2.2. Vaccines for substance use disorders

Addictive drugs are a chemically heterogeneous group of small molecules that are able to pass the blood‐brain barrier (BBB) and target the distinct neurotransmitter systems in the brain. Vaccines have the potential to induce anti‐drug antibodies that cannot cross the BBB but can potentially bind to the drug and inhibit its transport to the brain, without altering brain function. Because the addictive drugs are small non‐immunogenic molecules, a hapten‐carrier approach is used which requires conjugation to a carrier protein to enhance the induction of drug‐specific antibodies. For example, the key components of a conjugate nicotine vaccine are: a B cell epitope, in this case nicotine; a T cell epitope, provided by the carrier protein as used in polysaccharide conjugate vaccines for capsulated bacterial pathogens and an adjuvant that enhances vaccine immunogenicity. 37 Although the vaccine principle is the same for all drugs, when designing the hapten vaccine, it is important to consider drug properties such as size, chemical structure, metabolism, and biodistribution. 38 The efficacy of induced antibodies will depend on a number of variables including the carrier, hapten density, aggregates and adducts, and a choice of adjuvant. 38 , 39 , 40 , 41 , 42 , 43

Nicotine, a psychostimulant, is the main addictive component in tobacco products. Nicotine‐targeting vaccines have been the best studied of all addictive drug vaccines, with six candidates reaching clinical trials. 44 NicVax is the first‐generation anti‐nicotine vaccine that has demonstrated promising results in phase 2 trials, but has failed to show improvement in smoking cessation compared to placebo in phase III trials. 45 , 46 , 47 Esterlis et al 48 have shown that the vaccine resulted in only a 12.5% decrease of nicotine concentration in the brain. NicVAX consists of a hapten, 3’aminomethylnicotine, conjugated to the exoprotein A from Pseudomonas aeruginosa and alum as an adjuvant. 44

Improved responses have been seen with new hapten designs involving conjugation to cross‐reactive material 197 (CRM197), a non‐toxic derivative of diphtheria toxin (DT), and addition of CpG adjuvant (TLR agonist) in addition to alum. 41 , 49 , 50 , 51 Indeed, this formulation induced higher titers of nicotine‐binding antibodies in rats and non‐human primates (NHPs), and the study showed that a combination of alum and CpG adjuvants can enhance both the antibody titer and affinity. 49 Because of the positive preclinical results, the vaccine (NIC7‐001) is currently being tested in a phase I clinical study; however, the results are not yet available. The N4N vaccine is another second‐generation vaccine that has shown promise for nicotine vaccination. 52 The N4N hapten is a covalent modification of pyridine and has much higher nicotine affinity than 3’aminomethylnicotine from the NicVax vaccine. The N4N hapten is conjugated to flagellin but has not yet been tested clinically.

A different vaccine approach for inducing drug‐specific antibody responses involves particle‐based vaccines, which are built from either polymers, liposomes, peptides, virus‐like particles, or other combinations. 53 , 54 , 55 These self‐assembling particle vaccines are anticipated to enhance the activation of antigen‐presenting cells (APC), to promote stronger T‐helper cell responses, and to stimulate the differentiation of memory B cells. 56 , 57 Additionally, the hapten load can be controlled and the delivery of adjuvants and other immunomodulators to APCs made more efficient. 42 The nanoparticle‐based vaccine SEL‐068 from Selecta Bioscience consists of nicotine bound to the surface of polymers, a synthetic TLR ligand, and a T‐cell helper peptide. In preclinical studies in non‐human primates, the vaccine blocked the development of nicotine discrimination, a behavioral experimental procedure to test the effect of nicotine. 58 The Selecta group showed that codelivery of an antigen with a TLR7/8 or TLR9 agonist in synthetic polymer nanoparticles increased drug immunogenicity with minimal systemic production of inflammatory cytokines. 59 SEL‐068 is currently being evaluated in phase 1 clinical trials.

Another particle‐like vaccine in preclinical studies incorporates a synthesized short trimeric coiled‐coil peptide (TCC) that creates a series of B and T cell epitopes with uniform stoichiometry and high density. 60 Vaccination with this antigen and alum and a TLR4 agonist (GLA‐SE) could prevent 90% of a nicotine dose equivalent to three smoked cigarettes from reaching the brain. The TLR4‐based adjuvant, as a potent stimulator of T cell–mediated antibody responses, has shown superiority compared to alum, with higher antibody titers and improved antibody affinities.

