How Long Will It Take to Launch an Effective Helicobacter pylori Vaccine for Humans?

Abstract

Helicobacter pylori infection often occurs in early childhood, and can last a lifetime if not treated with medication. H. pylori infection can also cause a variety of stomach diseases, which can only be treated with a combination of antibiotics. Combinations of antibiotics can cure H. pylori infection, but it is easy to relapse and develop drug resistance. Therefore, a vaccine is a promising strategy for prevention and therapy for the infection of H. pylori. After decades of research and development, there has been no appearance of any H. pylori vaccine reaching the market, unfortunately. This review summarizes the aspects of candidate antigens, immunoadjuvants, and delivery systems in the long journey of H. pylori vaccine research, and also introduces some clinical trials that have displayed encouraging or depressing results. Possible reasons for the inability of an H. pylori vaccine to be available over the counter are cautiously discussed and some propositions for the future of H. pylori vaccines are outlined.

Graphical Abstract

Keywords:

• Helicobacter pylori vaccines
• antigen
• adjuvant
• delivery system
• clinical trials

Introduction

Helicobacter pylori became a concern in 1979 when John Warren, a pathologist in Perth, Western Australia, discovered that the inflamed gastric mucosa was covered with bacteria that looked like Campylobacter.¹ Warren and his colleague Barry Marshall cultured the first batch of these bacteria, newly named Helicobacter pylori, subsequently from 11 patients with gastritis in 1982.² H. pylori is a spiral and slightly aerobic pathogenic bacteria that can survive in the stomach and colonize for life through contact transmission from the oral cavity or feces.³ It may lead to gastritis, digestive tract ulcers, lymphoproliferative gastric lymphoma, and even gastric cancer.⁴ Nearly 50% of the global population is infected, and the infection rate is even higher in developing countries.^5,6 The pathogenic mechanism of H. pylori is a complex process that has been reviewed in detail by a considerable number of articles.

Currently, there are four main first-line treatment regimens for H. pylori: clarithromycin-containing triple therapy, sequential therapy, concomitant therapy, and bismuth quadruple therapy. Quadruple therapy is the recommended first-line treatment. In areas where the incidence of clarithromycin resistance is low and in patients who have not previously used macrolides, a 14-day triple therapy containing clarithromycin is recommended.⁷ The anti-inflammatory and antioxidant mechanisms of probiotics can improve intestinal microecology and general health, but cannot increase the eradication rate of H. pylori infection.⁸ Therefore, probiotic therapy can only be used as an adjunctive therapy to reduce antibiotic-related adverse events. Although clarithromycin-containing triple therapies initially achieve a radical cure rate of >90%,⁹ due to the increase in macrolide drug resistance, mainly clarithromycin, the efficacy of these therapies has been reduced to unacceptably low levels (≤80%) in most parts of the world.¹⁰ Vaccination is effective in the prophylaxis and therapy of infectious diseases and has been verified after hundreds of years of application practice. The focus has shifted to H. pylori infection, even though there is no available vaccine as yet. In 1990, Pallen et al thought that an anti-urease vaccine was promising,¹¹ and now urease is one of the most important vaccine antigens. Czinn et al proposed in 1991 that oral H. pylori antigen plus cholera toxin could increase the level of antibodies in serum and intestinal tract.¹² Then, in 1993 Czinn et al proved that the resistance to H. pylori comes from IgG and IgA produced by oral immunization. Active oral or passive immunization of IgA can provide resistance to H. pylori in mice.¹³ Since then, the search for an H. pylori vaccine has entered a period of rapid development. In the process, there have been >10 kinds of candidate antigen identified to use alone or in combination, nearly 10 kinds of adjuvant that have been tested to enhance immunoresponse, and various delivery systems introduced to help antigen presentation. Several H. pylori vaccines have been undergoing or completed clinical trials.^14–26 This review focuses on an H. pylori vaccine, provides references to counterparts, summarizes the progress of candidate antigens, immunoadjuvants, delivery systems, and clinical trials, analyzes the current situation cautiously, and looks forward to the future confidently.

Antigens

Urease

H. pylori urease is a polyenzyme composed of 12 UreA and UreB heterodimers, accounting for 10%–15% of the total protein content of bacteria.²⁷ Urease can catalyze the hydrolysis of urea to CO₂ and NH₃. NH₃ can neutralize excess gastric acid, inhibit the function of neutrophils,²⁸ promote the formation of cytotoxic NH₃-derived compounds,²⁹ and destroy the integrity of gastric epithelial cell connections,^30,31 thus providing conditions for the colonization of pathogens in the stomach. CO₂ can protect bacteria from ONOO^– cytotoxicity, thereupon then promoting the colonization of pathogens.³² The discrete surface of urease complex can directly interact with host components involved in immunomodulatory activities, thereby ensuring the continuous colonization of H. pylori.³³ Inhibition of urease activity will impair the ability of H. pylori to colonize in the stomach, and plays a role in the prevention and treatment of H. pylori.³⁴ Most studies have used urease as a candidate antigen, such as Nasr-Esfahani et al, who showed that injecting the recombinant plasmid pcDNA3.1 (+)-ureA into mice can stimulate an immunoresponse in an animal model infected with H. pylori.³⁵ Intranasal immunization with recombinant UreB vaccine with plant polysaccharides as adjuvants protected mice from H. pylori infection, which may be related to increased gastrointestinal specific SIgA and Th1/Th17 CD4⁺ T-cell response.³⁶ Most of the vaccines that have entered the clinical research stage contain urease antigen.^{14–17,19–23,25} It is worth mentioning that the H. pylori vaccine with urease and heat-labile enterotoxin subunit B (LTB) developed by Zeng et al in 2015 has achieved gratifying results in phase III clinical trials.²⁵

