Vaccine 33S (2015) B40–B43

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Vaccine journal homepage: www.elsevier.com/locate/vaccine

Review

Gaps in knowledge and prospects for research of adjuvanted vaccines Robert Seder a , Steven G. Reed b,∗ , Derek O’Hagan c , Padma Malyala c , Ugo D’Oro d , Donatello Laera d , Sergio Abrignani e , Vincenzo Cerundolo f , Lawrence Steinman g , Sylvie Bertholet d a

Cellular Immunology Section, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, Bethesda, MD, USA Infectious Disease Research Institute, Seattle, WA, USA Novartis Vaccines, Cambridge, MA, USA d Novartis Vaccines, Via Fiorentina 1, Siena, Italy e Instituto Nazionale Genetica Molecolare, Milano, Italy f Oxford University, Oxford, England, United Kingdom g Stanford University, School of Medicine, Palo Alto, CA, USA b c

a r t i c l e

i n f o

Keywords: Vaccine Adjuvant

a b s t r a c t A panel of researchers working in different areas of adjuvanted vaccines deliberated over the topic, “Gaps in knowledge and prospects for research of adjuvanted vaccines” at, “Enhancing Vaccine Immunity and Value” conference held in July 2014. Several vaccine challenges and applications for new adjuvant technologies were discussed. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction From a historical perspective, vaccines have been instrumental in eradicating or drastically reducing the incidence of numerous dreaded infectious diseases such as small pox, polio, tetanus, diphtheria, cholera, rabies and typhoid [1]. Vaccines to combat two major deadly infections, human papillomavirus [2] and meningococcal disease [3], were also introduced in the recent past, and ongoing research aims to eliminate many more diseases. The need for optimizing adjuvant formulations for new vaccines presents a number of challenges, though for most of these paths forward are becoming evident. This workshop addressed issues and potential solutions focusing on developing vaccines for HIV, malaria, tuberculosis (TB), and cancer. Although certain challenges are specific for each of these vaccine targets, some basic issues hold constant. These include adjuvant access, the importance of formulation, and the need for adjuvants that promote appropriate T cell responses, whether as effectors or helpers of effective and durable antibody responses. A radical change has taken place in the evolution of vaccines since Edward Jenner’s inoculation of cowpox matter as a small pox vaccine in the 1800s in an eight-year-old boy [4]. This seminal example using an attenuated viral vaccine to elicit long lived and broad based humoral and cellular immunity highlights the

∗ Corresponding author. Tel.: +1 206 381 0883. E-mail addresses: [email protected], [email protected] (S.G. Reed). http://dx.doi.org/10.1016/j.vaccine.2015.03.057 0264-410X/© 2015 Elsevier Ltd. All rights reserved.

efficiency by which antigens and innate immunity induced by the virus delivered synchronously is so effective. Since this discovery many vaccine approaches have become more selective where the science has emerged to encompass antigens in the form of conjugates, toxoids and recombinant vectors, inactivated and live attenuated vaccines and subunit vaccines [5]. In terms of proteins or inactivated viral productions, adjuvants have been used to improve the efficiency, potency and durability of immune responses [6]. There is extensive history of the use of vaccine adjuvants, mostly inadvertently through the inclusion of natural products in the form of live or inactivated virus or microbial products, but more recently through intentional efforts to develop vaccine components to enhance immune responses. Two such components in wide use are aluminum salts, commonly referred to as alum, and oil in water emulsions such as MF59 [7]. In addition to the empirical first generation adjuvants such as Alum, MF59 and AS03 emulsions, second generation adjuvants are typically agonists of toll like receptors (TLRs) and act by triggering signal transduction pathways that lead to activation of innate and adaptive immune cells. Though widely used as standalone adjuvants, both alum and emulsions may be used with TLR (and other) ligands as important formulation components. The most advanced example of this is the TLR4 ligand MPL® which has been combined with alum in the GSK Cervarix® vaccine [8]. MPL® is purified from endotoxin (lipopolysaccharide, LPS) derived from Gram-negative bacteria [9]. Endotoxin has been known as a potent stimulus of antibody responses, and extensive biochemical, biophysical, and immunological studies were