More recently, a hybrid nanoparticle‐based nicotine vaccine (NanoNiccine) has been developed with an aim to improve specificity and induce more sustained responses. 61 NanoNiccine is composed of a poly(lactide‐co‐glycolide) acid (PLGA) core, keyhole limpet hemocyanin (KLH) as an adjuvant protein enclosed within the PLGA core, a lipid layer, and nicotine haptens conjugated to the outer surface of the lipid layer. The vaccine showed superior immunogenicity compared to traditional nicotine‐protein conjugate vaccines. The particles were efficiently taken up by dendritic cells, and the principal adaptive immune response detected was the induction of antigen‐specific IgG antibodies. The same group have demonstrated that the immunogenicity of the NanoNiccine vaccine can be further improved by modulating factors such as particle size, 62 , 63 hapten localization, and density, 62 , 63 combinations of adjuvants, 64 , 65 conjugation of potent carrier proteins, 64 , 65 and degree of pegylation. 66 In addition to the great potential of novel adjuvant approaches to enhance the magnitude and quality of the anti‐drug antibody response, the nature of the hapten used and the degree to which it can trigger high affinity anti‐drug antibodies is pivotal. However, detailed discussion of this topic is outside the scope of this review.

2.3. Vaccines against chemical hazards

DDT (1,1,1‐trichloro‐2,2‐bis (p‐chlorophenyl) ethane) has been used widely as a pest control agent but was banned in 1970 because of its harmful effects on wildlife and human health. 67 This compound is therefore a chemical hazard for both human health and the environment. The biodegradation of DDT is very slow, 68 and in animals and humans, the DDT accumulates and is stored in adipose tissue. 69 When an organism is a part of the food chain, a biomagnification occurs, where the hazard is accumulated. 70 Although the liver is able to transform some of the DDT to DDE or DDD, 71 these degradation compounds are not eliminated, but are stored even more avidly. 72

Research has been conducted into the potential of the immune system to eliminate DDT following vaccination. The toxin DDT was made immunogenic by conjugating it with keyhole limpet hemocyanine (DDT‐KLH). Mice were immunized subcutaneously using aluminum hydroxide adsorbed DDT‐KLH conjugate, where the second group only received KLH adsorbed to aluminum hydroxide, and the third group was used as a control to provide information on DDT levels in serum in untreated animals. The mice were then fed with chow containing 40 mg/kg of DDT for 45 days. 73 The concentration of DDT and its metabolites (DDE and DDD) was analyzed in various tissues, and DDT‐specific antibody titers were determined. Higher antibody responses were detected in mice vaccinated with the DDT‐KLH conjugate, and DDT, DDE, and DDD levels in adipose tissue, blood, brain, and spleen were significantly reduced, compared to the group that received native unconjugated DDT. 73 This demonstrates that immunization against a chemical hazard may be used to treat animals or humans exposed to high levels of such toxic compounds, as well as prevent them from having these compounds accumulating in their body.

2.4. Alzheimer's disease vaccines

Alzheimer's disease (AD) is a progressive neurodegenerative disorder, characterized by plaques of misfolded amyloid β (Aβ) and tau protein aggregates in neural tissue, and cerebrovascular dysfunction resulting from damaged small blood vessels in the brain, 74 which eventually leads to dementia in the elderly population. Currently, it is considered an incurable disorder with limited treatment options. The mechanism(s) underlying the cognitive decline in Alzheimer's disease still has not been clearly unraveled, which makes it difficult to judge whether current therapies target the symptoms, rather than the key molecule(s) causing the pathology. In AD transgenic animal models, immunotherapeutic targeting of B cell epitopes of (misfolded) Aβ and/or tau seemed a most logical strategy to target neural plaques or vascular aggregates. Indeed, both vaccines, and passive antibody‐based interventions, resulted in a clear reduction of Aβ pathology and cognitive benefits, with limited local inflammatory adverse effects. However, despite these promising preclinical results, such approaches in human studies showed limited evidence of significant clinical benefits, even despite the postmortem observed clearance of amyloid pathology, and acceptable tolerability. 75 , 76 , 77

Several redesigned vaccines against the (modified) variants of soluble or aggregated Aβ and tau protein (with or without protein carrier Qβ or KLH, lacking common T cell epitopes) formulated with saponin QS‐21, liposomes plus TLR4 agonist, or KLH‐alum, as immune modulating adjuvants, are being evaluated in early‐stage trials.

