monovalent A/Hong 

Kong/125/2017, with seasonal 

IIV4

Phase II

NCT03318315 

AS03

Influenza 

A/H7N9 Monovalent A/H7N9 

A/Shanghai/2/2013 

Phase II

NCT02921997 

MF59®

Influenza 

A/H7N9

Monovalent A/H7N9 

A/Shanghai/2/2013, with live 

attenuated H7N9

Phase I

NCT02251288 

GLA-SE vs. 

AP10-602 

M. tuberculosis ID93 antigen Phase I

NCT02508376 

Alhydrogel

+/− GLA-AF

N. americanus Co-administered Na-GST-1 and 

Na-APR-1 antigens

Phase Ib

NCT02476773 

Alhydrogel

+/− GLA-AF or 

CpG 10104

N. americanus Na-GST-1 antigen Phase II

NCT03172975 

Alhydrogel

+/− GLA-AF 

(AP 10-701)

Schistosoma 

spp. Sm-TSP-2 antigen Phase Ia

NCT02337855 

Alhydrogel®

+/− GLA-AF 

(AP 10-701)

Schistosoma 

spp. Sm-TSP-2 antigen Phase Ib

NCT03110757 

Alum Zika Zika virus purified inactivated 

vaccine 

Phase I

NCT02963909 
34 

6.4. APPENDIX D: NIAID-SPONSORED CLINICAL TRIALS FOR HIV 

ADJUVANTED VACCINES, COMPLETED OR ONGOING (AS OF APRIL 

2018) 

Adjuvant HIV Vaccine Antigen/Approach Phase

ClinicalTrials.gov Identifier

Adju-Phos Ad26.Mos.HIV or Ad26.Mos4.HIV 

boosted by gp140 Clade C protein

Phase I

NCT02788045 

Adju-Phos Ad26.Mos4.HIV boosted by gp140 

Clade C protein

Phase I

NCT03060629 

Alhydrogel gp145 Clade C Env protein Phase I

NCT03382418 

GLA-SE EnvSeq1: CH505 TF gp120 series Phase I

NCT03220724 

GLA-SE Polyvalent DNA boosted by gp120 (A, 

B, C, AE) 

Phase I

NCT03409276 

pIL-12 PENNVAX®-GP DNA Phase I 

NCT02431767 

MF59® ALVAC (vCP2438) boosted by 

bivalent subtype C gp120 

Phase I/II 

NCT02404311 

MF59® ALVAC (vCP2438) boosted by 

bivalent subtype C gp120 

Phase IIb/III

NCT02968849 

MF59® DNA-HIV-PT123 boosted by bivalent 

subtype C gp120 

Phase I

NCT02997969 

MF59® gp140 Clade C boost to HVTN073 Phase I 

NCT01423825 

MF59® gp140 Clade C Phase I 

NCT01376726 

MF59®

MVA-C boosted by gp140, DNA-C2 

boosted by MVA-C, or DNA-C2 

boosted by MVA-C and gp140 

Phase I 

NCT01418235 

Rehydragel DNA-HIV-PT123 or NYVAC-HIVPT1/PT4 prime with AIDSVAX B/E 

boost 

Phase I 

NCT01799954 

Rehydragel ALVAC (vCP1521) boosted by 

AIDSVAX gp120 B/E 

Phase I 

NCT02109354 

Rehydragel DNA-HIV-PT123 boosted by 

AIDSVAX gp120 B/E

Phase I

NCT02207920
35 

Adjuvant HIV Vaccine Antigen/Approach Phase

ClinicalTrials.gov Identifier

Rehydragel ALVAC (vCP1521) boosted by 

AIDSVAX gp120 B/E

Phase III 

NCT00223080 

pIL-12 and pIL-15 PENNVAX-B DNA Phase I 

NCT00528489 

MF59® vs. AS01B DNA-HIV-PT123 boosted by bivalent 

subtype C gp120 

Phase I

NCT02915016 

MF59® vs. AS01B ALVAC (vCP2438) boosted by 

bivalent subtype C gp120 

Phase I/IIa

NCT03122223 

MF59® vs. 

Rehydragel 

ALVAC (vCP2438) boosted by 

bivalent subtype C gp120 

Phase I

NCT03284710