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Review

Probiotic-Based Vaccines May Provide Effective Protection against COVID-19 Acute Respiratory Disease

by
Sedigheh Taghinezhad-S
1,
Amir Hossein Mohseni
1,*,
Luis G. Bermúdez-Humarán
2,
Vincenzo Casolaro
3,
Naima G. Cortes-Perez
4,
Hossein Keyvani
5 and
Jesus Simal-Gandara
6
1
Department of Microbiology, Faculty of Basic Sciences, Science and Research Branch, Islamic Azad University, Tehran 1477893855, Iran
2
Université Paris-Saclay, INRAE, AgroParisTech, Micalis Institute, 78350 Jouy-en-Josas, France
3
Department of Medicine, Surgery and Dentistry “Scuola Medica Salernitana”, University of Salerno, Baronissi, 84081 Salerno, Italy
4
Université Paris-Saclay, INRAE, AgroParisTech, UMR 0496, 78350 Jouy-en-Josas, France
5
Department of Virology, Faculty of Medicine, Iran University of Medical Sciences, Tehran 1449614535, Iran
6
Nutrition and Bromatology Group, Department of Analytical Chemistry and Food Science, Faculty of Food Science and Technology, University of Vigo—Ourense Campus, E32004 Ourense, Spain
*
Author to whom correspondence should be addressed.
Vaccines 2021, 9(5), 466; https://doi.org/10.3390/vaccines9050466
Submission received: 8 March 2021 / Revised: 29 March 2021 / Accepted: 24 April 2021 / Published: 6 May 2021
(This article belongs to the Section COVID-19 Vaccines and Vaccination)
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Abstract

:
Severe acute respiratory syndrome coronavirus 2 virus (SARS-CoV-2) infection, the causative agent of COVID-19, now represents the sixth Public Health Emergency of International Concern (PHEIC)—as declared by the World Health Organization (WHO) since 2009. Considering that SARS-CoV-2 is mainly transmitted via the mucosal route, a therapy administered by this same route may represent a desirable approach to fight SARS-CoV-2 infection. It is now widely accepted that genetically modified microorganisms, including probiotics, represent attractive vehicles for oral or nasal mucosal delivery of therapeutic molecules. Previous studies have shown that the mucosal administration of therapeutic molecules is able to induce an immune response mediated by specific serum IgG and mucosal IgA antibodies along with mucosal cell-mediated immune responses, which effectively concur to neutralize and eradicate infections. Therefore, advances in the modulation of mucosal immune responses, and in particular the use of probiotics as live delivery vectors, may encourage prospective studies to assess the effectiveness of genetically modified probiotics for SARS-CoV-2 infection. Emerging trends in the ever-progressing field of vaccine development re-emphasize the contribution of adjuvants, along with optimization of codon usage (when designing a synthetic gene), expression level, and inoculation dose to elicit specific and potent protective immune responses. In this review, we will highlight the existing pre-clinical and clinical information on the use of genetically modified microorganisms in control strategies against respiratory and non-respiratory viruses. In addition, we will discuss some controversial aspects of the use of genetically modified probiotics in modulating the cross-talk between mucosal delivery of therapeutics and immune system modulation.

1. Introduction

The end of 2019 was marked with the beginning of the COVID-19 outbreak caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) [1]. As of March 2021, more than 115 million cases and 2.5 million SARS-CoV-2-associated deaths have been reported worldwide (WHO COVID-19 Disease Dashboard, 2020). SARS-CoV-2 is a positive-sense, single stranded RNA virus that replicates in the cytoplasm and encodes several structural and non-structural proteins with a genome size of around 29.9 kb (accession code MN908947) [2]. The high rates of SARS-CoV-2 transmission between humans [3], and the current lack of fast-paced, comprehensive vaccination strategies to contain the spread of this infection, make this pandemic a major international public health concern [4]. Therefore, studies are ongoing to find effective drugs to treat and prevent COVID-19. It is generally accepted that vaccination is the most effective approach to control the spread of SARS-CoV-2 transmission [5].
The four major structural proteins of SARS-COV-2 are the spike (S) glycoprotein, and the nucleocapsid (N), membrane (M) and envelope (E) proteins [6]. The S protein is responsible for attachment to the host cell following recognition of the human angiotensin-converting enzyme 2 (hACE2) receptor, which plays a pivotal role in provoking the immune response during the progression of disease and is targeted by host neutralizing antibodies (Figure 1) [7,8]. Therefore, the S protein serves as a key target in the assessment of SARS-CoV-2-reactive IgG antibodies, as well as the development of COVID-19 vaccines. Additional studies have identified a number of CD4+ and CD8+ T-cell epitopes within the amino acid sequences of the S protein, stressing their potential roles in inducing T-cell responses [9,10,11]. In addition, current studies suggest that the N protein of SARS-CoV-2 is also a suitable candidate for vaccine development given its high potential to trigger both a humoral and a T-cell immune response in humans [12,13].
Unlike the S and N proteins, the M and E proteins are poorly immunogenic and are not targeted by immune responses against coronaviruses, possibly owed to their small ectodomains and small overall molecular sizes [14], as was demonstrated in animal models adoptively transferred with sera from donors vaccinated with a virus vector delivering these proteins [15].
The essential roles of the upper respiratory and/or gastrointestinal tracts as the main routes of SARS-CoV-2 transmission in humans strongly suggest that mucosal delivery of SARS-CoV-2 antigens may represent an attractive and effective strategy for the development of a mucosal vaccine to control COVID-19. Lactic acid bacteria (LAB) are a group of Gram-positive bacteria widely used in industrial food fermentation processes. The most studied genera belonging to LAB are Lactobacillus, Lactococcus, Streptococcus, Enterococcus, and Pediococcus [16]. Thus, these microorganisms are Generally Recognized As Safe by health authorities, earning a GRAS status by the FDA (USA) [17] and a qualified presumption of safety (QPS) by the EFSA (Europe) [18]. When these microorganisms are ingested live in adequate amounts, they can survive in the host digestive tract, where they are likely to exert various beneficial actions on the host, an effect known as probiotic [19,20]. In addition, some genera, such as lactobacilli, are commensal bacteria and considered thus as part of the intestinal microbiota [21].
The ability of these microorganisms to survive and colonize the host mucosal surfaces and their immunomodulatory capabilities (i.e., probiotic effects) make them promising vehicles for the delivery of heterologous antigens via mucosal routes [22,23,24,25]. In addition, these vectors, which are easier and safer to administer and cheaper to produce than traditional, injectable vaccines, could be well suited to mass vaccination campaigns in developing countries [26,27,28]. Over the last two decades, research on the use of LAB as live vectors in the development of mucosal vaccines has focused on the construction of genetically modified (or recombinant), strains of the species: Lactobacillus plantarum, Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus delbrueckii, and Lactococcus lactis able to produce numerous respiratory and non-respiratory virus-derived antigens (among others) (Table 1). Based on these pioneering studies, we can envisage that recombinant LAB-based vaccines may be an attractive option to deliver SARS-CoV-2 antigens to mucosal surfaces and evoke a protective immune response. However, despite numerous reports on the use of recombinant LAB and their demonstrated potential in inducing mucosal immune responses and the prevention of respiratory viral infections, to our knowledge no studies to date have explored the use of recombinant probiotics in the development of vaccines to treat SARS-CoV-2 infections and help control the COVID-19 outbreak. Certainly, more research is needed to demonstrate the full potential of recombinant probiotics. Here, we will discuss the potential antiviral efficiency of a recombinant probiotics-based vaccine, with a special emphasis on SARS-CoV-2 infection. Our goal is to provide a global overview on the use of recombinant LAB in vaccine development, which includes a full understanding of the mechanisms involved in the crosstalk between mucosally delivered therapeutics and the host immune system. This knowledge will be key in the design of future in vitro and preclinical studies as well as subsequent clinical trials.