2.5. Atherosclerosis vaccines

Accumulation of leukocytes in the intima of the arterial wall and subsequent uptake of lipids in the form of low‐density lipoprotein (LDL) by macrophages initiate the formation of atherosclerotic lesions in large and medium sized arteries. As lesions grow, they become increasingly unstable, ultimately causing them to rupture, which can result in thrombotic occlusion of blood vessels. As such, atherosclerosis is the major underlying pathology of cardiovascular events like myocardial infarction or stroke and is the leading cause of death in the Western world. Although current treatment strategies focus on controlling LDL levels (eg, by statins, PCSK9 inhibitors), immunomodulatory interventions, including vaccination, are currently in preclinical and clinical investigation.

Development of atherosclerotic plaques is driven by an inability to clear LDL from the lesion, resulting in a chronic inflammatory reaction involving both innate and adaptive immunity, and therefore, LDL has been the most intensively studied antigen. Indeed, LDL‐specific T cells have been identified in atherosclerotic lesions 78 and circulating auto‐antibodies against LDL or modified LDL can be readily detected in most individuals. 79 Interestingly, induction of anti‐LDL antibodies by immunizing rabbits with chemically modified LDL particles suspended in the water‐in‐oil emulsion, Complete Freund's adjuvant (CFA), or AdjuPrime had a protective effect with less prominent atherosclerotic lesions in the vaccinated animals. 80 , 81 Similarly, vaccination with peptides derived from the largest protein in LDL, ApoB100, formulated with aluminum phosphate and cationized BSA, or CFA, reduced atherosclerosis in mice. 82 , 83

3.2. Therapeutic cancer vaccines

Anti‐tumor therapeutic vaccination should generate not only tumor‐specific CD8⁺ T cells but also tumor‐specific tissue‐resident memory T cells (Trm) at the tumor site. 119 Indeed, Trm are emerging as an essential actor in tissue immunosurveillance due to their specific expression of defined adhesion molecules (CD103 or CD49a integrins, chemokine receptors) facilitating their retention in tissues and consequently into the tumor. Their well‐positioned location in close contact with tumor cells and their higher cytotoxic capacities explain why their tumor infiltration is correlated with good clinical outcomes in many cancers. Accordingly, it appears that eliciting Trm represents a key target for the success of cancer vaccines.

The phenomenon of antigen spreading, which corresponds to the secondary CD8⁺ T cell response against antigen epitopes that are not present in the vaccine but likely result from local release after the first wave of tumor‐specific CD8⁺ T cells, provides additional evidence for the important role of these effector cells. 120 Indeed, in human clinical case studies, it was reported that tumor regression may be dependent on CD8⁺ T cells directed against tumor antigens not present in the vaccine, and thus, the induction of CD8⁺ T cells might be a suitable surrogate biomarker based on immune‐related response criteria to evaluate therapeutic vaccines. 121

One of the most critical issues for efficient priming of naïve CD8⁺ T cells relates to their contact with mature antigen‐presenting DCs. Some vaccine formulations including DNA‐, RNA‐, or DC‐based vaccines exhibit direct immunostimulatory properties and have been shown to elicit CD8⁺ T cells in mice and in humans. 122 However, some discrepancies between results obtained in mice and humans, especially for DNA vaccines, have to be addressed. 123 The plasmid‐based DNA vaccines were thus very efficient in many species including mice and non‐human primates, but early human studies failed due to low efficacy. The plasmid technology has greatly improved over the years, both when it comes to codon optimization increasing expression of the antigens, and plasmid delivery within the body. So, it will be interesting to follow these techniques in the future.

Immunostimulatory adjuvants such as TLR ligands, CD40 agonists, cytokines, or activators of stimulator of IFN genes (STING) are indicated to potentiate vaccine immunogenicity and to break self‐TAA tolerance. 124 Interestingly, some adjuvants can also preferentially polarize the immune cells to a Tc1‐type CD8⁺ T cell response. For example, vaccination with CpG and Poly I:C increased the ratio between effector CD8⁺ T cells and T_REG. 125 , 126 Activators of STING delivered via a particle platform to reach the cytosol also favor the induction of CD8⁺ T cells. 127 To date, relatively few immunostimulants have been approved for human use, although several are under evaluation in clinical trials. 128