VacA

Vacuolating cytotoxin A (VacA) is a protein component that causes vacuolization of eukaryotic cells.³⁷ All H. pylori strains contain the vacA gene, while only about 50% of H. pylori strains produce cytotoxin encoded by the vacA gene.^37,38 VacA is a kind of secretory pore-forming toxin, which can form anion-selective channels in planar lipid bilayers and have extensive effects on host cells,³⁸ such as leading to epithelial vacuolization, enhancing H. pylori colonization in the stomach, and interfering with MHC II antigen presentation.³⁹ Recently, using a polysaccharide adjuvant (PA) containing Lycium barbarum polysaccharides (LBPs) and chitosan as adjuvant, Guo et al designed a vaccine FVpE containing Th1 immunoadjuvant NAP, functional fragments of CagA and VacA, and urease multiepitope peptides. Compared with natural urease vaccine, FVpE can induce higher levels of antigen-specific antibodies and significantly reduce the number of H. pylori in infected mice.⁴⁰ Phase II clinical trials have been carried out on the multiepitope vaccine containing vacuolating toxin. Compared with the placebo group, this vaccine can enhance the systemic humoral response to key H. pylori antigens, which have fully demonstrated the potential of vacuolating toxins as vaccine candidates.^24,41

cagPAI

The cag pathogenicity island (cagPAI) is a chromosome region of 40 kb encoding a functional type IV secretory system, which plays an important role in the pathogenesis of H. pylori.⁴¹ The three genes cagA, cagL, and cagW encoded by this region can be used as candidate antigens.

CagA

Cytotoxin-associated gene A (cagA) is located at one end of cagPAI, which is closely related to the production of VacA.^41,42 The protein encoded by cagA is a bacterial surface protein with a relative molecular weight of 120–128 kDa containing a carboxyl terminal variable region.⁴³ The expression rate of CagA in strains found in Western countries is about 60%, while almost all East Asian strains express CagA.⁴⁴ It has been indicated that the fragments of CagA can produce immunogenicity in human models.⁴⁵ Shapouri et al successfully expressed the 38 kD recombinant protein CagA (rCagA) in Escherichia coli BL21, which was able to bind to human antiserum.⁴⁶ After mice were immunized with rCagA and lipopolysaccharide, compared with the control group, the combined-immunization group produced a strong Th1 immunoresponse. Importantly, the number of H. pylori cells decreased sixfold.⁴⁷ Similarly, Paydarnia et al proposed that a CpG adjuvant carrying H. pylori lipopolysaccharide and rCagA protein not only maintained the antigenicity of the recombinant protein throughout the experiment but also induced a strong Th1-biased immunoresponse.⁴⁸ CagA has also been used as a candidate antigen in clinical experiments and has been proved to be effective in stimulating immunoresponses.^24,26

CagW

CagW is the key subunit of the protein transport type IV secretory system, which is the VirB6-like protein of the Cag system.^49,50 The interaction between CagW and CagA provides conditions for CagA to pass through the bacterial membrane barrier.^49,50 Chehelger et al successfully prepared high-quality chitosan nanoparticle (NP)-encapsulated cagW gene DNA vaccine-pcDNA3.1 (+)-cagW-CS-NPs, which induced high mucosal and humoral immunoresponses in mice.⁵¹

CagL

CagL is a highly conserved protein encoded by cagPAI located on the surface of bacterial type IV secretory pili.^52,53 It has been shown to bind integrin receptor α₅β₁, activate EGFR, and mediate virulence-factor infusion into host target cells, so it has the potential to be used as a candidate antigen.⁴⁵ Aliramaei et al cloned the cagL gene in a pAMJ2008 vector and transferred the recombinant plasmid into Lactococcus lactis MG1363. Mice immunized with recombinant L. lactis MG1363/pAMJ2008 cagL can produce specific anti-cagL IgA.⁵⁴

Catalase

Catalase (CAT) is a ubiquitous enzyme in eukaryotes and most prokaryotes to protect bacteria from hydrogen peroxide.⁵⁵ H. pylori CAT is a homologous tetramer protein with an isoelectric point of 9.0–9.3, which accounts for about 1% of the total protein of H. pylori.^55–57 CAT protects bacteria from reactive oxygen species produced by the host and helps bacteria escape the phagocytosis of macrophages.^57,58 Recently, the immunodominant Th1 epitopes of CAT have been fully identified. Seven new epitopes of CAT can produce significant Th1 response by expressing IFNγ.⁵⁹ Three Th-cell epitopes and five B-cell epitopes from H. pylori antigen (HpaA, UreB and CAT), adjuvant LTB, and an appropriate linker were selected to form the multivalent epitope vaccine LHUC. After oral administration of fusion peptide LHUC, antigen-specific antibodies were detected in mouse serum, and the secretion of IFNγ, IL4, and IL17 by lymphocytes increased significantly. LHUC can significantly prevent C57BL/6 mice from being infected with H. pylori.⁶⁰

HpaA Adhesion

H. pylori adhesion A (HpaA), which is described as hemagglutinin, belongs to the H. pylori outer-membrane proteins and the adhesin family. It is a highly conserved H. pylori-specific lipoprotein, which can bind to neuraminyllactose and various glycosylated components on the surface of gastric epithelial cells to ensure H. pylori attachment to the surface of gastric mucosa.^61,62 Additionally, HpaA is highly conserved and is expressed on the surface and intracellular regions of bacteria.^63,64 Also, it mediates binding with sialic acid in vitro, participates in the maturation of dendritic cells in vivo, and may affect antigen presentation.^65,66 HpaA with deletion of the amino-terminal region may lose immunogenicity, and consequently the ability of lipoprotein to activate TLR2 depends on its N-terminal lipid part.⁶⁷ Xue et al synthesized two new lipopeptides (LP1 and LP2) by predicting the lipid-modification sites of natural HpaA and simulating the terminal structure of natural HpaA. The results showed that LP2 could enhance the protective effect of rHpaA against H. pylori infection, which might be closely related to its ability to simulate the terminal structure of natural HpaA to activate TLR2.⁶⁸