2. Recombinant Probiotics as Inducers of Humoral Immune Responses

Mucosal vaccination is advocated in several extensive studies as one of the most effective approaches to control and prevent respiratory viral infections [28,29,30]. In view of the fact that the mucosal surfaces of the respiratory tract are the major portal of entry and initiation of respiratory viral infections, it would be desirable to design a vaccine able to elicit specific functions of the mucosal immune system, such as the production of secretory IgA (sIgA) dimers. sIgA are a critical component of mucosal immunity in the respiratory tract, in that they can inhibit the entrance and proliferation of respiratory viruses in the airway mucosa [69,70]. In fact, detailed and in-depth research studies found that sIgA play a more critical role than IgG antibodies in the prevention of viral infections in the upper respiratory tract, including the nose and trachea, due to their ability to decrease virus attachment and avoid virus internalization at the mucosal surfaces [31,71,72]. Besides their role in the prevention of infection, recent work also suggests a putative role of sIgA in the maintenance of microbiota homeostasis [73]. Therefore, stimulation of mucosal immunity has received particular attention in the development of strategies to fight pathogenic microorganisms. Among the different formulations for mucosal immunization, genetically modified LAB have been explored as effective vehicles for antigen delivery due to their safety. Among these, L. lactis, L. plantarum, and L. casei have received special attention due to their superior effects relative to other LAB strains in comparative studies [32,33,74,75,76].
Several L. plantarum-based model vaccines against viral disease have been constructed and tested in animal studies, such as L. plantarum carrying the hemagglutinin–neuraminidase protein (HN) of Newcastle disease virus (NDV) [31], the hemagglutinin (HA) gene of H9N2 avian influenza virus (AIV) [28,34], and different proteins of influenza virus [33,35,36], all of which effectively inducing measures of mucosal immunity (sIgA) as well as serum IgG antibody responses, indicating an encouraging gut-lung axis for orally administered vaccines to combat respiratory viral infections.
More recently, L. lactis was also extensively used to develop various oral-based mucosal vaccines. In vivo studies showed that oral administration of L. lactis displaying various viral antigens can stimulate robust mucosal and systemic immunity. In addition, several studies have demonstrated that oral immunization with a recombinant probiotic could result in the secretion of sIgA at sites besides the gastrointestinal tract, as these were detected in bronchoalveolar lavage fluids (BALF), ophthalmic and vaginal washings, consistent with acquired resistance to respiratory, gastrointestinal, and genital tract infections [22,29,77,78].
Subsequent studies focused on the induction of mucosal immune responses by another probiotic, L. casei. A surface antigen display system was designed using anchoring matrix such as poly-γ-glutamate synthetase A (pgsA) protein of Bacillus subtilis, which could effectively express different viral antigens at the surface of L. casei, including conserved matrix protein 2 of divergent influenza subtypes [37], HA2 and sM2 influenza antigens [38], N protein of TGEV [39], and HPV-16 L2 protein [40]. Oral and/or nasal administration of these recombinant L. casei preparations in mice resulted in stronger induction of serum IgG as well as sIgA against the displayed antigens. Similar results were observed in mice and other animal models following oral administration of recombinant L. casei harboring major protective antigen VP4 of porcine rotavirus [23], recombinant protein of TGEV [41], or co-expressing epitopes of porcine parvovirus (PPV) and classical swine fever virus (CSFV) [42], indicating an efficient induction of protective immunity against various viral infections. In light of the valuable insights provided by these studies with genetically engineered L. casei carrying viral antigens, more in vivo studies focused on the expression of VP2 protein from infectious pancreatic necrosis virus (IPNV) in recombinant L. casei, which resulted in the stimulation of systemic and local mucosal immune responses, high-level production of IgM and IgT, and reduction of viral load in orally immunized rainbow trouts [26,43].
Three human clinical trials in support of these pre-clinical findings were launched, in which oral immunization of recombinant L. lactis and L. casei carrying HPV-16 antigens induced high levels of specific serum IgG and vaginal IgA in volunteers who completed the vaccination schedules [27,44,45]. Interestingly, oral administration in mice of L. acidophilus carrying Gag antigen from human immunodeficiency virus 1 (HIV-1) [46] or protein HA1 from highly pathogenic avian influenza (HPAI) virus (H5N1) [22] could only stimulate local sIgA production in the digestive tract, while expression of protein HA1 of HPAI virus by recombinant L. delbrueckii subsp. lactis could provoke a mucosal immune response in both the gastrointestinal and the respiratory tract [22]. The first-in-human study of an orally delivered probiotic-based SARS-CoV-2 vaccine, called bacTRL-Spike-1, has been designed by Symvivo Corp. (Melbourne, Victoria, Australia), which makes use of engineered Bifidobacterium longum to deliver plasmids harboring a full-length S protein gene (Figure 2). To functionally characterize the safety, tolerability, and immunogenicity of the vaccine for the prevention of COVID-19 in healthy adults, three different oral doses of live recombinant Bifidobacterium longum, 1 billion (Group 1), 3 billion (Group 2) or 10 billion (Group 3) colony-forming-units (cfu), will be evaluated in subjects 18 years of age and older during a Phase 1, randomized, observer-blind, placebo-controlled trial (NCT number: NCT04334980). The final results of this trial will be made available on 28 February 2022.

