Mucosal Lactobacillus vectored vaccines

Abstract

Traditional non-gastrointestinal vaccines can prevent effectively the invasion of pathogens; however, these vaccines are less effective against mucosal infections because there is not a sufficient immune response at the mucosa. Most pathogens invade via a mucosal pathway (oral, intranasal, or vaginal).¹ It is widely accepted that Lactobacillus species play a critical role as commensals in the gastrointestinal (GI) tract.² Their ability to survive in the digestive tract, their close association with the intestinal epithelium, their immunomodulatory properties and their safety even when consumed in large amounts make lactobacilli attractive candidates for live vehicles for the delivery of immunogens to the intestinal mucosa.³ The oral or intranasal administration of Lactobacillus-based vaccines is a promising method to control mucosal infection because these vaccines could induce strong humoral and cellular immune responses both in the blood and at mucosal sites.

Overcoming Harsh Conditions Using Lactobacillus Isolated From Mucosa to Enhance the Ability of the Bacteria to Adhere to the Epithelium

Lactobacillus and Lactococcus lactis are both GRAS (generally regarded as safe) bacteria that are widely used in the food industry and as a platform for the delivery of vaccines, especially via mucosal routes. However, the vaccination efficacy varies significantly depending on a variety of factors, including the route of administration, the expressed antigen and the lactic acid bacterium used. Some Lactococcus lactis strains survive poorly in the harsh physicochemical environment of the stomach and do not produce a sufficient amount of antigen.¹ Some Lactococcus lactis live in the gastrointestinal tract for only a short time and thus cannot induce a sufficient mucosal immune response. The selection of a Lactobacillus strain with good stress resistance properties and the ability to adhere will to the epithelium is important to achieve good immunoprotection at mucosal sites. Bacteria derived from the indigenous gut flora of termites are well adapted to the living conditions and selective pressures in the gut of termites.⁴ Lactobacilli isolated from human or animal gut should also be well adapted to the conditions in the intestines of the original host. The future of using lactobacilli as carriers of heterogenetic antigens for oral vaccination is promising.

Two recombinant strains, Lactobacillus acidophilus ATCC 4356 (LA4356-pH) and Lactobacillus delbrueckii subsp lactis D17 (DLD17-pH), both of which express hemagglutinin 1 (HA1) of HPAI virus, were successfully constructed. Following oral administration to BALB/c mice, DLD17-pH was more effective than DLD17-pH at inducing systemic and mucosal immune responses, with higher anti-HA1-specific IgA and IgG levels.⁵ DLD17, isolated from the gut of a chicken, is well adapted to the conditions in the intestines and can colonize the digestive tract.⁶ Therefore, the recombinant strain DLD17-pH would likely survive better in chicken intestines and should be able to stimulate a persistent intestinal mucosal immune response. LA4356 originated from the human pharynx, and thus, the adhesion to the chicken intestinal epithelium may be weak, resulting in a weaker mucosal immune response.

Lactobacillus species isolated from the vaginal mucosa of women are efficient colonizers of the vaginal mucosa and likely exist in a natural “biofilm” composed of bacteria and extracellular matrix materials.^7-9 Recombinant Lactobacillus expressing two-domain CD4 (2D CD4) proteins can inhibit HIV-1 entry into target cells in a dose-dependent manner.¹⁰ Lactobacillus jensenii isolated from vaginal mucosa was also used to express the potent HIV inhibitor cyanovirin-N (CV-N). This bacterial strain was capable of colonizing the vagina and producing full-length CV-N when administered intravaginally to mice during estrus.¹¹ These studies represent a major step toward the development of engineered commensal bacteria that can survive within the vaginal microflora and inhibit the heterosexual transmission of HIV.

Enhancing the immune efficiency of Lactobacillus vaccine vectors

To elicit a strong mucosal immune response against pathogens, several strategies have been used to genetically modify Lactobacillus, including the coexpression of antigens and bacterial toxins, dendritic cell (DC) target proteins or microfold cells (M cell) target proteins.

