Summary of ICMS Seminar Club presentation on Friday, January 28, 2022

Summary of ICMS Seminar Club presentation on Friday, January 28, 2021.

Title: The organization of the US National Institutes of Health (NIH) and an introduction to the NIAID Vaccine Adjuvant Program

Speaker: Dr. Wolfgang W. Leitner, Chief of the Innate Immunity Section at the National Institute of Allergy and Infectious Diseases (NIAID/NIH), USA.

On Friday, January 28, Dr. Wolfgang W. Leitner gave a presentation to the ICMS Seminar Club at Fujita Health University.  He told us about the organization of the National Institutes of Health (NIH) in the US, and how we, as foreign researchers, may apply for its grants. He also told us about the Vaccine Adjuvant Program of which he is in charge, which has a worldwide impact on vaccine development, including against COVID-19.

For members of Fujita Health University, a recording of the meeting (without the discussion part) will soon be available at our “Manabi” site. Unfortunately, we cannot open the recording for a wider audience.

There were 24 participants. Several to whom I talked afterward felt that they had been listening to a great presentation but found some parts difficult to grasp. On the other hand, Professor Emeritus Teruyuki Nakanishi, who was my first boss in Japan and is now involved in vaccine development in Japanese industry, told me how he had enjoyed the talk and how much he had learned. Indeed, this was a top-level presentation delivered in a pleasant and accessible manner (you may realize by replaying the recording, possibly after reading this text). That, for non-specialists, it may have nevertheless been difficult to grasp is probably related to some difficulties of concept intrinsic to the field of vaccinology (and immunology).

Therefore, before I go to summarizing Dr. Leitner’s presentation, I first want to describe some general seemingly “paradoxical” concepts in vaccinology.

Paradox 1:

• We don’t know the details of how vaccines work. Ultimately, decisions are made based on trial-and-error experiments.
• We know how some parts of the immune system work. Therefore, educated strategies can be chosen to improve the chance of developing good and safe vaccines (by, ultimately, trial-and-error experiments).

Paradox 2:

• Immune memory is only induced if there is immune stimulation (which requires a “danger” signal). Thus, injecting people with only an antigen (e.g., a part of a virus against which one wants to induce immune memory) is not enough if that antigen is not “immunogenic.” Therefore, most vaccines include “adjuvants” for helping to stimulate the immune response.
• Severe immune stimulation (inflammation), especially if systemic (through the whole body), is accompanied by severe side effects. This problem is not so easy to circumvent and until this century only a single type of adjuvants was allowed in human vaccines, namely several kinds of aluminum salts (examples in Fig. 1). 

Paradox 3:

• The best possible science requires full disclosure of data.
• Companies, which spend loads of money on vaccine development, often do not want to disclose the precise vaccine contents. The result is that, generally, we know what is approximately in a vaccine but often not the details, and that Dr. Leitner in some instances could not provide detailed explanations.

Then, there are some general facts about the immune system (beyond standard knowledge such as, e.g.,  understanding what B cells and T cells are) that one probably needed to know for easily understanding this presentation. Dr. Leitner mentioned them shortly, but it may have been too fast, and therefore I repeat:

Immune polarization

The immune system is a myriad of interacting cells and molecules, and we only understand some parts of it. However, we know that the immune system must continuously choose between competing strategies that mutually suppress each other. Major strategies that can be distinguished are (simplified):

Type 1 immunity (activated NK cells, CD8⁺ cytotoxic T cells, and Th1 CD4⁺ helper T cells; marker cytokines interferon gamma and IL-12; important for killing virus-infected cells and cancer cells)

Type 3 immunity (activated neutrophils and Th17 CD4⁺ helper T cells; marker cytokines IL-17, IL-6, IL-21, and IL-23; important for killing bacteria)

Regulatory immunity (activated Treg CD4⁺ regulatory T cells; marker cytokines IL-10 and TGFb; important for downregulation of other immune responses and protection of homeostasis)

Type 2 immunity (activated eosinophils, basophils, mast cells, and Th2 CD4⁺ helper T cells; marker cytokines IL-4, IL-5, and IL-13; important for IgA and IgE production by B cells and for fighting parasites)

These immune strategies are deliberately listed in this order, because Type 1 and Type 2 immunity are the most opposing to each other. It is important to understand that vaccines should not just stimulate any type of immunity but the right kind of immunity. Against viruses, which are intracellular pathogens, it usually is important that not only B cells (for antibody production) but also CD8⁺ cytotoxic T cells (aka cytotoxic T lymphocytes or CTLs) are stimulated because the latter can kill virus-infected cells and have immune memory. During the presentation, you could sometimes hear the issue raised of whether a certain adjuvant also helped to stimulate CTLs. For doing that efficiently, the vaccine should be delivered to within the cells (as the antigen needs to be loaded onto MHC class I molecules) and stimulate Type 1 immunity.