3. Recombinant Probiotics as Inducers of Cell-Mediated Immune Responses

In recent years, it has become clear that studies aimed at addressing the induction of T cell-mediated immune responses combined to the humoral immune response could offer a much broader scope of protection against invading pathogens such as respiratory viruses. Major progress has been made to define the potential of recombinant probiotic vaccines in the stimulation of T cell-mediated immune responses in addition to IgG and sIgA production, which, combined, could open up new opportunities in the fight against viral infection [47,79,80]. Therefore, uncovering the ability of recombinant probiotics to elicit T cell-mediated immune responses of proper sign and intensity might help predict their likely impact on the ongoing COVID-19 pandemic.

3.1. T Helper

CD4+ T cells exposed to diverse pathogens can express diverging effector phenotypes, the best studied of which are characterized by predominant production of the cytokines, interferon (IFN)-γ or interleukin (IL)-4 and have been termed T helper (Th) 1 and Th2 cells, respectively. Substantial production of Th2-type (IL-4) and Th1-type (IFN-γ) cytokines was reported in response to mucosal administration of L. casei and L. lactis carrying viral antigens in immunized mice, variably contributing to host defense against viral infection [37,48,49,50]. In agreement with earlier studies, oral vaccination of mice with recombinant Lactobacillus strains and L. lactis expressing viral antigens could induce IL-4 production and provoke a proliferative response of splenic lymphocytes, raising the possibility that common mucosal immunization might stimulate Th2-like cell-mediated immunity [22,51,52]. However, it is well known that IFN-γ, produced by NK cells and T lymphocytes, and tumor necrosis factor (TNF)-α, produced by T lymphocytes and monocytes, play major roles in antiviral immunity. It has long been known that IFN-γ plays an important role in promoting phagocytic activity against viral and bacterial infection and TNF-α is a major mediator of inflammatory responses [34,81]. Production of TNF-α and IFN-γ by Th1 cells has a significant role in the activation antiviral responses via stimulating macrophages and cells associated with cell-mediated cytotoxicity [36,82]. In fact, IFN-γ and TNF-α levels could significantly increase in mice receiving L. plantarum, L. casei, and L. lactis expressing viral genes, implying that recombinant probiotics could modulate adaptive immunity by up-regulating the effector responses of CD8+ T cytotoxic cells and CD4+ Th cells [27,34,48]. Consistent with this, in vivo administration of recombinant L. plantarum expressing influenza virus H9N2 protein resulted in the generation of protective immune responses through the expansion of IFN-γ-expressing CD8+ T cells and Th1 cells [28,34,36] (Figure 3).

3.2. T Killer

Studies in mice orally administered with L. lactis expressing HPV-16 antigens showed the expansion of specific IL-2-secreting CD4+ T cells and IFN-γ-secreting CD8+ T cells in the intestinal mucosa, and of vaginal and splenic lymphocytes, resulting in protective and therapeutic anti-tumoral responses against challenge with an E6/E7-expressing tumor cell line (TC-1) [32,53]. Recent findings also show that expansion of IFN-γ-secreting CD4+ and CD8+ T cells, and stimulation of mucosal Th1 immune responses by recombinant L. casei could elicit confer a substantial level of protection against viral infections in humans [44,54]. Clinical studies found that oral vaccination with recombinant L. lactis containing viral antigens could stimulate production of high amounts of IFN-γ at the intestinal mucosal inductive sites (Peyer’s patches). By contrast, the poor ability of recombinant L. lactis to induce systemic immunity has been documented elsewhere [27,45]. Together, these observations suggest that mucosal T cells stimulated by recombinant L. lactis in the gut, while initially moving to the peripheral circulation, will eventually home and settle in the specific mucosa. In any instance, these studies strongly point to the role of CD8+ cytotoxic T lymphocytes (CTLs) induced by recombinant probiotics in promoting viral clearance. For instance, recombinant L. casei can efficiently stimulate CSFV-specific CD8+ CTL responses to protect pigs against CSFV challenge [42]. This notion is further confirmed in studies showing that recombinant L. plantarum could provoke the expansion of CD8+ CTLs conferring protection and increased survival against lethal influenza virus challenge [36]. Additionally, we found preclinical evidence that recombinant L. casei and L. lactis expressing viral antigens could sustain long-lasting immune responses, which were observed at least 2–6 months after the last vaccine boost [37,38,55]. These data are supported by clinical evidence showing long-term specific CTL responses against HPV-16 during 6-month follow-ups in healthy females, further demonstrating that recombinant LAB can elicit long-lasting immunity against viral pathogens [27,45].

3.3. Dendritic Cells (DCs)

Dendritic cells (DCs) were characterized as the bridge between host innate and adaptive immunity. DCs can efficiently trap self and foreign antigens and present them to naïve T cells in secondary lymphoid tissues. DCs isolated from mucosal tissues and the spleen could favorably stimulate Th2 and Th1 responses, respectively [34,83]. The potential role of DCs in promoting strong cellular immunity toward genetically modified L. lactis and L. plantarum-derived antigens was shown in some in vivo studies. Consistent with this, it was long believed that the oral administration of these strains could induce Peyer’s patch (PP) DCs activation [33,56]. Compelling evidence in animal studies shows DC stimulation in the small intestine mucosa and the mesenteric lymph nodes (MLNs) by probiotic strains, which would support pathogen’s killing. Owing to the distinct ability of DCs to elicit an immune response, DC targeting strategies have received more attention in vaccinology. A specific DC-targeting peptide (DCpep) was utilized in some studies to enhance the robustness of immune responses [31,36]. For example, Wang et al. fused the S gene of SARS-CoV-2 with DCpep and reported the successful expression of recombinant S protein on the surface of L. plantarum [57]. As a result, a significant increase in the percentages of CD4+ T cells was observed in the spleen and peripheral blood of mouse and chicken models after immunization with L. plantarum expressing DCpep fused with viral antigens. In contrast, such a response was not observed in animals immunized with recombinant L. plantarum not expressing DCpep. Along the same line, the few in vivo studies conducted to date showed that L. plantarum expressing viral antigens attached to DCpep could effectively stimulate DCs activation in PPs, MLNs, and the small intestine. These findings indicate the potential usefulness of DCpep fusion antigens to provide an effective immune adjuvant in the development of a mucosal vaccine [36,58,84]. Building on these studies, Jiang et al. have provided evidence of an association between DC activation and the promotion of T-cell differentiation, both contributing to pathogen clearance in animal models [31]. Moreover, probiotics-based vaccines can regulate the elicited immune responses by interacting with Toll-like receptors (TLRs) on macrophages and DCs [85]. As well, one in vivo study indicated that inhibiting TLR expression could be achieved by a Lactobacillus-vaccine; thus neutralization of viruses will occur [41].