NAP

H. pylori neutrophil activating protein is a highly conserved spherical dodecameric protein with molecular weight of 17 kDa.⁶⁹ It is a member of the adhesion family and exists in almost all H. pylori isolates.^70,71 NAP has the ability to specifically bind to high-molecular-weight mucins and mediate the adhesion of H. pylori to host cells.^70,72 NAP can also protect bacteria from DNA oxidative damage by inducing the host to produce oxidative stress.⁷³ NAP has proinflammatory and immunomodulatory effects, which play an important role in the pathogenesis of H. pylori.^74,75 Recently, new advances have been made in the study of NAP as a candidate antigen of H. pylori. After oral administration of H. pylori multicomponent subunit vaccine composed of NAP, UreA, UreB, and double-mutant heat-labile toxin (dmLT) from E. coli, mice produced significant Th1/Th17 immunoresponse and antigen-specific antibodies.^76,77 Jafari et al screened three main CD4⁺T cell epitopes of H. pylori antigens UreB, HpaA, and NapA by immunoinformatics. After ligation with a suitable linker, the fusion protein was successfully expressed in E. coli BL21(DE3), but the effectiveness of the vaccine needs to be further studied.⁷⁸ Clinical studies have shown that NAP combined with other antigens can enhance the systemic humoral immunoresponse.^24,26

OipA

H. pylori outer-membrane inflammatory protein A (OipA) is one of the outer-membrane proteins with relative molecular weight of 34 kDa, which is one of the important virulence factors of H. pylori.⁷⁹ A functional OipA should have the ability to promote the production of proinflammatory cytokines, participate in the attachment of bacteria to host cells, and help the host adapt to the environment.^80,81 Soudi et al proved that both oral and injection of recombinant OipA can induce mice to produce IFNγ and promote Th1 immunoresponse.⁸²

HcpD

Cysteine-rich H. pylori protein (Hcp) is a unique protein of H. pylori expressed in the natural environment.⁸³ HcpD (HP0160) is also a member of the Hcp family, which can covalently bind to penicillin derivatives.⁸⁴ Nasr et al prepared recombinant plasmid pCDNA3.1 (-)-hcpD and synthesized chitosan NPs by ion gelation. The results showed that pCDNA3.1 (-)-hcpD vaccine stimulated the immune system of vaccinated mice, either alone or in combination with chitosan NPs.⁸⁵

Flagellin

FlaA

Flagella play a major driving role in the movement of bacteria, which means that flagellin is essential for H. pylori infection and continuous colonization.^86,87 H. pylori flagellin consists of two subunits, FlaA, the main flagellin of 53 kDa, and FlaB, the secondary flagellin of 54 kDa.^86,87 In view of the important role of the two flagellins in gastric mucosal injury, FlaA and FlaB may be used as candidate antigens against H. pylori infection.^88,89 Recombinant expression vector pBudCE4.1-flaA was successfully expressed in human dermal fibroblasts, and immunoresponse was activated by intramuscular injection of the vaccine in mice.⁹⁰ Hamzehloo et al constructed the recombinant plasmid pET32a-flaA-ureB. The recombinant protein can be recognized in the sera of patients with H. pylori infection with high sensitivity and specificity.⁹¹

FliD

At the distal end of flagellar filaments, there is a flagellar cap protein (FliD) of 56 kDa encoded by the fliD gene, which participates in the growth of flagellar filaments and protects the structure of flagellar tip from destruction.^92–94 The fliD gene encodes the structural component hook-associated protein 2 (HAP2) of the flagellum cap at the end of the filament, which is involved in the assembly of flagella.⁹² FliD has been proved to be an antigen that can induce immunoresponse to H. pylori.⁹⁵ Recently, Zhao et al studied the crystal structure of HpFliD at the molecular level. Serological tests showed that the D4 and D5 domains of HpFliD had stronger antigenicity than the D2 and D3 domains and could stimulate stronger immunoresponse, so the D4 and D5 domains were expected to be targets for an H. pylori vaccine or diagnosis of related diseases.⁹⁴

Lipopolysaccharide

H. pylori lipopolysaccharide is a glycolipid component on the cell surface of H. pylori, and it is also an endotoxin.⁹⁶ It consists of the three different regions: O-chain polysaccharide (PS) with variable structure, conservative core oligosaccharide, and lipid A.⁹⁶ H. pylori lipopolysaccharide has been proved that it performs an important role in the pathogenic mechanism of H. pylori and it has the ability to proliferate gastric epithelial cells.⁹⁷ It can also bind to the protein receptor on the mucous membrane and enhance the binding ability of bacteria to the gastric mucosa.⁹⁸ O-chain polysaccharides have an aspecific immunostimulatory effect, and play a part in inducing immunoresponse and gastric injury in the host.⁹⁹ Tian et al synthesized a unique α-(1→3)-linked tri-D-glycero-D-manno-heptose antigen from lipopolysaccharide of H. pylori serogroups O3 and O6 and strains MO19, D2, D4, and D5. This antigen can induce very strong T cell–dependent antigen-specific immunoresponse and induce the body to produce high titers of the antigen-specific antibodies IgG₁ and IgG_2b.¹⁰⁰

Lpp20

Lipoprotein 20 (Lpp20) is a conserved lipoprotein with apparent molecular weight of 18 kDa, which is unique to H. pylori and related to membrane.¹⁰¹ Lpp20 is expressed in almost all tested H. pylori strains.¹⁰¹ By virtue of its strong immunogenicity, many studies have identified it as a dominant candidate for H. pylori vaccine.^101–104 Ning et al identified two dominant epitopes of Lpp20, and proved that these two epitopes can stimulate the proliferation of CD4⁺ T cells.¹⁰⁵ Sun et al successfully expressed Lpp20 in recombinant L. lactis strains. Oral administration of the engineered bacteria increased serum IgG and decreased gastric urease activity in mice.¹⁰⁶