Toll-like receptors

The immune system uses a series of leucine-rich repeat molecules called “toll-like-receptors (TLRs)” which can recognize molecular motifs conserved within classes of pathogens, also known as “PAMPs (pathogen-associated molecular patterns).” The binding of PAMPs by TLRs induces an immune response. As Dr. Leitner described, intelligent vaccine adjuvant development has targeted the receptors TLR4, TLR7, TLR8, and TLR9. TLR4 is stimulated by (amongst other molecules) LPS, which is a constituent of the wall of gram-negative bacteria; TLR7 and TLR8 are stimulated by single-stranded RNA if that doesn’t have the typical modifications of the host cell RNA; and TLR9 can bind unmethylated CpG oligodeoxynucleotide DNA as found in bacteria and DNA viruses.

TLRs are considered part of the “innate immune system,” and since Dr. Leitner is Chief of the Innate Immunity Section of the NIAID/NIH, it makes perfect sense that he is also in charge of the NIAID Vaccine Adjuvant Program.

No summary of the Discussion section

We had a long and interesting discussion about the NIH and about the latest developments in vaccine development, including in relation to the COVID-19 pandemic. However, because of the sensitivity of the topics, I decided not to summarize that discussion for this blog.

The contents of the presentation

The presentation, as is the below, was divided into (I) Introduction of the NIH, (II) Explanation of how researchers (also at foreign institutes) can apply for NIH grants, (III) NIH resources (also for non-US researchers), and (IV) The NIAID Vaccine Adjuvant Program.

(I) Introduction of the NIH

The NIH was founded in 1887 and is primarily located in Bethesda near Washington (Fig. 2). The NIH is the largest “extramural” (outside the NIH) research funding agency in the world and —after Harvard — the second-largest contributor, through “intramural” research at the NIH itself, of papers in leading journals.

The NIH consists of 24 institutes involved in extramural funding and three centers without funding authority (Fig. 3). The National Institute of Allergy and Infectious Diseases (NIAID) is one of the bigger NIH institutes. In accordance with its name, the NIAID plays an important role in fighting the COVID-19 pandemic.

Dr. Leitner explained that the research conducted within the NIH also includes clinical trials that are commercially not interesting (e.g., for rare diseases) (Fig. 4). As for extramural research, the NIH does not only provide support with grants but also in various other ways such as, for example, by offering training, logistics, resources, organizing contacts between researchers, etc. (Fig. 4).

(II) Explanation of how researchers (also at foreign institutes) can apply for NIH grants

If researchers want to apply for an NIH grant, the steps are (Figs. 5 and 6):

• Register at the electronic Research Administration (eRA) site (https://era.nih.gov/)
• Find out about what the NIH is currently funding at the RePORTER site (https://reporter.nih.gov/)
• Look for funding opportunities and regulations (e.g., deadlines) at the news site of the respective NIH institute; for example, the NIAID Funding News site (https://www.niaid.nih.gov/grants-contracts/funding-news). Anyone can sign up for the NIAID funding newsletter which describes new funding opportunities but also discusses, e.g., how to write a good grant application and answers questions about funding from investigators.
• Reach out to an NIH program officer (PO). To find out which PO may help you, use the Matchmaker function of the RePORTER site (https://reporter.nih.gov/matchmaker). If you would experience any difficulties, Dr. Leitner has kindly offered that the members of our audience can directly contact him (!!).

As an example, to show how the above-mentioned RePORTER site works, and that the NIH is also funding researchers in Japan, I entered “Japan” as a search word (Fig. 7).

The NIH supports foreign researchers if that is considered to be of interest to the US (for which they use a broad definition) (see this article by Fauci and Collins in 2015).