4. Optimization of the Immune Response Induced by Recombinant Probiotic-Based Vaccines

Studies performed to date show that heterologous proteins in some recombinant probiotics may be expressed at low levels, likely due to the intrinsic low-copy number of a shuttle vector. Given this limitation, the appearance of weak signals in the subsequent IFA tests and/or Western blot experiments would be the main drawback of recombinant probiotic-based vaccines [24,86]. Attempts have been made to produce higher recombinant protein levels by optimizing nutrients, such as protein or sugar sources, in a fermenter/bioreactor under controlled pH conditions to prevent batch-to-batch variability [87]. In some cases production of recombinant proteins was increased by optimizing the temperature at the induction point to prevent protein degradation in probiotics [48,88,89]. Codon optimization in probiotics such as L. casei and L. lactis has been shown to be an important factor to optimize the translational efficiency of heterologous proteins and dramatically enhance the overall yield of recombinant proteins [90,91,92]. These measures dramatically reduce the number of non-matching genes containing native codons, which enables to select codons corresponding to those of the probiotic hosts which generate a higher level of recombinant expression of the protein [93]. It is documented that recombinant L. lactis harboring codon-optimized oncogenes of HPV-16 had an improved inhibitory effect on tumor size progression and tumor growth, thus resulting in better survival rates in vivo compared to those with native codons [32,52]. Using a similar approach, successful expression of influenza virus genes was reported in L. casei in vitro [24,91]. These optimized responses were postulated to result from substantial enhancement in humoral and cellular immunity elicited after administration of recombinant probiotics.
Data collected over the past decade indicate that optimizing vaccine dose during dose-escalation studies may be considered one of the most important factors to properly stimulate a mucosal immune response in animals and humans [59,94]. This point was supported in a few clinical studies showing that the number of viable colonies (colony-forming units: CFU) of recombinant probiotics correlates with the efficacy of immune responses. Consistently, Mohseni et al. and Taghinezhad et al., in a Phase I, proof-of-concept clinical trial, showed that the induction of humoral and cell-mediated immune responses in volunteers who received 5,000,000,000 CFU/mL of recombinant L. lactis were more robust than in those receiving 1,000,000,000 CFU/mL, a parameter clearly depending on the dose of this strain administered for mucosal immunization [27,45]. Nevertheless, no studies have systematically investigated the impact of dose escalation on the expression of respiratory viral genes in probiotics and the ensuing immune responses to fight off these pathogens. Clearly, additional studies will be needed to confirm this theory.
This information would also assist greatly in interpreting the effects of specific adjuvants on the robustness of protective responses [35]. Concerning this aspect, different adjuvants have been used to properly enhance the immune response to recombinant probiotics. A number of adjuvants have been used in these studies, including heat-labile toxin B subunit (LTB) [23,42], heat-labile toxin LT (R192G/L211A) [60], FliC [46], CTA1 [38], Gram-positive enhancer matrix (GEM) [95,96], AcmA [29], DC-targeting peptides (DCpep) [31], the nontoxic B subunit of cholera toxin (CTB) [36,50], MDP, and tuftsin [41]. In addition, results from other studies suggest that the provision of definite amounts of IL-2, IL-18, IL-1, and IL-10 as adjuvants may further improve the elicited immune response [97,98]. However, it is essential to emphasize that LAB possess inherent adjuvant characteristics, sufficient to properly induce the host immune system thanks to their intrinsic immunomodulatory properties [34,99,100]. The potential adjuvant effects of LAB could be attributed to the systemic release of specific cytokines which stimulate innate immunity [101,102,103]. According to this paradigm, striking results from in vitro and in vivo studies provide evidence that probiotic strains could exert their adjuvant functions by up-regulating DC and Th1 cytokines and down-regulating Th2 activity [104,105]. However, the exact mechanisms mediating these functions are not fully elucidated.