Heat-Shock Protein

Heat-shock protein A (HspA) is a homologue of GroES chaperone protein family with relative molecular weight of 13 kDa and is found in H. pylori.^107,108 Its C-terminal domain is rich in histidine and cysteine, which is different from the GroES protein family.¹⁰⁸ This unique domain is conducive to the binding of proteins to nickel ions.¹⁰⁹ Some researchers have studied it as a vaccine component of H. pylori and proved its potential as a candidate antigen.^110,111 Zhang et al successfully identified two highly conserved B-cell epitopes of HspA with good immunogenicity.¹¹² The recombinant measles virus (MV) vaccine expressing H. pylori HspA antigen has strong antitumor activity and strong immunogenicity.¹¹³

GroEL

GroEL is a chaperone protein secreted by H. pylori, which belongs to the molecular chaperone family, which is also described as a homologue of Hsp60.¹¹⁴ It is highly conserved and has been identified as a virulence factor of H. pylori and a risk predictor of gastric cancer.^115,116 It has been reported that GroEL antigen was found in all tested strains of H. pylori.¹¹⁷ Khan et al designed a new type of H. pylori multiepitope subunit vaccine containing helper T-lymphocyte epitopes (HTL) of CagA, OipA, GroEL, and VacA antigens using a variety of immunoinformatic methods and other computational methods. Although the stability and immunity of the vaccine have been verified, its effectiveness vaccine still needs to be tested in further clinical trials.¹¹⁸

52 kDa H. pylori Membrane Peptide

The isolation and purification of Hcp is facing great challenges, so the synthetic peptide designed by immunogenic proteins has become an alternative for diagnosis and prevention.¹¹⁹ Espinosa-Ramos et al designed and synthesized an H. pylori 50–52 kDa immunogen-derived peptide antigen with the sequence Met-Val-Thr-Leu-Ile-Asn-Asn-Glu (MVTLINNE). Compared with the untreated group, the MVTLINNE polypeptide vaccine mediated the body to produce specific antiserum IgG and IgA, reduced H. pylori colonization significantly in mice, and combated the occurrence of gastric ulcer effectively.¹¹⁹

Adjuvants

LTB

Heat-labile enterotoxin (LT) is a diarrhea-causing toxin produced by E. coli (ETEC) and a member of the AB₅ bacterial toxin family.^120,121 LT is related to the cholera toxin in chemical characterization and immunology.¹²² It consists of one enzyme active subunit (LTA) and five subunits (LTB).¹²⁰ LTB has been widely used in immunity experiments owing to the fact that it is no catalytic activity or toxicity. There is considerable evidence that vaccines with LTB as an immunoadjuvant can stimulate the body to produce immunoresponse.^123–125 Recently, Peng et al expressed the napA gene and LTB through Lactobacillus lactis, and proposed that LTB can enhance the protective effect induced by oral vaccine by aggravating mucosal inflammatory injury and leukocyte leakage.¹²⁶ LTB has been selected as an immunoadjuvant in most clinical studies of H. pylori vaccine and has good immunoeffect, but it has some side effects on the human body.^{15,18,20,21,25} A clinical study has shown that low-dose LTB can reduce toxicity and maintain immunogenicity.²¹

CpG ODNs

The interaction between CpG oligodeoxynucleotides (CpG ODNs) and the expression of Toll-like receptor 9 triggers a cascade of signals to activate B and T lymphocytes, monocytes, natural killer cells, macrophages, and dendritic cells.¹²⁷ It can also improve the host’s ability to resist pathogen attacks by initiating immunomodulatory cascades.¹²⁸ CpG ODNs have been widely used as a vaccine adjuvant and have been proved to be safe for humans.^129,130 Paydarnia et al demonstrated that the rCagA protein carried by CpG adjuvant not only maintains the antigenicity of the recombinant protein throughout an experiment but also induces a strong Th1-biased immunoresponse.⁴⁸

cGAMP

cGAMP is the first endogenous cyclic dinucleotides discovered. It is synthesized by cyclase in response to the stimulation of DNA ligands, which can directly bind to STING receptor protein and activate STING to trigger downstream signal cascade, including TBK1, IRF3 activation, IFNβ, and other cytokines.^131,132 STING agonists have proved to be a promising immunoadjuvant, including cGAMP.^133,134 In a recent study, H. pylori UreA, UreB, and NAP adjuvanted with cGAMP induced immunoresponse in mice. The reaction of antigen-specific immunoglobulin and mucosal IgA in serum of mice immunized after nasal cavity and subcutaneous immunization increased significantly, while gastric mucosal colonization decreased significantly.¹³⁵

Plant Polysaccharides

Plant polysaccharides (PPSs) are active components extracted from natural plants, and have unique characteristics and low toxicity.¹³⁶ Many studies have shown that polysaccharide adjuvants are effective vaccine adjuvants that can improve humoral or cellular immunity, such as in Astragalus polysaccharides, Epimedium polysaccharides, LBPs, chitosan, Taiwan Ganoderma formosanum polysaccharides PS-F2, Chinese yam polysaccharides, isatis root polysaccharides, and Achyranthes bidentata polysaccharides.^137–140 Liu et al have proved that Astragaluspolysaccharides combined with rUreB can induce mixed Th1 and Th17 immunoresponses, which may help mice to resist H. pylori infection.¹⁴¹ Similarly, Guo et al showed that the combination of polysaccharide mucosal adjuvant-containing LBPs and chitosan with a multivalent epitope vaccine enhanced the protective effect of this vaccine.⁴⁰

α-GalCer

α-Galactosylceramide (α-GalCer) is a kind of glycolipid originally extracted from marine sponge.¹⁴² α-GalCer is considered an effective multifunctional vaccine adjuvant, and can induce humoral and cellular immunoresponses.^143,144 The results of prophylactic intragastric immunization with whole-cell inactivated antigen of H. pylori combined with nontoxic oral adjuvant α-GalCer showed strong intestinal and systemic Th1 cellular immunoresponse, as well as significant antigen-specific mucosal and systemic antibody response. The effect of α-GalCer adjuvant is similar to that of the standard adjuvant cholera toxin.¹⁴⁵