One of the most promising strategies for development of therapeutic vaccines against tumor targets uses recombinant, attenuated live vectors derived from viruses or bacteria as a vehicle for the tumor target antigen. Because of their intrinsic capacity to reach the cytosol of the cells, these recombinant pathogen‐derived delivery systems favor MHC class I‐restricted peptide presentation and generally induce a better CD8⁺ T cell response than free proteins or peptide vaccines. 129 In addition, they are highly immunogenic as they express PAMPs and can be engineered to also express activating molecules like cytokines (IL‐2) or immunomodulatory molecules (B7.1; or a triade of costimulatory molecules called TRICOM: B7.1, ICAM‐1, and LFA‐3) to further amplify the vaccine response. 130 However, it has to be considered that the efficacy of live vectors can be reduced after repeated vaccinations due to anti‐vector antibody responses. A diversified, prime‐pull strategy using distinct attenuated pathogens is required to overcome the neutralization of the vector bacteria or virus by host immunity. A recently described deep learning approach to neoantigen identification 131 offers promise for more rapid and targeted tumor antigen discovery, and novel adjuvant and delivery strategies are being evaluated. Several non‐replicating live vectors have been tested in clinical trials with minimal toxicity and the ability to generate antigen‐specific CD8⁺ T cells. 132 Adjuvant systems such as ISCOMS, saponins, emulsions, liposomes, virosomes, or nanoparticles (Table 3) can also favor cross‐presentation to CD8⁺ T cells in parallel with delivering immune signals. Allostatine is a short peptide derived from Alloferons, a group of naturally occurring peptides isolated from bacteria‐challenged larvae of the blow fly Calliphora vicina. These larvae are well known for medical use in wound healing: Napoleon's army was using them on wounded soldiers (a.k.a. “Surgical maggots”). Allostatine is a synthetic linear peptide having amino acid sequence HGVSGWGQHGTHG. It demonstrated strong adjuvant properties in a mouse P388/DBA2 tumor transplantation model when combined with a vaccine consisting of X‐ray inactivated tumor cells. 133 Although the vaccine alone demonstrated only weak tumoristatic effect in a quarter of the recipients, its combination with allostatine caused a tumoristatic effect in 65% of recipients and prevented tumor appearance in another 30% (the overall positive effect was 95%). The anti‐tumor effect of allostatine "alone" (tumor‐free animals two months after tumor implantation) has been detected in only 9% of the animals. Alloferons are antiviral and anti‐tumor peptides isolated from insects, exhibiting stimulatory effect on the cytotoxic activity of human and mouse NK cells. 134 , 135 The Ig site is an evolutionarily conserved pattern in all human and mouse immunoglobulins. Even very low concentration of allostatine in culture medium (at ng/ml scale) causes rearrangement of NK and T cell receptors, stimulation of NK cell cytotoxic activity against cancer cells, and an increased number of IFN‐γ and IL‐2‐producing cells. Allostatine is non‐toxic to immune cells, even at high dose, water soluble, stable in aqueous solutions and can be produced in large quantities at relatively low cost.

Short CD8⁺ peptides can bypass the need for cross‐presentation by directly binding to MHC‐I molecules on the surface of the APC, but they show low efficiency due to the lack of CD4⁺ T cell help, illustrating the importance of cross‐presentation. It is well‐established that CD4⁺ T cells and especially Th1 cells play a key role in promoting anti‐tumor cellular immunity, mediated via their “helper” function. 136 Activation of CD4⁺ T cells is essential for optimal CD8⁺ T cell priming, by enhancing antigen presentation and favoring cross‐priming of tumor antigen by DC 137 (see also Figure 4). CD4⁺ T cells also support recruitment of CD8⁺ T cells to the tumor site, maintenance, and expansion of a CD8⁺ T cell memory response. 138 Besides their “help” to CD8⁺ T cell responses, CD4⁺ T cells can mediate other anti‐tumor effects such as direct killing of tumor cells, recruitment toward and activation of innate immune cells (eg, NK cells or M1 macrophages) at the tumor site, and modulation of the tumor microenvironment by anti‐angiogenic effects. 139 , 140 CD4⁺ Th1 cells also promote vessel normalization leading to better intratumoral CD8⁺ T cell infiltration. 141

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FIGURE 4

Adjuvants shape CD4⁺ and CD8⁺ T cell immunity through direct and indirect effects on dendritic cells (DCs)

Certain DC subpopulations, especially human CD141⁺ DC designated as cDC1, are thought to be specialized for cross‐presentation. 142 Recent studies revealed that antibody‐based targeting of antigens to specific DC receptors can enhance antigen uptake and anti‐tumor vaccine efficiency. 143 Alternative targeting strategies are also possible based on the fusion of tumor antigen(s) to a protein that can engage with receptors on the DC membrane in situ. For example, the group of Eric Tartour demonstrated that targeting DC in vivo with the B subunit of Shiga toxin (STxB), a non‐toxic vector, which binds to Gb3, preferentially expressed by DCs, significantly increased antigen‐specific CD8⁺ T cell responses when coupled to various antigens. 144 From this pioneering study, a number of cancer vaccines have been developed based on the targeting of surface molecules preferentially expressed on DC (DEC‐205, Clec9a/DNGR, XCR1, CD11c). 145 Antibodies against human DC receptors such as DEC‐205 and Clec9a coupled with model antigens reported an increase of cross‐presentation, resulting in enhanced induction of antigen‐specific CD8⁺ T cells. 146 Alternatively, tumor antigen may be formulated and administered ex vivo to DCs in the form of a cell therapy product. 147 However, the choice of the subpopulation of DC to be selected, the maturation signal to be used and the optimal formulation of antigen remain a matter of debate. 148 While this type of cancer vaccine has been shown to induce specific CD8⁺ T cells, it has had variable clinical impact, 149 although the approach remains under evaluation. In this context, specific adjuvants may have potential to optimally activate DC in vitro before adoptive transfer into patients.