5. Discussion

Experience in the past decades have clearly demonstrated that a shift from traditional needle-based immunization to a needle-free one can overcome a number of limitations, thus accelerating large vaccination programs, particularly in resource-limited developing countries. In particular, studies within this area of investigation have led to discover that mucosal immunization, which entails the delivery of heterologous proteins to mucosal surfaces, is one of the few needle-free approaches that can exert significant prophylactic and therapeutic effects [106,107,108]. Mucosal vaccines have rapidly raised considerable practical and conceptual interest due to their easy administration, low cost, the ability to provoke mucosal, humoral, and systemic immune responses, the negligible risk of blood-borne infections, and the convenient distribution, not requiring a cold chain [109,110,111]. Intensive efforts have been carried out by many groups over the past years to develop mucosal vaccines against an expanding range of pathogens, and their results indicate that delivery of immunogenic molecules to the mucosa via recombinant probiotics administered through nasogastric or orogastric routes is a promising non-invasive way for protection against various infections by improving humoral, mucosal and T-cell-mediated immune responses [44,45,49]. These discoveries sparked a raised level of attention from the scientific community, leading to an ever expanding bulk of studies aimed at defining the best strategies for efficient, high-level expression of heterologous proteins in probiotics to improve the therapeutic effects of probiotic-based vaccines [79,88,89]. Along this line, studies conducted over the last few years have brought substantial insights into the efficacy of prophylactic or therapeutic probiotic-based vaccines against respiratory and non-respiratory viral agents. Vaccines based on LAB, especially L. plantarum, L. casei, and L. lactis, have shown promising beneficial effects, in particular when administered to overcome infections from emerging respiratory viruses, including SARS and influenza viruses [112,113].
Increasing evidence indicates that the cellular localization of viral antigens plays a crucial role in the susceptibility of antigens to environmental control and proper recognition by the immune system [32,114]. Heterologous proteins harbored in recombinant probiotics can be expressed in the cytoplasm, anchored to the cell wall, or secreted. It is documented that the expression on the cell wall can generally stimulate more robust host immune responses following immunization with recombinant probiotics compared to preparations resulting in cytoplasmic or secreted expression [115,116]. Several methods exist for anchoring proteins to probiotics, of which the inclusion of a LPXTG anchor motif and poly-γ-glutamic acid synthetase A (pgsA) have been the most commonly used for producing viral antigens [30,38,40,61]. Theoretically speaking, exposure of bioactive protein molecules on the surface of probiotics could resist harsh conditions such as proteolysis, improve the antigen’s stability, facilitate antigen presentation, and subsequently provide an effective means for eliciting protective immune response, thus ensuring a higher therapeutic efficacy in challenge experiments than intracellular antigens [116,117].
Due to the increasing challenges regarding the safety of probiotic-based vaccines for human health, several studies suggest that a biological containment system represents the best way to prevent the survival of probiotic-based vaccines in the environment outside the host [118,119]. This concept is reinforced by the results obtained in human clinical trials using recombinant probiotic-based vaccines against viruses, which confirm that these vectors have no side effects in humans [27,45,120,121,122]. It is also generally accepted that the use of heat-attenuated probiotic-based vaccines can decrease the spread of antibiotic-resistance genes in humans and in the environment, but this assumption is still awaiting definitive evidence [54,123].
Pre-clinical and clinical studies document that among different routes for mucosal immunization, oral immunization offers several advantages, including facilitated stimulation of gut-associated lymphoid tissue (GALT), enhanced production of anti-viral IgA, effective overall induction of mucosal immune responses, decreased risk of contamination, cost-effectiveness, easy self-administration or administration to animals, and antigen access to a larger mucosal area for a prolonged duration [22,23,27,45,50]. It has been speculated that oral vaccination, compared to the nasal route, can significantly increase DC activation, specific sIgA production, CD8+ T-cell induction, and cross-protection against viral challenge in vivo [36]. In line with this view, in vivo studies showed that oral intake of recombinant LAB can provide higher neutralizing antibody activities compared to intraperitoneal injection [55,62]. Moreover, oral immunization with recombinant Lactobacillus is more effective than the intranasal route in eliciting neutralizing antibodies, including sIgA, in the respiratory tract [124]. It is worth emphasizing that the elicited antibodies in these models exerted potent neutralizing activities against SARS pseudoviruses [30]. By contrast, some of the advantages of intranasal administration relative to oral vaccination would include a reduced frequency of administration, lower inoculation dose, and administration in the same location as the natural infection [106,125]. Using this route to expose immune cells to high concentrations of vaccine would contribute to inhibiting viral colonization in the respiratory tract by effectively inducing sIgA production. In fact, intranasal administration can also induce greater quantities of IgG in serum and of some cytokines in epithelial cells in the lung alveoli than oral administration, thus increasing the speed of the immune and antibody response to viral antigens. These elements may therefore lead to conclude that intranasal inoculation could be a more efficient route for mucosal immunization [35,37,38]. In the end, it is generally accepted that oral immunization could be particularly beneficial for extensive immunization of farm animals where the recombinant probiotic could be administered in drinking water or food. Conversely, since the main infection route of respiratory viruses is nasal, and nasal immunization is more likely to induce high titers of specific antibody titers (mainly sIgA), it is currently reasonable to opt for nasal administration of recombinant probiotics to combat respiratory viral infections.
In the face of extensive studies in different infection models, still little is known about the potential effect of a probiotic-based vaccine against SARS-CoV-2. However, during the COVID-19 outbreak, all therapeutic options tested against this disease originated mainly from indirect observations and previous knowledge generated in studies of the new influenza A (H1N1) virus, the middle east respiratory syndrome (MERS), and the severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1), among others. Lessons learned from earlier studies of recombinant probiotics to treat other viral infections (both in vitro and in preclinical models), allow us to infer that similar strategies might be devised for the development of a probiotic-based SARS-CoV-2 vaccine. Therefore, it is likely that mucosal immunization, mainly through the nasal route, with a probiotic-based vaccine may strongly inhibit SARS-CoV-2 infection. In conclusion, future efforts are warranted in this area of investigation, in the setup of aptly designed pre-clinical and clinical studies, to explore the potential benefits of mucosal delivery of therapeutics in the fight against COVID-19 pandemic.

Author Contributions

Conceptualization, S.T.-S., A.H.M., L.G.B.-H., H.K., J.S.-G.; writing—original draft preparation, S.T.-S., A.H.M., L.G.B.-H., N.G.C.-P., H.K., J.S.-G.; writing—review and editing, S.T.-S., A.H.M., L.G.B.-H., V.C., N.G.C.-P., H.K., J.S.-G. All authors have read and agreed to the published version of the manuscript.

Funding

Not applicable.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

This work was supported by the Keyvan Virology Specialty Laboratory (KVSL), Tehran, Iran.

Conflicts of Interest

The authors declare that they have no conflict of interest.

Availability of Data and Material

Not applicable.

Code Availability

Not applicable.