Cytokines

Many studies have shown that the combination of cytokine protein or cytokine gene-encoding plasmid with DNA vaccine can make DNA vaccine prefer to trigger Th1 immunoresponse, such as IL2, IL1, IL6, IL15, and IL12.¹⁴⁶ Nemattalab et al proposed for the first time that the use of IL18, IL17A, and IL22 as molecular adjuvants in a DNA vaccine regimen can change immunoresponse and improve the efficacy of DNA vaccine. It was proved that the coexpression of oipA gene and IL17A molecular adjuvant can effectively combat H. pylori infection.¹⁴⁷

Propolis

Propolis, a kind of resin compound collected by bees from developing flowers, has complex chemical composition and different biological and pharmacological properties.¹⁴⁸ Propolis also shows the characteristics of immunostimulation and immunomodulation.¹⁴⁸ It has been proved to be an adjuvant in mouse-model vaccination.^149,150 Soudi et al found that the recombinant OipA vaccine with propolis as adjuvant can induce more IFNγ and induce stronger cellular immunoresponse than the recombinant OipA vaccine without adjuvant.⁸²

Chemically Synthesized Adjuvants

An important intracellular signal molecule of bacteria, 3’,5’-cyclic diguanylic acid (c-di-GMP) plays an important role in bacterial movement, adhesion, and virulence, so it has been considered an effective immunostimulator and a useful mucosal adjuvant.^151,152 Introduction of fluorine into therapeutic agents has been well recognized as a useful modification to modulate pharmacological properties.¹⁵³ The chemically synthesized bis-(3’-5’)-cyclic dimeric 2’-deoxy-2’-fluoroguanosine monophosphate (20-F-c-di-GMP) as an adjuvant in combination with the ultrasonic extract of H. pylori can induce the production of antigen-specific antibodies in mice and reduce the colonization of H. pylori in the stomach of immunized mice.¹⁵⁴

Vaccine-Delivery Vectors

Bacterial Carrier-Delivery System

Live bacteria, including attenuated bacteria and probiotics, can be modified into delivery systems for delivering target antigens. The advantage of these live bacterial vaccine vectors is that they stimulate long-lasting humoral and cellular immunity.¹⁵⁵ Pathogens such as bacteria and viruses are usually pathogenic to humans, but their pathogenic genes can be mutated by chemical or molecular biological techniques, which can reduce their toxicity while maintaining their invasiveness to the mucosa. The application of bacterial delivery systems in H. pylori vaccine will be briefly described below.

Lactic Acid Bacteria

Lactic acid bacteria (LAB) are nonpathogenic, non-colonized, and Gram-positive and rated Generally Recognized as Safe (GRAS) by the US Food and Drug Administration.¹⁵⁶ Target proteins delivered by LAB via mucosal routes can successfully induce systemic humoral immunoresponses.¹⁵⁷ Recently, there have also been some new developments in LAB as vaccine carriers. Recombinant Lb. acidophilus expressing H. pylori adhesin Hp0410 can induce high levels of mucosal SIgA antibody and enhance the effect of mice against H. pylori infection.¹⁵⁸ Gou et al designed an M cell–targeting L. lactis surface display system — plSAM. The system can successfully deliver recombinant antigen, promote the phagocytosis and transport of antigens by M cells in the gastrointestinal tract, and induce protective immunoresponse.¹⁵⁹ Likewise, recombinant L. lactis MG1363 carrying antigen CagL can induce antigen-specific antibodies in mice.⁵⁴

Saccharomyces cerevisiae

Saccharomyces cerevisiae is a single-celled eukaryotic microorganism with a size of about 5–10 μm that was awarded GRAS organism status by the US Food and Drug Administration.¹⁶⁰ S. cerevisiae has achieved results in the vaccine research on diverse viruses, bacteria, parasites, cancers.¹⁶¹ In H. pylori vaccine development, researchers applied S. cerevisiae to expressing recombinant UreB and VacA, and gained an oral vaccine against H. pylori, which showed significant humoral and mucosal immunoresponses and significantly reduced the colonization of H. pylori after vaccination in mice.¹⁶²

Listeria monocytogenes

Listeria monocytogenes is a Gram-positive, facultative anaerobe with a broad host spectrum, and is considered as a potential vaccine vector that can induce a wide range of immunoresponses.¹⁶³ With L. monocytogenes as the vaccine carrier, the attenuated live vaccine containing multiple epitope chimeric antigens (MECU) of H. pylori has been constructed. The results indicated that the vaccine can stably express and secrete MECU. After intragastric and intravenous immunization, it can significantly reduce the colonization of H. pylori and induce a high level of anti-H. pylori antibody.¹⁵⁵

Shigella

Shigella, also known as Shigella dysenteriae, is a non-capsular, non-athletic filamentum that is highly contagious. Noninvasive Shigella strains without plasmid invasion genes may be used as carriers of oral vaccines for short-term immunization.¹⁶⁴ Oral attenuated Shigella vector vaccine expressing the H. pylori fusion protein rUreB–HspA and subcutaneous injection of rUreB–HspA protein can induce antigen-specific serum immunoresponse, mucosal SIgA immunoresponse, and T-cell immunoresponse.¹⁶⁵

Nanometer Material-Delivery Systems

As the protein degrades after the oral vaccine enters the gastrointestinal tract, the antigen-uptake rate may be low, which leads to the oral vaccine having no immunogenicity or weak immunogenicity.¹⁶⁶ NP-delivery systems are increasingly used in vaccine design to overcome the weak immunogenicity of oral vaccines.¹⁶⁷ Some NPs themselves have adjuvant properties, which can improve vaccine efficacy.¹⁶⁸ D,L-lactide-co-glycolic acid (PLGA) NPs are qualified vaccine-delivery devices and drug controlled-release systems that have been widely used in vaccine delivery.¹⁶⁹ HP55 is a special type of enteric coating agent that can prevent drugs from being degraded by gastric acid.¹⁶⁶ PLGA-NPs can be modified with HP55 to obtain HP55/PLGA NPs, which are acid-resistant with excellent stability.¹⁶⁶ Tan et al encapsulated the H. pylori recombinant antigen in acid-resistant HP55/PLGA NPs, which protected the recombinant H. pylori antigen against the complex gastrointestinal environment and improved the protective effect of the recombinant vaccine.¹⁶⁶ As a kind of biocompatible carrier, solid-lipid NPs have been widely used in delivering siRNA and plasmid DNA in vitro.^170–172 MPL-A is an effective TLR4 agonist and an attenuated analogue of lipopolysaccharide that has been used as vaccine adjuvant in some studies.^173,174 SLN-A nanoliposome is made by mixing the adjuvant MPL-A into SLNs, which can effectively express recombinant H. pylori antigen UreA in mouse immune cells, stimulate human macrophage-like cells to express proinflammatory cytokine TNFα, and make cells absorb particles and localize in the intracellular cavity within 3 hours.¹⁶⁷