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(III) NIH resources (also for non-US researchers)

Dr. Leitner gave two examples of NIH resources that are provided all around the world against only shipping costs:

• MHC tetramers (Fig. 8). These are provided by the NIH Tetramer Core Facility (https://tetramer.yerkes.emory.edu/). MHC tetramers, for example, can play a role in the investigation of T cell memory against COVID-19.
• A large variety of biological resources for studying pathogens or other microorganisms (Fig. 9). These are provided by BEI Resources (the long name is “The Biodefense and Emerging Infections Research Resources Repository”) (https://www.beiresources.org/Home.aspx).

Dr. Leitner furthermore explained that the NIAID has a site for helping us to find the resources that it provides (https://www.niaid.nih.gov/research/resources) (Fig. 10).

(IV) The NIAID Vaccine Adjuvant Program

Dr. Leitner oversees this program.

General

A vaccine consists of several types of ingredients (Fig. 11). The function of the adjuvants is to optimize the immunity induced by the vaccine — the word adjuvant is derived from the Latin word adjuvare meaning “to aid.”

Adjuvants in vaccines provide the obvious advantage that less of the antigen (the pathogen, part of a pathogen, or a recombinant protein representing the pathogen) is needed (Fig. 12). Furthermore, adjuvants can help to stimulate immunity in people with a weakened immune system (such as the elderly), drive the immune system into the right type of immune polarization (Type 1 immunity for many viruses; see my explanation above), and induce a longer-lasting immune memory.

It is not easy to induce CTL (T cell responses for killing virus-infected cells; see my explanation above) and Type 1 (Th1) immune polarization if using a subunit vaccine. This is especially so because the traditional adjuvants, aluminum salts, drive immune polarization in the opposite direction, namely towards Type 2. On the other hand, adjuvants that stimulate TLR4, TLR7, TLR8, or TLR9 receptors, and thereby drive the immune response into a Type 1 polarization, have a tendency to be too reactogenic if not properly formulated. As explained below, some novel adjuvants have found ways around this concern.

The NIAID Vaccine Adjuvant Program has been supporting research to find novel adjuvants and improving adjuvants for vaccine makers. This also includes the combination of known adjuvants in order to keep their good while reducing their bad, or to achieve synergetic effects; Dr. Leitner argues that also during natural infection multiple immune stimulatory signals work together, and this may give better types of immune responses. Hurdles for companies to develop or use a new type of adjuvants are quite high, thus support in this field by the NIH makes a lot of sense from the viewpoint of public health. This supportive and intelligent approach has been working, as in the last few years a new set of approved adjuvants has rapidly been developed (examples at the end of this blogpost), whereas until 20 years ago the only approved adjuvants in the US were aluminum salts (Fig. 13).

The NIAID Vaccine Adjuvant Program can promote the various stages of adjuvant research, including the discovery, preclinical, and clinical stages (Fig. 14).

Modern ways to find new adjuvants

Aluminum salts have been discovered as vaccine adjuvants in the 1920s (Matsiko 2020) and licensed for use in human vaccines since 1932 (Di Pasquale et al. 2013). In the US, this has been the only licensed adjuvant until, in 2009, monophosphoryl lipid A (MPL), a detoxified form of lipopolysaccharide (LPS), was licensed for use in the Cervarix vaccine against human papillomavirus (HPV); in this vaccine the MPL is absorbed on aluminum salts (Pulendran et al. 2021). MPL has been known since the early 1980s, so the development of this second type of adjuvant has taken >25 years. Other examples of “second generation” adjuvants are squalene (an oil found in all plants and animals) used in Europe in some influenza vaccines during the flu pandemic in 2009-2010, and cytosine phosphoguanosine (CpG; a stimulator of TLR9) 1018 first used in a hepatitis B vaccine in 2017 (Pulendran et al. 2021). Developments of these adjuvants were initiated based on direct in vivo trial-and-error experiments using animals (aluminum salts and squalene) or known immune functions (MPL and CpG).

Nowadays, the development of adjuvants goes much faster. Adjuvants are found more efficiently by high throughput in vitro screening for (immune) functions, or even by computer predictions (Fig. 15). The NIAID Vaccine Adjuvant Program has supported large numbers of both types of screenings (Fig. 15). The computer predictions can help to select compounds for in vitro screening, which helps to select compounds for in vivo screening (Fig. 16).