References

  1. Chen, N.; Zhou, M.; Dong, X.; Qu, J.; Gong, F.; Han, Y.; Qiu, Y.; Wang, J.; Liu, Y.; Wei, Y.; et al. Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: A descriptive study. Lancet 2020, 395, 507–513. [Google Scholar] [CrossRef] [Green Version]
  2. Chen, L.; Liu, W.; Zhang, Q.; Xu, K.; Ye, G.; Wu, W.; Sun, Z.; Liu, F.; Wu, K.; Zhong, B.; et al. RNA based mNGS approach identifies a novel human coronavirus from two individual pneumonia cases in 2019 Wuhan outbreak. Emerg. Microbes Infect. 2020, 9, 313–319. [Google Scholar] [CrossRef] [PubMed]
  3. Mohseni, A.H.; Taghinezhad, S.S.; Xu, Z.; Fu, X. Body fluids may contribute to human-to-human transmission of severe acute respiratory syndrome coronavirus 2: Evidence and practical experience. Chin. Med. 2020, 15, 58. [Google Scholar] [CrossRef] [PubMed]
  4. Lai, C.-C.; Shih, T.-P.; Ko, W.-C.; Tang, H.-J.; Hsueh, P.-R. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and coronavirus disease-2019 (COVID-19): The epidemic and the challenges. Int. J. Antimicrob. Agents. 2020, 55, 105924. [Google Scholar] [CrossRef] [PubMed]
  5. Hu, B.; Guo, H.; Zhou, P.; Shi, Z.L. Characteristics of SARS-CoV-2 and COVID-19. Nat. Rev. Microbiol. 2021, 19, 141–154. [Google Scholar] [CrossRef] [PubMed]
  6. Thaiss, C.A.; Zmora, N.; Levy, M.; Elinav, E. The microbiome and innate immunity. Nature 2016, 535, 65–74. [Google Scholar] [CrossRef] [PubMed]
  7. Tai, W.; He, L.; Zhang, X.; Pu, J.; Voronin, D.; Jiang, S.; Zhou, Y.; Du, L. Characterization of the receptor-binding domain (RBD) of 2019 novel coronavirus: Implication for development of RBD protein as a viral attachment inhibitor and vaccine. Cell. Mol. Immunol. 2020, 17, 613–620. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  8. To, K.K.; Tsang, O.T.; Leung, W.S.; Tam, A.R.; Wu, T.C.; Lung, D.C.; Yip, C.C.; Cai, J.P.; Chan, J.M.; Chik, T.S.; et al. Temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by SARS-CoV-2: An observational cohort study. Lancet Infect. Dis. 2020, 20, 565–574. [Google Scholar] [CrossRef] [Green Version]
  9. Yu, J.; Tostanoski, L.H.; Peter, L.; Mercado, N.B.; McMahan, K.; Mahrokhian, S.H.; Nkolola, J.P.; Liu, J.; Li, Z.; Chandrashekar, A.; et al. DNA vaccine protection against SARS-CoV-2 in rhesus macaques. Science 2020, 369, 806–811. [Google Scholar] [CrossRef]
  10. Grifoni, A.; Weiskopf, D.; Ramirez, S.I.; Mateus, J.; Dan, J.M.; Moderbacher, C.R.; Rawlings, S.A.; Sutherland, A.; Premkumar, L.; Jadi, R.S.; et al. Targets of T Cell Responses to SARS-CoV-2 Coronavirus in Humans with COVID-19 Disease and Unexposed Individuals. Cell 2020, 181, 1489–1501. [Google Scholar] [CrossRef]
  11. Yang, J.; Wang, W.; Chen, Z.; Lu, S.; Yang, F.; Bi, Z.; Bao, L.; Mo, F.; Li, X.; Huang, Y.; et al. A vaccine targeting the RBD of the S protein of SARS-CoV-2 induces protective immunity. Nature 2020, 586, 572–577. [Google Scholar] [CrossRef] [PubMed]
  12. Mohseni, A.H.; Taghinezhad-Saroukalaei, S.; Su, B.; Wang, F. Inferring MHC interacting SARS-CoV-2 epitopes recognized by TCRs towards designing T cell-based vaccines. bioRxiv 2020. [Google Scholar] [CrossRef]
  13. Le Bert, N.; Tan, A.T.; Kunasegaran, K.; Tham, C.Y.L.; Hafezi, M.; Chia, A.; Chng, M.H.Y.; Lin, M.; Tan, N.; Linster, M.; et al. SARS-CoV-2-specific T cell immunity in cases of COVID-19 and SARS, and uninfected controls. Nature 2020, 584, 457–462. [Google Scholar] [CrossRef] [PubMed]
  14. Dai, L.; Gao, G.F. Viral targets for vaccines against COVID-19. Nat. Rev. Immunol. 2021, 21, 73–82. [Google Scholar] [CrossRef]
  15. Sun, J.; Zhuang, Z.; Zheng, J.; Li, K.; Wong, R.L.; Liu, D.; Huang, J.; He, J.; Zhu, A.; Zhao, J.; et al. Generation of a Broadly Useful Model for COVID-19 Pathogenesis, Vaccination, and Treatment. Cell 2020, 182, 734–743. [Google Scholar] [CrossRef]
  16. Landete, J.M. A review of food-grade vectors in lactic acid bacteria: From the laboratory to their application. Crit. Rev. Biotechnol. 2017, 37, 296–308. [Google Scholar] [CrossRef] [PubMed]
  17. Vijayakumar, P.P.; Muriana, P.M. A Microplate Growth Inhibition Assay for Screening Bacteriocins against Listeria monocytogenes to Differentiate Their Mode-of-Action. Biomolecules 2015, 5, 1178–1194. [Google Scholar] [CrossRef] [Green Version]
  18. Ricci, A.; Allende, A.; Bolton, D.; Chemaly, M.; Davies, R.; Girones, R.; Koutsoumanis, K.; Lindqvist, R.; Nørrung, B.; Robertson, L.; et al. Update of the list of QPS-recommended biological agents intentionally added to food or feed as notified to EFSA 7: Suitability of taxonomic units notified to EFSA until September 2017. EFSA J. 2018, 16, e05131. [Google Scholar]
  19. Mohseni, A.H.; Casolaro, V.; Bermúdez-Humarán, L.G.; Keyvani, H.; Taghinezhad, S.S. Modulation of the PI3K/Akt/mTOR signaling pathway by probiotics as a fruitful target for orchestrating the immune response. Gut. Microbes 2021, 13, 1–17. [Google Scholar] [CrossRef]
  20. LeBlanc, J.G.; Aubry, C.; Cortes-Perez, N.G.; de Moreno de LeBlanc, A.; Vergnolle, N.; Langella, P.; Azevedo, V.; Chatel, J.M.; Miyoshi, A.; Bermúdez-Humarán, L.G. Mucosal targeting of therapeutic molecules using genetically modified lactic acid bacteria: An update. FEMS. Microbiol. Lett. 2013, 344, 1–9. [Google Scholar] [CrossRef] [Green Version]