Measles Virus

MV is a pure human pathogen and an envelope virus belonging to the genus Morbillivirus of the paramyxovirus family.¹⁷⁵ MV has two surface glycoproteins — hemagglutinin (H) and fusion protein (F) — which are responsible for virus attachment and entry into host cells.¹¹³ MV is considered a promising carrier platform for vaccine development, owing to its stability, low cost, and high security.¹⁷⁶ Iankov et al constructed a recombinant MV Edmonston vaccine strain expressing HspA antigen of H. pylori. The results proved that the recombinant MV-HspA strain has strong antitumor activity and strong immunogenicity to H. pylori HspA antigen.¹¹³

Plant Expression System

Transgenic plants are used in the production of biological products and vaccines, and have been widely used in the development of edible vaccines.¹⁷⁷ Leafy crops, including vegetables, are considered suitable candidates for recombinant vaccines.¹⁷⁸

Outer-Membrane Vesicles

Outer-membrane vesicles (OMVs) are a spherical bilayer membrane structure naturally released from the outer membrane of Gram-negative bacteria with the participation of the VacJ/Yrb ATP-binding cassette xtrasport system.^179,180 The main components are periplasmic proteins, toxins, outer-membrane proteins, and lipids.^179,181 OMVs have been proved to play a significant role in the interaction between host and pathogen.¹⁸² Due to their nanometer size, good plasticity, and safety, OMVs have been described as a promising antigen-delivery system.^183,184 Chen et al successfully transported heterologous proteins to vesicles by fusing the natural bacterial protein ClyA with heterologous proteins.¹⁸³ Recently, Liu et al proved that oral administration of H. pylori OMVs can induce strong humoral immunoresponse and significant mucosal immunoresponse in mice.¹⁸⁵

Clinical Trials of H. pylori Vaccines

Currently, most candidate vaccines are in the early stage. According to reports, there are more than 10 H. pylori vaccines that have completed clinical trials or are undergoing clinical trials. A research summary of H. pylori vaccines entering the clinical experiment stage is shown in Table 1.

Table 1 List of vaccines and completed clinical trials

Antigen(s)	Adjuvant(s)	Delivery system	Route	Stage	Results	References
Urease	/	/	Oral	1	Good tolerance; cannot eradicate H. pylori infection.	Kreiss et al (1996)^Citation14
Urease	LT	/	Oral	1	Good tolerance and immunogenicity; cannot eradicate H. pylori infection.	Michetti et al (1999)^Citation15
UreA and UreB	/	PhoP/phoQ-deleted S. enterica Typhi	Oral	1	Cannot eradicate H. pylori infection.	DiPetrillo et al (1999)^Citation16
Urease	/	PhoP/phoQ deleted S. enterica Typhi	Oral	1	50% of vaccinated people developed a detectable immunoresponse to H. pylori urease.	Angelakopoulos et al (2000)^Citation17
Formalin-inactivated H. pylori whole-cell (HWC) vaccine	LTR192G	/	Oral	1	Immunogenic to humans; cannot eradicate H. pylori infection.	Kotloff et al (2001)^Citation18
Urease	/	S. enterica Typhi Ty21a (pDB1)	Oral	1	Three vaccinators showed T-cell response to H. pylori urease; no humoral response.	Bumann et al (2001)^Citation19
Urease	LT	/	Rectally	1	Rectal delivery of 5 μg LT is not only well tolerated, but can induce vigorous immunoresponses against LT.	Sougioultzis et al (2002)^Citation20
Urease	LT	/	Oral	1	Safe and immunogenic to humans; enteric coated urease may have more immunogenicity than soluble urease.	Banerjee et al (2002)^Citation21
UreA, UreB	/	S. enterica Typhi Ty21a (pDB1)	Oral	1	56% (five of nine) of vaccinators showed a cellular immunoresponse to urease.	Metzger et al (2004)^Citation22
UreA, UreB	/	S. typhi Ty21a	Oral	1	Good tolerance; cannot eradicate H. pylori infection.	Aebischer et al (2008)^Citation23
VacA, CagA, NAP	Aluminium hydroxide	/	IM	1	Safe and immunogenic; can produce specific antibodies and T-cell response.	Malfertheiner et al (2008)^Citation24
UreB	LTB	/	Oral	3	Effective, safe and immunogenic; can provide continuous protection against H. pylori infection for up to 3 years.	Zeng et al (2015)^Citation25
VacA, CagA, NAP	Aluminium hydroxide	/	IM	1/2	Systemic humoral response increases; cannot eradicate H. pylori infection.	Malfertheiner et al (2018)^Citation26

Among these candidate vaccines that have entered the clinical research stage, few have shown satisfactory immunization results. The earliest clinical trial, by Kreiss et al in 1996, showed that all volunteers were still infected with H. pylori after oral administration of recombinant H. pylori urease vaccine.¹⁴ Later, researchers speculated that adding an effective adjuvant might significantly enhance the immunoeffect. Michetti et al added LT to urease as an immunoadjuvant, and oral administration of this vaccine significantly reduced the density of H. pylori in the stomach, but could not eradicate H. pylori infection.¹⁵ These results mean more powerful or extensive antigens may be needed to participate in immunization. Banerjee et al proved that enteric coated urease is safe and may have more immunogenicity than soluble urease.²¹ Rectal administration of LT has a reliable safety profile, but immunoresponse to the target antigen urease is very poor and does not improve with increased LT dose. This may be due to urease degradation in the rectum, improper selection of urease dose levels, failure of the basic immunomechanism to coordinate the appropriate response, or inadequate antigen presentation and lack of appropriate T cells to help or induce tolerance.²⁰ A recombinant H. pylori oral vaccine combined with UreB and LTB has completed phase III clinical trials. It can significantly reduce the incidence of H. pylori infection in children and is fairly safe. One year after vaccination, the natural acquisition rate of H. pylori decreased by 71.8% (95% CI 48.2%–85.6%),²⁵ but the protection rate had dropped to 55% 2 years later. Unfortunately, the development of this vaccine has stopped.