The ideal setting for treatment with a therapeutic anti‐cancer vaccine is after surgical resection and/or chemo/radiotherapy, where the induction of anti‐tumor immunity can contribute to eliminate residual circulating cancer cells and micrometastasis. However, despite extensive efforts and promising preclinical studies, therapeutic cancer vaccines have shown limited objective tumor regression and therefore have mostly proven unsuccessful in the clinic. This may be explained by the state of immune suppression after chemotherapy and radiotherapy in the patient and the escape from immune surveillance of newly mutated tumor cells. These anti‐cancer vaccines have been hampered by poor immunogenicity and limited triggering of rare tumor‐specific immune cells, which makes the elicitation of both humoral and cellular immunity challenging and in most cases unsuccessful. 150

The range of tumor‐associated antigens recognized by T cells provides a variety of potential targets for cancer immunotherapy. In the case of TAAs targeted via cell‐mediated immunity, the activated T cells induced by vaccination recognize the MHC‐peptide complexes of the tumor antigen on the cell surface and not the surface protein itself. Thus, the optimal tumor‐associated antigens for triggering cellular immune responses are not necessarily cell surface proteins and can be classified in several categories 151 :

• Cancer‐testis antigens—normally expressed in germ cells but aberrantly expressed in tumor cells: BAGE, GAGE, MAGE (in melanoma, bladder cancers), and NY‐ESO1 (in melanomas and ovarian cancers; antibody and T‐cell responses have been observed in patients).

• Differentiation antigens—expressed on tumor cells and normal cells from the same tissue: Melan‐A/Mart‐1, gp100, tyrosinase (in melanomas), PSA (in prostate cancer), and CEA (colon).

• Overexpressed antigens—expressed on normal cells at low levels, but at higher levels on tumor cells: HER2 (breast), hTERT, p53, survivin (melanoma), and MUC1 (breast, prostate, colon).

Tumor‐associated peptides antigens, in particular MHC class I‐restricted peptides derived from cancer‐testis and differentiation antigens, have been used in cancer vaccine clinical trials as a means to generate anti‐tumor T‐lymphocyte responses. These vaccines have generated some cytotoxic T‐lymphocyte (CTL) activity; however, the frequency and duration of these responses have been uniformly low and the clinical outcomes have been limited. 150 Accordingly, the design of therapeutic vaccines against cancer, especially for treating early disease, should not focus only on skewing a response to a single T‐helper cell type (for instance, Th1) or one effector mechanism (for instance, CTL). Rather, the ideal cancer vaccine should seek the simultaneous activation and synergy of both arms of the immune system to generate as many effector cells as possible to eradicate the tumor. Instead of using single peptide, protein, or carbohydrate antigens as TAA for cancer vaccines, cancer antigens prepared from surgically removed autologous or allogeneic tumor tissue may provide a source of TAAs with all, or many, of the required antigens in one preparation. 152 , 153

Despite extensive efforts and clinical trials with vaccines targeting TAAs, the clinical success of anti‐cancer vaccine therapies remains limited, with the DC‐based prostate cancer vaccine Sipuleucel‐T being the first and only human therapeutic cancer vaccine approved by the US Food and Drug Administration (FDA). 154 To advance clinical development of cancer vaccines, further efforts are required, not only to optimize and discover new tumor‐associated antigens, but also to identify and develop improved vaccine adjuvants, delivery systems, and formulations, building upon an increased understanding of the mechanisms underlying the induction of the required immune responses. Moreover, the recent success of checkpoint inhibitors has set the stage for combination strategies and opened the door to revisit cancer vaccine design. Taken together, progress on this front will enable realization of the full potential of a patient's immune response to target and combat cancer.

This article/publication is based upon work from COST Action CA16231 ENOVA (European Network of Vaccine Adjuvants), supported by COST (European Cooperation in Science and Technology—www.cost.eu).