  21. Walter, J. Ecological role of lactobacilli in the gastrointestinal tract: Implications for fundamental and biomedical research. Appl. Environ. Microbiol. 2008, 74, 4985–4996. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  22. Wang, Z.; Yu, Q.; Gao, J.; Yang, Q. Mucosal and systemic immune responses induced by recombinant Lactobacillus spp. expressing the hemagglutinin of the avian influenza virus H5N1. Clin. Vaccine Immunol. 2012, 19, 174–179. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  23. Qiao, X.; Li, G.; Wang, X.; Li, X.; Liu, M.; Li, Y. Recombinant porcine rotavirus VP4 and VP4-LTB expressed in Lactobacillus casei induced mucosal and systemic antibody responses in mice. BMC Microbiol. 2009, 9, 1471–2180. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  24. Engchanil, C. Construction of the Recombinant Probiotic Lactobacillus casei and Lactobacillus fermentum Expressing the Codon-Optimized M2e: HBc Fusion Gene. J. Med. Assoc. Thail. 2016, 99, S9–S18. [Google Scholar]
  25. Peirotén, Á.; Landete, J.M. Natural and engineered promoters for gene expression in Lactobacillus species. Appl. Microbiol. Biotechnol. 2020, 104, 3797–3805. [Google Scholar] [CrossRef] [PubMed]
  26. Zhao, L.L.; Liu, M.; Ge, J.W.; Qiao, X.Y.; Li, Y.J.; Liu, D.Q. Expression of infectious pancreatic necrosis virus (IPNV) VP2-VP3 fusion protein in Lactobacillus casei and immunogenicity in rainbow trouts. Vaccine 2012, 30, 1823–1829. [Google Scholar]
  27. Mohseni, A.H.; Taghinezhad, S.S.; Keyvani, H. The First Clinical Use of a Recombinant Lactococcus lactis Expressing Human Papillomavirus Type 16 E7 Oncogene Oral Vaccine: A Phase I Safety and Immunogenicity Trial in Healthy Women Volunteers. Mol. Cancer Ther. 2020, 19, 717–727. [Google Scholar] [CrossRef] [Green Version]
  28. Shi, S.H.; Yang, W.T.; Yang, G.L.; Cong, Y.L.; Huang, H.B.; Wang, Q.; Cai, R.P.; Ye, L.P.; Hu, J.T.; Zhou, J.Y.; et al. Immunoprotection against influenza virus H9N2 by the oral administration of recombinant Lactobacillus plantarumNC8 expressing hemagglutinin in BALB/c mice. Virology 2014, 465, 166–176. [Google Scholar] [CrossRef] [Green Version]
  29. Jee, P.F.; Tiong, V.; Shu, M.H.; Khoo, J.J.; Wong, W.F.; Abdul Rahim, R.; AbuBakar, S.; Chang, L.Y. Oral immunization of a non-recombinant Lactococcus lactis surface displaying influenza hemagglutinin 1 (HA1) induces mucosal immunity in mice. PLoS ONE 2017, 12, e0187718. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  30. Lee, J.S.; Poo, H.; Han, D.P.; Hong, S.P.; Kim, K.; Cho, M.W.; Kim, E.; Sung, M.H.; Kim, C.J. Mucosal immunization with surface-displayed severe acute respiratory syndrome coronavirus spike protein on Lactobacillus casei induces neutralizing antibodies in mice. J. Virol. 2006, 80, 4079–4087. [Google Scholar] [CrossRef] [Green Version]
  31. Jiang, Y.; Hu, J.; Guo, Y.; Yang, W.; Ye, L.; Shi, C.; Liu, Y.; Yang, G.; Wang, C. Construction and immunological evaluation of recombinant Lactobacillus plantarum expressing HN of Newcastle disease virus and DC-targeting peptide fusion protein. J. Biotechnol. 2015, 216, 82–89. [Google Scholar] [CrossRef] [PubMed]
  32. Mohseni, A.H.; Razavilar, V.; Keyvani, H.; Razavi, M.R.; Khavari-Nejad, R.A. Oral immunization with recombinant Lactococcus lactis NZ9000 expressing human papillomavirus type 16 E7 antigen and evaluation of its immune effects in female C57BL/6 mice. J. Med. Virol. 2019, 91, 296–307. [Google Scholar] [CrossRef] [PubMed]
  33. Yang, W.T.; Yang, G.L.; Wang, Q.; Huang, H.B.; Jiang, Y.L.; Shi, C.W.; Wang, J.Z.; Huang, K.Y.; Jin, Y.B.; Wang, C.F. Protective efficacy of Fc targeting conserved influenza virus M2e antigen expressed by Lactobacillus plantarum. Antivir. Res. 2017, 138, 9–21. [Google Scholar] [CrossRef]
  34. Liu, Y.Y.; Yang, W.T.; Shi, S.H.; Li, Y.J.; Zhao, L.; Shi, C.W.; Zhou, F.Y.; Jiang, Y.L.; Hu, J.T.; Gu, W.; et al. Immunogenicity of recombinant Lactobacillus plantarum NC8 expressing goose parvovirus VP2 gene in BALB/c mice. J. Vet. Sci. 2017, 18, 159–167. [Google Scholar] [CrossRef]
  35. Yang, W.T.; Yang, G.L.; Zhao, L.; Jin, Y.B.; Jiang, Y.L.; Huang, H.B.; Shi, C.W.; Wang, J.Z.; Wang, G.; Kang, Y.H.; et al. Lactobacillus plantarum displaying conserved M2e and HA2 fusion antigens induces protection against influenza virus challenge. Appl. Microbiol. Biotechnol. 2018, 102, 5077–5088. [Google Scholar] [CrossRef]
  36. Yang, W.T.; Shi, S.H.; Yang, G.L.; Jiang, Y.L.; Zhao, L.; Li, Y.; Wang, C.F. Cross-protective efficacy of dendritic cells targeting conserved influenza virus antigen expressed by Lactobacillus plantarum. Sci. Rep. 2016, 6, 39665. [Google Scholar] [CrossRef]
  37. Chowdhury, M.Y.; Li, R.; Kim, J.H.; Park, M.E.; Kim, T.H.; Pathinayake, P.; Weeratunga, P.; Song, M.K.; Son, H.Y.; Hong, S.P.; et al. Mucosal vaccination with recombinant Lactobacillus casei-displayed CTA1-conjugated consensus matrix protein-2 (sM2) induces broad protection against divergent influenza subtypes in BALB/c mice. PLoS ONE 2014, 9, e94051. [Google Scholar] [CrossRef]
  38. Li, R.; Chowdhury, M.Y.; Kim, J.H.; Kim, T.H.; Pathinayake, P.; Koo, W.S.; Park, M.E.; Yoon, J.E.; Roh, J.B.; Hong, S.P.; et al. Mucosally administered Lactobacillus surface-displayed influenza antigens (sM2 and HA2) with cholera toxin subunit A1 (CTA1) Induce broadly protective immune responses against divergent influenza subtypes. Vet. Microbiol. 2015, 179, 250–263. [Google Scholar] [CrossRef] [PubMed]
  39. Wang, G.H.; Hou, X.L.; Yu, L.Y.; Liu, J.K.; Wei, C.H. Studies on Mucosal Immunity Induced by Transmissible Gastroenteritis Virus Nucleocapsid Protein Recombinant Lactobacillus casei in Mice and Sow. Agric. Sci. China 2009, 8, 231–237. [Google Scholar] [CrossRef]
  40. Yoon, S.W.; Lee, T.Y.; Kim, S.J.; Lee, I.H.; Sung, M.H.; Park, J.S.; Poo, H. Oral administration of HPV-16 L2 displayed on Lactobacillus casei induces systematic and mucosal cross-neutralizing effects in Balb/c mice. Vaccine 2012, 30, 3286–3294. [Google Scholar] [CrossRef]
  41. Jiang, X.; Hou, X.; Tang, L.; Jiang, Y.; Ma, G.; Li, Y. A phase trial of the oral Lactobacillus casei vaccine polarizes Th2 cell immunity against transmissible gastroenteritis coronavirus infection. Appl. Microbiol. Biotechnol. 2016, 100, 7457–7469. [Google Scholar] [CrossRef] [Green Version]