A series of clinical trials have also been carried out on the use of S. enterica Typhi as a vaccine-delivery carrier. PhoP/phoQ-deleted S. enterica Typhi Ty800 vaccine strains expressing UreA and UreB of H. pylori could not stimulate humoral or mucosal immunoresponse to urease. The reason may be that the amount of antigen inoculation is small and needs to be taken orally many times. Angelakopoulos et al thought that enhanced plasmid stability can prolong antigen presentation and enhance immunogenicity. A more highly attenuated S. enterica serovar Typhi strain given at larger doses could result in fewer adverse events and more consistent and vigorous immunoresponses to urease. This may be related to the enhancement of plasmid stability and the prolongation of intestinal colonization time.¹⁷ Bumamn et al speculated that the reason that S. enterica serovar Typhi Ty21a expressing H. pylori urease could not stimulate humoral immunoresponse to urease may be related to the low expression of heterologous antigen, and also the good immunoresponse of bacterial carrier itself may make the weak humoral or mucosal response of antigen ignored.¹⁹ Follow-up clinical trials further proved that attenuated S. enterica Typhi vaccine did not show a satisfactory protective effect,²² so Aebischer et al proposed that prime–boost regimen combined with live vaccine and other vaccine preparations may be more effective.²³

Malfertheiner et al designed an H. pylori vaccine containing three antigens (VacA, CagA, and NAP) associated with H. pylori virulence and aluminium hydroxide adjuvant. The vaccine had good tolerance and strong immunogenicity, but did not provide additional protection against H. pylori infection.²⁴ H. pylori colonization usually occurs in early childhood, and the immune system of adults is more likely to eliminate H. pylori spontaneously than children. Therefore, the challenge experiment may be more suitable for children. The research on whole-cell inactivated vaccine of H. pylori has also been going on for some time. Oral formalin inactivated whole-cell vaccine (HWC) of H. pylori can stimulate mucosal and systemic immunoresponse to H. pylori antigen in humans, but there is no evidence that vaccination can eradicate H. pylori in infected volunteers.¹⁸

Discussion

Reasons for the Lack of Clinical Trials on H. pylori Vaccine

Although putative H. pylori vaccines have been studied for 30 years, none has been put on the market, and most of the published clinical trials have ended at phase I. There are many reasons for this phenomenon, which are discussed below.

Immunotolerance Mechanism of H. pylori

T-regulatory (Treg) cells can maintain the benign interaction between H. pylori and host to some extent.¹⁸⁶ Treg-cell response can be observed after H. pylori infection, and can drive immunotolerance and inhibit the activity of Th1 and Th17 cells.¹⁸⁷ Therefore, the immunoregulation and -tolerance mechanism caused indirectly by the activation of Treg cells may ensure the continuous colonization of H. pylori.

Genetic Diversity of H. pylori

The high spontaneous mutation rate and recombination frequency of the genome leads to 20%–30% genomic variation among different H. pylori strains.⁴ One study showed that not only were H. pylori isolates from different individuals highly polymorphic but that variation in H. pylori strains was observed in the same host.¹⁸⁸

Presence of H. pylori in Cells

It has been reported that after extracellular H. pylori is cleared by gentamicin, the bacteria will re-gather in the extracellular environment, indicating that H. pylori may be a facultative intracellular bacteria that can exist and hide in the cells.¹⁸⁹ H. pylori can exist in the gastric lamina propria, gastric epithelial cells, and immune cells of patients with gastric diseases.^190–192 The number of H. pylori cells is often more than the number of phagocytes, resulting in the persistence of H. pylori in gastric epithelial cells.¹⁹³ All in all, the presence of H. pylori in cells may be one of the reasons for the persistence of bacteria in the body.

Limitations of Mouse Model

Although it must be admitted that the mouse H. pylori model has some value for the preliminary evaluation of the immunoefficacy of candidate vaccines, it has limitations, because mice are not natural hosts of H. pylori.¹⁹⁴ Therefore, the immunoeffect observed in mouse models may not reflect the level of protection of the vaccine in humans.⁴ Studies have shown that H. pylori carried by captive rhesus monkeys is the same as that found in humans, so it may indicate that large animals such as rhesus monkeys are more suitable for vaccine efficacy testing than mice.^195,196

Lack of Attention and Investment

Despite the high coverage of H. pylori and the huge burden of disease, as far as we know, there are no large biological companies that have plans to develop H. pylori vaccines. The urgency of vaccine demand, public attention, and capital investment may also affect the speed of the vaccine reaching the market. From this point of view, an H. pylori vaccine is different from other much-needed infectious disease vaccines (such as COVID-19 vaccines). These reasons can lead to poor immunoeffect of vaccines, low efficiency of vaccine development, and difficulty in marketing a vaccine, so researchers need to overcome these difficulties in the future.