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undertaken to define its effects on the immune system and to attempt to disassociate molecules responsible for toxic properties of endotoxin from its adjuvant activity. MPL® have been developed and incorporated into approved vaccines including GlaxoSmithKline’s (GSK) Cervarix® vaccine against human papilloma virus (HPV) and Fendrix® , a hepatitis B vaccine. Both vaccines contain alum-based formulations of MPL (AS04), illustrating the point discussed above, i.e. the utility of alum as a formulation component. A next generation TLR 4 agonist (GLA) has been developed by IDRI and Immune Design Corp. Alum and emulsions based formulations containing GLA (a.k.a. MPLA) has been clinically evaluated with a variety of vaccine candidates (HIV, influenza, schistosomes, hookworm, malaria, leishmaniasis, tuberculosis, and cancer) [10]. Other TLR agonists, including CpG, and a novel TLR 7 agonist developed by Novartis are now being formulated resulting in maintained or enhanced potency with reduced dose. Several other delivery systems like liposomes, PLG and ISCOMs are currently in early clinical trials while non-TLR immune potentiators such as NOD, CLR, RIG-I, and STING agonists are still in early stages of vaccine research. As we step into the 21st century, identifying the gaps in knowledge and outlining pathways that researchers in vaccine adjuvant research could adopt would steer the field in the right direction and be of immense benefit to public health [11]. As new molecules are being discovered specifically for vaccines, the onus is on the vaccine researchers to safeguard the quality of drug product before embarking on safety and efficacy trials in humans. Key questions to focus on would be robustness, reproducibility and scalability of the drug product. The rationale for inclusion of these new molecules needs to be addressed too; superiority over existing vaccine products and ability to use in a wide range of vaccines would pose distinct advantages. Formulation plays a major role with the introduction of delivery systems and adjuvants to enable the antigen reach the targeted site of action as well as elicit a strong long term immune response. The role of formulation becomes even more prominent as one considers the type of release that is desired for the antigen and immune potentiator and controlled release formulations are more relevant for modulating the release of these active molecules. Consequently, the complexities in formulation would largely depend on the nature of interaction between the antigens, immune potentiators and delivery systems. While co-administration might be the simplest approach to adopt, association with the delivery system might necessitate techniques such as complexation, conjugation, encapsulation and adsorption. As formulation of adjuvants will play a major role in the future of next generation of vaccines, the gaps in knowledge in this field were discussed in the workshop. Additionally, although the list of alarming diseases that have been conquered by vaccines is impressive, there are many that remain elusive. A few areas that can benefit with concentrated efforts would be HIV, malaria, tuberculosis (TB), chronic diseases, cancer and vaccines for the elderly and the gaps in these disease areas were also discussed along with steps to reduce these barriers.

2. Gaps in knowledge 2.1. Disease areas As mentioned in the preceding section, the working group started to define the unmet medical needs that require concentrated efforts in the future: HIV, Malaria, TB, chronic diseases, vaccines for the elderly, and cancer. Each of these disease areas requires induction of a different specific type of immune response by a potentially efficacious vaccine. Looking at the gaps from a

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fundamental mechanistic view, durability, magnitude and breadth of the immune response are major issues for all these diseases. Additional scientific hurdles identified for HIV, Malaria and TB were maintaining adaptive immunity in specific secondary lymphoid or non-lymphoid organs may be critical. As such for TB, malaria and HIV, protective immune responses would be required in the lungs, liver, and mucosa, respectively, the sites where initial infection occurs. Aside from these diseases in which there are no effective vaccines, an additional challenge is to improve the efficacy of existing vaccines in the elderly. In the elderly population, a limited naïve T cell repertoire and the use of chronic medications (such as steroids and other anti-inflammatory drugs, statins, antihypertensive treatments, and antibiotics) potentially affects their innate and adaptive immunity. Therefore, novel vaccines or improved adjuvants for this population may be needed to overcome these age related deficiencies. The group also deliberated over disease areas such as Alzheimer’s and autoimmune diseases and established them as challenging future vaccine targets due to a lack of clear cut outcomes and the potential long-term design required for Alzheimer’s disease. In contrast, it was agreed that therapeutic vaccines for cancer could likely work as recent data with immune check-point blockade pathways in vivo have clearly shown potent anti-tumor effects by harnessing host immunity. Therefore, using improved vaccine adjuvant formulations that can favorably alter the tumor microenvironment and/or be used to deliver tumor specific antigens in combination with check point inhibitors, radiation or chemotherapy may be a promising immunotherapeutic strategy in the oncology and an area that needs to be explored extensively. Another related point of discussion was on the modalities for increasing durability of the adaptive immune response. This relates to inducing and sustaining long-lived T cell and antibody responses. The ability to induce and maintain both CD4 and CD8 immunity sufficient to mediate protection in humans remains a major hurdle. Most vaccine platforms can induce CD4 responses and protein/adjuvant vaccines offer a particularly useful platform since the dose of antigen and type of adjuvant can influence the magnitude and quality of the response. Based on the enormous heterogeneity of CD4+ T cell immunity (e.g. Th1, Th17, Tfh), it may be desirable to induce a particular response. For CD8+ T cells, currently this is most efficiently induced by recombinant viral vectors. To sustain T cell immunity above a protective threshold may require boosting throughout life. In terms of humoral immunity, most current vaccines induce long lived antibody responses sufficient to mediate protection. The requirement for sustaining high level antibodies is most evident for protection against pre-erythrocytic malaria infection. Thus, subjects vaccinated with the RTS, S + AS01, the most well formulated vaccine adjuvant to date show high level shortterm protection but this wanes after 6–12 months [12]. Antibody responses are 50–100 ␮g/ml after 3 immunizations, which would be well above a protective threshold for most other current vaccines. Thus, for this approach to work, improving the durability of the responses will be required. Finally, a critical question here is if a long lasting response is a simple consequence of a strong initial burst in immune response or if it is dependent on other qualitative aspects of the triggered immune response as well as independent of the magnitude of initial response. It was pointed out that perhaps an initial response with the highest magnitude, high avidity and high breath of coverage would be beneficial for an extended duration of the response; however, on the other side it was also agreed that a too vigorous stimulation of CD4T cell could potentially lead to their exhaustion which would be detrimental to obtain an extended durability of the immune response. The panel also discussed the types of responses desirable for each of the disease areas considered as unmet medical needs and is outlined in Table 1.