The intragastric immunization of mice with recombinant Lactobacillus expressing HIV-1 Gag fused with Salmonella enterica serovar Typhimurium flagellin (FliC) resulted in the strong activation of TLR5 and induced an increase in number of Gag-specific IgA-secreting cells in the local mucosa, indicating that the presence of FliC conferred an adjuvant effect.^12,13 Compared with mice orogastrically immunized with L. casei expressing the core neutralizing epitope (COE) region of porcine epidemic diarrhea virus (PEDV), mice immunized with recombinant Lactobacillus expressing Escherichia coli heat-labile enterotoxin B (LTB) fused with COE produced higher levels of sIgA, IgG, IL-4 and IFN-γ and had a greater ability to neutralize PEDV, indicating that Lactobacillus expressing LTB-COE more effectively induced local and systematic immune responses. Therefore, this bacterium is a better candidate for a live bacterial vaccine against PEDV infection.¹⁴ LTB was also fused with the protective antigen of porcine rotavirus, VP4, and expressed in recombinant L. casei. These bacteria were used to orally immunize mice, which produced higher levels of mucosal IgA than mice immunized with L. casei expressing VP4.¹⁵

The ability of M cells in Peyer's patches to take up and transcytose antigens to DCs has made M cells an ideal target for vaccine delivery to the mucosal immune system.^16,17 Targeting specific receptors on the apical surface of M cells may promote an increase in the uptake and presentation of antigens, consequently initiating the immune response and inducing protection against an infectious challenge.¹⁸ In addition to M cells, professional antigen-presenting DCs located in or beneath the epithelium can sample and capture various bacterial antigens that cross the epithelial layer through M cells.^19,20 Lactobacillus expressing antigens fused to DC-binding peptide may enhance the rate and efficiency of uptake by DCs, thus inducing an effective immune response. This novel and highly innovative vaccine strategy has various benefits, including the specific activation of DCs, the directional elicitation of humoral and T cell-mediated immunity by these cells, and the use of a delivery system that can serve as a safe and potent adjuvant.²¹ Greatly increased immunity was achieved when the antigen expressed by the recombinant Lactobacillus was fused to a dendritic cell (DC)-targeting peptide. The oral administration of Lactobacillus acidophilus NCFM expressing the DC-targeting peptide fused to the Bacillus anthracis protective antigen provided 100% protection of mice against anthrax and was significantly better than simple antigen delivery.²² The oral administration to mice of L. gasseri expressing the B. anthracis protective antigen (PA) genetically fused to a DC-binding peptide (DCpep) elicited robust PA-neutralizing antibody and T-cell mediated immune responses against anthrax Sterne challenge, resulting in the survival of all animal.²³

Mode of Antigen Presentation by Lactobacillus Vaccine Vectors

The presentation of antigens by Lactobacillus can be divided into three types: (1) cytoplasmic, which allows the protein to escape digestion in the gastrointestinal tract but requires cellular lysis for protein release and delivery; (2) secreted, in which the expressed antigen is secreted into the intestinal tract and contacts the mucosal epithelium directly; and (3) cell surface associated, in which the expressed protein is anchored to the cell membrane to prevent proteolytic degradation.^24,25 The protective immune response depends not only on the mode of antigen presentation but also on the Lactobacillus strain, amount of antigen produces and the vaccination route.

The oral administration of recombinant L. lactis expressing VP4 of rotavirus (cell surface associated) or VP7 (secreted) could elicit viral neutralizing antibodies.^26,27 L. lactis expressing the S protein of transmissible gastroenteritis virus in two ways (cell surface associated or secreted) has been used to orally vaccinate mice, and both local mucosal (intestinal IgA) and systemic immune (serum IgG) responses against TGEV were induced.^28,29 Helicobacter pylori urease subunit B (UreB) has been expressed in both L. lactis (secreted) and L. plantarum (cytoplasmic), which were orally administered to mice.^30,31 Serum IgG and IgA were detected after vaccination with recombinant L. plantarum (cytoplasmic), which caused a reduction in the Helicobacter felis load following an intragastric H. felis challenge.³⁰ The recombinant L. lactis (secreted) could induce protection against intragastric H. pylori challenge by eliciting the production of IgG in the serum and IgA in the feces.³¹

Related studies have demonstrated that compared with intracellular antigens, exported antigens (those secreted into the medium or anchored to the cell surface) induce stronger immune responses, a difference that can be attributed to the direct contact between the antigen and the immune system.^24,32 However, exported antigens are more susceptible to the proteolytic degradation when they are exposed to the host’s body fluids.³³

Conclusion

The next generation of mucosal vaccines should ideally be administered in a single, tolerable, efficacious dose that induces robust neutralizing humoral and cellular immunity against specific microbial pathogens. Lactobacillus strains are widely used for the manufacturing of foodstuffs and have been consumed in large amounts by humans for thousands of years without causing any known health problems. Therefore, these bacteria are classified as “generally regarded as safe” (GRAS) organisms. Safe and highly efficient vaccines based on lactobacilli are an attractive alternative to induce mucosal immune responses to protection against infective agents. During the past decade, recombinant lactobacilli have been successfully engineered to produce a wide variety of antigens from microorganisms and have proven to be effective. Lactobacillus strains maybe considered promising vehicles not only for antigens but also for biologically active compounds such as immunomodulators, antibodies, enzymes and peptides.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.