An unexpected and successful example of the high throughput in vitro screenings was Fos47-adjuvant, which is a combination of two small compounds, one activating TLR7/8, and one activating TLR4 (actually, the TLR4/MD2 complex). The latter compound is very different from the large lipid structures, such as MPL, which are the more natural targets for having their chains recognized by TLR4/MD2, but was found to be a good binder for the same TLR4/MD2 pocket (Fig. 17).

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The NIAID adjuvant database and its use as a “dating site” for adjuvant makers and vaccine makers

Last year, the NIAID adjuvant database was launched (https://vac.niaid.nih.gov) (Fig. 18). On the site, various types of information (e.g., pathogen, receptor, immune profile, clinical trials) can easily be retrieved about adjuvants supported by the NIAID (Fig. 19; an example of available information is shown in Fig. 20). A big advantage to most other adjuvants databases is that these adjuvants are accessible to vaccine developers for preclinical and clinical studies through the NIAID. Adjuvant makers can deposit the information on their new adjuvants here and vaccine makers can easily find that information.

For convenient use, the NIAID Adjuvant Database is linked with the Vaccine Ontology site of the Ontobee project of the University of Michigan (http://www.ontobee.org/) (Fig. 21). This helps with the harmonization of the terminology, but also helps  with the identification of adjuvants (for example in publications) since  each adjuvant gets a unique code (Fig. 22).

COVID-19 vaccine adjuvants developed with support from the NIAID adjuvant program

The NIAID adjuvant program has supported the development of several adjuvants that now are part of some licensed COVID-19 vaccines. Dr. Leitner has explained about this extensively. However, here I provide only a very short summary. I like to stress that Dr. Leitner, even when asked, did not express preference for any type of vaccine. The target of the NIAID adjuvant program is to provide a wide repertoire of possible adjuvants to the research community. Dr. Leitner emphasized that the ideal vaccine and adjuvant depend on many factors (such as the disease and the persons to be vaccinated).

Two adjuvants developed with support of the NIAID adjuvant program and now used in COVID-19 vaccines are Alhydroxiquim-II and Advax/CpG 55.2 (Fig. 23).

Alhydroxiquim-II

As the adjuvant component of “Covaxin,” Alhydroxiqum-II has been used already in >100 million COVID-19 vaccinations in India and other countries (https://www.nih.gov/news-events/news-releases/adjuvant-developed-nih-funding-enhances-efficacy-indias-covid-19-vaccine). Alhydroxiquim-II is an interesting non-covalent combination of alum (alhydrogel) and an imidazoquinoline class molecule (looks a bit like an oversized nucleobase) that stimulates TLR7 and TLR8. The non-covalent binding is very strong and based on the same chemistry that mussels use to attach themselves to a substrate, i.e., wet stones (Waite 2008). If they would be alone as an adjuvant, TLR7/8 agonists disperse through the body and cause unacceptable immune side effects (they are too “reactogenic”). Binding to the alum, however, causes them to stay local, and to concentrate in the draining lymph nodes of the injection site. There, because the binding to alum is non-covalent, eventually the imidazoquinoline will come off and can stimulate TLR7/8; because of the local nature of the delivery and the small amounts of the TLR agonist that is used since it is delivered in a targeted manner, the side effects are minimal. I has been shown that the immune profile of imidazoquinoline (which induces Type 1 immune polarization) overrides that of alum (which induces Type 2/Reg immune polarization) so that Alhydroxium-II induces the desired Type 1 immune polarization.

Advax/CpG 55.2

Advax/CpG is the adjuvant component of the “Spikogen” (also named CoVax-19) COVID-19 vaccine used in Iran (https://covid19.trackvaccines.org/vaccines/8/). This is also a combination of a particulate structure, Advax, with a highly immunogenic molecule, CpG oligodinucleotide. Advax is a crystal structure of “inulin,” a poly-fructose which is very common and — for example — used as sweetener in yoghurts. Similar to what alum does in Alhydroxium-II, the Advax component of the combination adjuvant also helps keeping the CpG molecule close to the vaccine injection site. Moreover, Advax has an anti-inflammatory activity, which is counterintuitive for a desirable adjuvant property but appears to soften the reactogenicity of CpG. The CpG nucleotide sequence used has an interesting history as it was initially predicted in silico (Khanna et al. 2019). The sequence was chosen because it was—which is not common as CpG sequences are species-specific—predicted to stimulate TLR9 in both humans and mice, so that mice could be used as a preclinical model.