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Table 1 Considerations for vaccines for various target diseases. Disease

Considerations discussed by the working group

HIV

A broad and persistent neutralizing antibody response would be optimal for inducing high-level protection. The fact that antibodies from infected people can neutralize more than 90% of strains indicates that the human immune response is capable of generating a protective antibody response. Some of the unique features of these antibodies include extensive somatic hypermutation. A challenge will be to identify the appropriate conditions (antigen structure, formulation or vector) able to generate these antibodies A robust and durable CD8 T cell response would be required to control HIV infected cells. This would likely require viral vectors A CD4 T cell response is needed to help the antibody responses and enhance the function and durability CD8 responses Stimulating trafficking of immune T cells and/or antibodies to mucosal sites will be critical for protection from infection via mucosa, as obtained with HPV vaccine

Malaria

Based on the results of phase 3 clinical trial with RTS, S + AS01 vaccine showing a 30–50% efficacy, which declined over time, an increased durability of antibody response would be highly desirable to improve vaccine protection against clinical disease Trafficking of immune T cells to the liver is likely needed to block infection at this stage Breadth of antigens may be critical for inducing high-level protection A strong memory response is required for long term protection High and sustained antibody titers will also be required to obtain blocking of transmission

TB

Triggering of a CD4T cell response in the lung appears to be crucial Polarization of the immune response toward a Th1, Tfh or Th17 profile may be necessary Trafficking of immune cells to the lung is then important for efficient protection Vaccine delivery directly to the lung to induce tissue specific cells

Cancer

A strong T cell response localized to the tumor site is critical While there is some evidence that CD4+ T cells can mediate anti tumor immunity, it would be most desirable to induce CD8+ T cells against tumor specific antigens. Unfortunately cross priming in humans has been very difficult to obtain so far with protein vaccine approaches Immunization with long peptides may be preferable and recent work from sequencing tumors may lead to individualized therapies with such peptides. While viral vectors are clearly more potent for inducing CD8 responses, individualized therapies based on tumor sequencing of antigens may depend on peptides on adjuvant platforms or RNA delivery It would be important to identify the best option for a booster immunization after priming with viral vectors, as well as the best priming immunization before a boost by vectors Many different types of effector immune responses may be required for complete efficacy

3. Adjuvant formulations This area was the second major gap identified in current research. Existing protein based vaccines can induce antibody and low-level CD4/Th1 responses with limited priming of CD8 T cells. Thus optimization of formulations and vaccine delivery with adjuvants would aid in developing effective vaccines for infections requiring broad based immunity. A major advantage of using protein based vaccines is that they can be given repeatedly to maintain or boost immunity for infections or therapeutically for cancer which is a potential limitation of viral vectors. Despite the broad knowledge of the many of the innate pathways for how current adjuvants mediate their effects, an intricate knowledge of the in vivo mechanisms vis-a-vis antigen presentation is still an unexplored territory. For instance, it would be desirable to evaluate if different adjuvants alter the differentiation of B cells in short or long-lived plasmablasts, compared to memory B cells. Based on similarities in the tissue specific expression of TLR ligands with