Improvement of Vaccine-Design Strategy

Many studies have shown that immunity against H. pylori induced by a single antigen does not produce effective protection, and effective immunity against H. pylori infection is often achieved by the combination of various antigens.¹⁹⁷ Therefore, many research groups are committed to developing H. pylori vaccines based on multiple antigens. Epitope-based vaccines are more cost-effective than mixed proteins and can cover more protein targets, so multiepitope vaccines receive more attention.⁹¹

Clinical studies have proved that most of the adverse reactions in tested populations are caused by adjuvants,^15,21 so it is necessary to explore an a toxic or low-toxicicty but effective adjuvant. Aluminum hydroxide and LT are widely used adjuvants of H. pylori vaccine in clinical research, but aluminum hydroxide does not enhance cellular immunity. It is well known that LT is toxic, so some studies choose to reduce the dosage of LT to reduce the toxic side effects and maintain the adjuvant activity. Some studies have also proposed using LT mutants, such as double-mutant LT,¹⁹⁸ but the production of mutant toxins requires sophisticated gene manipulation. Cytokines such as IL18, IL17A, and IL22 as molecular adjuvants can improve the efficacy of DNA vaccine.^146,147 α-GalCer as a mucosal adjuvant has been proved to be as effective as the standard adjuvant cholera toxin.^145,199 cGAMP can enhance antigen-specific cellular immunoresponse and is a promising molecular adjuvant.^133,200 When CpG is used as an adjuvant of H. pylori vaccine, it can induce biased Th1 immunoresponse.⁴⁸ Fluorinated c-di-GMP analogues have been shown to be excellent adjuvants both intranasally and orally.¹⁵⁴ However, the safety and efficacy of these adjuvants in humans need to be evaluated. In addition, some PPSs and propolis are considered candidates for a low-toxicity H. pylori vaccine adjuvant because of their safety.^{40,82,136,141} However, the related immunostimulation mechanism of PPSs needs further research. Propolis can indeed improve the effectiveness of the vaccine to a certain extent, but it can be used as adjuvant only after its chemical composition is analyzed and its quality-control process checked. OMV is more effective than cholera toxin adjuvant in eradicating H. pylori infection, and compared with toxic adjuvants such as CpG, OMV has the advantage of natural atoxicity, but the production of OMV requires certain technology, so its application is limited. These findings provide more ideas and innovations for the development of vaccine adjuvants in the future.

Live pathogens cannot only be used as vaccine vectors but also stimulate long-lasting humoral and cellular immunity. Although the use of live vectors for antigen delivery is a promising method, the stability of antigen-delivery vectors is an important variable of human response to carrier antigens. Prolonging the intestinal colonization time of live vaccine allows for longer antigen transmission,¹⁷ and studies have shown that some antigens may be more immunogenic when secreted from S. enterica Typhi than expressed in cells, because the transmission time of the surface antigen may be longer than that of the plasma antigen.¹⁶ The expression level of heterologous antigen in S. enterica Typhi may play a vital part in the strength of immunoresponse, and less antigen expression may mean more regular vaccination.

Nanometer material-delivery systems have good tolerance and biocompatibility, can prolong antigen contact time, and overcome the shortcomings of weak immunogenicity of oral vaccines. Therefore, the nanopreparation-delivery system has been used by researchers to treat a variety of diseases, including H. pylori-related diseases.^166,167 The shape and size of NPs usually change with the loading of antigen, and the stability of NPs is usually independent of temperature, but related to time, so improving the uniformity and stability of NPs is a direction for future study. At the same time, accurate control of antigen release and cell targeting should also be paid attention.

Prospects

An effective H. pylori vaccine for human should not only be effective and safe but also needs good patient compliance and a long period of protection. With regard to candidate antigens, subsequent scientific research can be devoted to the development of multiepitope vaccines with multiple targets, and some advanced modern immunoinformatics methods can also be used in the design of multiepitope vaccines. Due to a variety of side effects of adjuvants, the safety of adjuvants has always been an issue of concern to researchers. Excellent adjuvants should be stable, cheap, easy to prepare, without unacceptable side effects, and compatible with vaccine ingredients. We should continue to develop safe and atoxic adjuvants to improve the immunoeffect of the vaccine.

Many live-vaccine bacterial vectors have the problem of low expression, and secretory antigens may allow longer antigen transmission than cytoplasmic antigens, so maybe we can try to explore a vector system that can efficiently secrete and express foreign antigens. NPs are also a promising antigen protection strategy, but the uniformity and stability of NPs need to be further optimized. MV-attenuated strains are also a good vaccine platform for expressing bacterial protective antigens that can not only induce anti-MV immunoresponse but also target H. pylori antigen, but the safety and efficacy of this vaccine in humans need to be further studied.

Many studies on H. pylori vaccine have also revealed problems in the dosage form of the vaccine. H. pylori infection usually occurs in early childhood, so vaccine development should be targeted more at children. We also need to consider children’s medication, such as the oral dose of the vaccine not being too high. Otherwise, it is difficult for children to take. Therefore, it is necessary for us to study other better formulations or more convenient vaccination methods. We may be able to develop vaccines with higher antigen loads to solve the problem of high oral doses. In addition to oral and intravenous injections, nasal and rectal administration have been shown to be at least as effective as oral administration. Oral enhanced vaccination after parenteral injection may be a more effective scheme.²⁰

In conclusion, through the efforts of researchers in recent years, some discoveries have been made in the screening and discovery of candidate antigens. The exploration of delivery systems and adjuvants provides novel ideas for the development of an H. pylori vaccine. However, the vast majority of H. pylori vaccine research is still in the early stages. The development of an H. pylori vaccine that can be used in humans still needs much exploration and effort. We need to know more about the mechanism of immunosuppression in order to better design vaccine strategies, and exploring the optimal combination of antigens, adjuvants, and delivery carriers may be the key to vaccine design. In addition, we believe there is a need to raise awareness of H. pylori infection in low- and middle-income countries to scale up the introduction of talent and funding so as to better promote the development of an H. pylori vaccine.

Disclosure

Songhui Li and Wenfeng Zhao are-co-first authors for this study. Lingyi Kong and Lei Yang are co-correspondence authors for this study. Lei Xia is affiliated with Bloomage Biotechnology Corporation Limited. The authors report no other conflicts of interest in this work.