humans, non-human primate (NHP) provide a useful and potentially predictive model for assessing the in vivo mechanisms by which adjuvants mediate their effects. Another relevant question to answer would be if a chronic infection could be mimicked using adjuvant formulations or sequential immunizations. Some adjuvants (for example CAF01) induce depot effect and may be useful to obtain continuous antigen stimulation [13]. Likewise, micro particles that allow controlled release of antigen may be useful to induce a long-term antibody response [14]. Nevertheless, only continuous exposure to small amount of antigen would be useful as a massive exposure could lead to exhaustion of antigen specific immune cells. Also, antigen exposure in the absence of inflammation may lead to tolerance while a prolonged inflammatory stimulation may have downside effects. Other required attributes of the adjuvant induced immune response, like IgG isotypes required for efficacy, may be pathogen specific. Yet another unanswered question would be the expectations from adjuvants on induction of CD8 T cell response since no adjuvant has shown any promising effect on this aspect in humans. Maybe nothing is expected, as in adjuvants will not induce CD8+ , but maybe a boost in CD8+ responses is expected. As life expectancy increases across the world, it is important to establish that adjuvants are efficacious on the aging immune system. These subjects may have reduced naïve T cells and a restricted innate immune response. In addition, this category of population may be under constant anti-inflammatory therapy, which may affect the vaccine response. Also, metabolism of T cells may be altered in aged people who may adversely impact the vaccine response. Other drugs that are chronically used by elderly such as anti-hypertensives and statins may possibly affect vaccine response and if this could be demonstrated, a strategy of interrupting therapy (wash period) before vaccination could be assessed to increase vaccine responsiveness. Also, change in microbiota can potentially influence vaccine response in elderly people and a study to probe for differences in microbiome by comparing subjects that respond positively to vaccine with those that do not would be of value. Other chronic disease states (diabetes, hepatitis, etc.) represent other opportunities for adjuvanted vaccine development.

4. Reducing barriers The group continued the discussion analyzing how to reduce the gaps that were identified in the previous part of the workshop. It was agreed that building appropriate pre-clinical models for protective outcomes and immune mechanism will provide vital information to proceed towards human clinical trials. Emphasis should then be put on enhancement of data analysis from past, ongoing and future clinical studies to increase outcomes from these trials. Samples need to be collected from blood and, when possible, from relevant additional tissues (lymph nodes, bone marrow, BAL) for detailed analysis of the immune response using appropriate “omics” techniques. In both prophylactic and therapeutic applications (such as tumors, malaria and HSV), the presence of a pre-existing immunity should be taken in consideration and different options should be evaluated such as immunization with homologous vs heterologous proteins, application of specific dosing regimens such as primeboost schedule or repeating boosting throughout life to maintain protective threshold. Boostability of the appropriate response may indeed be dependent on many factors such as the cell phenotypes, antibody subclasses or the quality of desired response, or the magnitude of a central memory cells reservoir and of the effector population. Variability in the response amongst individuals is another factor that needs to be considered. It should be expected that sub-optimal priming would also result in poor boosting.

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Selection criteria of patient population in clinical trials are another important factor that could impact the outcome of a trial. In particular the group discussed the feasibility and usefulness of screening patients in order to exclude from the trial subjects who are seropositive for the target infection: it was agreed that this option may be taken in consideration in some case although it could raise ethical and legal issues that need to be taken into consideration and might limit the generalizability of the results. In terms of forward thinking studies, the likely interference by ‘drug load’ in elderly patients could be easily addressed with studies aimed to evaluate the impact of chronic drug treatments with anti-inflammatory or other type of drugs in this patient population. Since different prophylactic and therapeutic vaccines against infections or tumors may require either only an antibody response, or mainly induction of T cell immunity, or they may need both, selection of the right adjuvant and the correct formulation is key to obtain the specific required response. Indeed, optimization of vaccine formulations may allow obtaining the appropriate innate immune stimulation and the desired increase of the adaptive responses. Moreover, selection of the most suitable adjuvant for each specific vaccine antigen should allow selection of the right combinations of proteins/adjuvants, leading to an improved vaccine efficacy. 5. Preclinical guidelines Researchers across different groups conduct studies in preclinical animal models and oftentimes, these published studies are very hard to reproduce due to the use of ill-defined constituents and methods. More attention needs to be focused on using wellcharacterized vaccines to obtain reproducible and meaningful data. The first generation adjuvants came into being as these materials had approvals in pharmaceutical applications, wherein all the materials were validated. There needs to be a shift in paradigm and pharmaceutical aspects has to be incorporated to advance the development of adjuvants and vaccines into drug products. The in vivo studies should be well designed; dosing the correct volumes at the right sites, defined readouts, systematic evaluation of all tissues and benchmarking with defined adjuvants would enable the initial proof of concept studies to be more robust. Selecting the right in vivo model is another important criterion for assessment of protective efficacy as well as to establish correlates with larger animal models. The findings need to be validated in non-human primate animal models by adopting the same criteria. An improved understanding of drug substance and drug product will go a long way to bridge the gaps in comprehension of how the vaccines

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impact the safety and efficacy at a molecular level. Furthermore, as the vaccine moves into the clinic, regulatory authorities would welcome the holistic approaches adopted for vaccine development and this would help facilitate faster approvals. Adjuvant development has suffered from, in many cases, a failure to consider lowering regulatory hurdles by using minimal doses of novel molecules in acceptable formulations. Addressing unmet needs for including adjuvants in new vaccines will require attention to a variety of factors, including cost, risk:benefit, and minimizing untested components in the final product. Conflict of interest statement None declared. References [1] Ehreth Jenifer. The global value of vaccination. Vaccine 2003;21.7:596–600. [2] Harper Diane M, Franco Eduardo L, Wheeler Cosette, Ferris Daron G, Jenkins David, Schuind Anne, et al. Efficacy of a bivalent L1 virus-like particle vaccine in prevention of infection with human papillomavirus types 16 and 18 in young women: a randomised controlled trial. Lancet 2004;364(9447):1757–65. [3] Pizza Mariagrazia, Scarlato Vincenzo, Masignani Vega, Giuliani Marzia Monica, Arico Beatrice, Comanducci Maurizio, et al. Identification of vaccine candidates against serogroup B meningococcus by whole-genome sequencing. Science 2000;287(5459):1816–20. [4] Riedel Stefan. Edward Jenner and the history of smallpox and vaccination. Proc (Baylor Univ, Med Center) 2005;18(1):21. [5] Rappuoli Rino, Black Steven, Lambert Paul Henri. Vaccine discovery and translation of new vaccine technology. Lancet 2011;378(9788):360–8. [6] Cox John C, Coulter Alan R. Adjuvants—a classification and review of their modes of action. Vaccine 1997;15(3):248–56. [7] Podda Audino. The adjuvanted influenza vaccines with novel adjuvants: experience with the MF59-adjuvanted vaccine. Vaccine 2001;19(17):2673–80. [8] McKeage Kate, Romanowski Barbara. AS04-adjuvanted human papillomavirus (HPV) types 16 and 18 vaccine (Cervarix® ). Drugs 2011;71(4):465–88. [9] Thompson Bruce S, Chilton Paula M, Ward Jon R, Evans Jay T, Mitchell Thomas C. The low-toxicity versions of LPS, MPL® adjuvant and RC529, are efficient adjuvants for CD4+ T cells. J Leukoc Biol 2005;78(6):1273–80. [10] Pantel Austin, Cheong Cheolho, Dandamudi Durga, Shrestha Elina, Mehandru Saurabh, Brane Luke, et al. A new synthetic TLR4 agonist, GLA, allows dendritic cells targeted with antigen to elicit Th1 T-cell immunity in vivo. Eur J Immunol 2012;42(1):101–9. [11] Levitz Stuart M, Golenbock Douglas T. Beyond empiricism: informing vaccine development through innate immunity research. Cell 2012;148(6):1284–92. [12] Alonso Pedro L, Sacarlal Jahit, Aponte John J, Leach Amanda, Macete Eusebio, Milman Jessica, et al. Efficacy of the RTS, S/AS02A vaccine against Plasmodium falciparum infection and disease in young African children: randomised controlled trial. Lancet 2004;364(9443):1411–20. [13] Nordly Pernille, Rose Fabrice, Christensen Dennis, Nielsen Hanne Mørck, Andersen Peter, Agger Else Marie, et al. Immunity by formulation design: induction of high CD8+ T-cell responses by poly (I:C) incorporated into the CAF01 adjuvant via a double emulsion method. J Control Release 2011;150(3):307–17. [14] O’Hagan Derek T, Singh Manmohan, Ulmer Jeffrey B. Microparticle-based technologies for vaccines. Methods 2006;40(1):10–9.