  42. Xu, Y.; Cui, L.; Tian, C.; Zhang, G.; Huo, G.; Tang, L.; Li, Y. Immunogenicity of recombinant classic swine fever virus CD8(+) T lymphocyte epitope and porcine parvovirus VP2 antigen coexpressed by Lactobacillus casei in swine via oral vaccination. Clin. Vaccine Immunol. 2011, 18, 1979–1986. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  43. Duan, K.; Hua, X.; Wang, Y.; Chen, Y.; Shi, W.; Tang, L.; Li, Y.; Liu, M. Oral immunization with a recombinant Lactobacillus expressing CK6 fused with VP2 protein against IPNV in rainbow trout (Oncorhynchus mykiss). Fish Shellfish. Immunol. 2018, 83, 223–231. [Google Scholar] [CrossRef] [PubMed]
  44. Park, Y.C.; Ouh, Y.T.; Sung, M.H.; Park, H.G.; Kim, T.J.; Cho, C.H.; Park, J.S.; Lee, J.K. A phase 1/2a, dose-escalation, safety and preliminary efficacy study of oral therapeutic vaccine in subjects with cervical intraepithelial neoplasia 3. J. Gynecol. Oncol. 2019, 30, e88. [Google Scholar] [CrossRef]
  45. Taghinezhad, S.S.; Mohseni, A.H.; Keyvani, H.; Razavi, M.R. Phase 1 Safety and Immunogenicity Trial of Recombinant Lactococcus lactis Expressing Human Papillomavirus Type 16 E6 Oncoprotein Vaccine. Mol. Ther. Methods Clin. Dev. 2019, 15, 40–51. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  46. Kajikawa, A.; Zhang, L.; Long, J.; Nordone, S.; Stoeker, L.; LaVoy, A.; Bumgardner, S.; Klaenhammer, T.; Dean, G. Construction and immunological evaluation of dual cell surface display of HIV-1 gag and Salmonella enterica serovar Typhimurium FliC in Lactobacillus acidophilus for vaccine delivery. Clin. Vaccine Immunol. 2012, 19, 1374–1381. [Google Scholar] [CrossRef] [Green Version]
  47. Hanniffy, S.B.; Carter, A.T.; Hitchin, E.; Wells, J.M. Mucosal delivery of a pneumococcal vaccine using Lactococcus lactis affords protection against respiratory infection. J. Infect. Dis. 2007, 195, 185–193. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  48. Liu, D.Q.; Ge, J.W.; Qiao, X.Y.; Jiang, Y.P.; Liu, S.M.; Li, Y.J. High-level mucosal and systemic immune responses induced by oral administration with Lactobacillus-expressed porcine epidemic diarrhea virus (PEDV) S1 region combined with Lactobacillus-expressed N protein. Appl. Microbiol. Biotechnol. 2012, 93, 2437–2446. [Google Scholar]
  49. Wang, Z.; Gao, J.; Yu, Q.; Yang, Q. Oral immunization with recombinant Lactococcus lactis expressing the hemagglutinin of the avian influenza virus induces mucosal and systemic immune responses. Future Microbiol. 2012, 7, 1003–1010. [Google Scholar] [CrossRef]
  50. Lei, H.; Sheng, Z.; Ding, Q.; Chen, J.; Wei, X.; Lam, D.M.; Xu, Y. Evaluation of oral immunization with recombinant avian influenza virus HA1 displayed on the Lactococcus lactis surface and combined with the mucosal adjuvant cholera toxin subunit B. Clin. Vaccine Immunol. 2011, 18, 1046–1051. [Google Scholar] [CrossRef] [Green Version]
  51. Jiang, Y.; Yang, G.; Wang, Q.; Wang, Z.; Yang, W.; Gu, W.; Shi, C.; Wang, J.; Huang, H.; Wang, C. Molecular mechanisms underlying protection against H9N2 influenza virus challenge in mice by recombinant Lactobacillus plantarum with surface displayed HA2-LTB. J. Biotechnol. 2017, 259, 6–14. [Google Scholar] [CrossRef]
  52. Gao, S.; Li, D.; Liu, Y.; Zha, E.; Zhou, T.; Yue, X. Oral immunization with recombinant hepatitis E virus antigen displayed on the Lactococcus lactis surface enhances ORF2-specific mucosal and systemic immune responses in mice. Int. Immunopharmacol. 2015, 24, 140–145. [Google Scholar] [CrossRef]
  53. Taghinezhad, S.S.; Mohseni, A.H.; Keyvani, H.; Razavilar, V. Protection against human papillomavirus type 16-induced tumors in C57BL/6 mice by mucosal vaccination with Lactococcus lactis NZ9000 expressing E6 oncoprotein. Microb. Pathog. 2019, 126, 149–156. [Google Scholar] [CrossRef]
  54. Kawana, K.; Adachi, K.; Kojima, S.; Taguchi, A.; Tomio, K.; Yamashita, A.; Nishida, H.; Nagasaka, K.; Arimoto, T.; Yokoyama, T.; et al. Oral vaccination against HPV E7 for treatment of cervical intraepithelial neoplasia grade 3 (CIN3) elicits E7-specific mucosal immunity in the cervix of CIN3 patients. Vaccine 2014, 32, 6233–6239. [Google Scholar] [CrossRef]
  55. Sim, A.C.; Lin, W.; Tan, G.K.; Sim, M.S.; Chow, V.T.; Alonso, S. Induction of neutralizing antibodies against dengue virus type 2 upon mucosal administration of a recombinant Lactococcus lactis strain expressing envelope domain III antigen. Vaccine 2008, 26, 1145–1154. [Google Scholar] [CrossRef]
  56. Xin, K.Q.; Hoshino, Y.; Toda, Y.; Igimi, S.; Kojima, Y.; Jounai, N.; Ohba, K.; Kushiro, A.; Kiwaki, M.; Hamajima, K.; et al. Immunogenicity and protective efficacy of orally administered recombinant Lactococcus lactis expressing surface-bound HIV Env. Blood 2003, 102, 223–228. [Google Scholar] [CrossRef] [Green Version]
  57. Wang, M.; Fu, T.; Hao, J.; Li, L.; Tian, M.; Jin, N.; Ren, L.; Li, C. A recombinant Lactobacillus plantarum strain expressing the spike protein of SARS-CoV-2. Int. J. Biol. Macromol. 2020, 160, 736–740. [Google Scholar] [CrossRef]
  58. Shi, S.H.; Yang, W.T.; Yang, G.L.; Zhang, X.K.; Liu, Y.Y.; Zhang, L.J.; Ye, L.P.; Hu, J.T.; Xing, X.; Qi, C.; et al. Lactobacillus plantarum vaccine vector expressing hemagglutinin provides protection against H9N2 challenge infection. Virus. Res. 2016, 211, 46–57. [Google Scholar] [CrossRef]
  59. Komatsu, A.; Igimi, S.; Kawana, K. Optimization of human papillomavirus (HPV) type 16 E7-expressing lactobacillus-based vaccine for induction of mucosal E7-specific IFNγ-producing cells. Vaccine 2018, 36, 3423–3426. [Google Scholar] [CrossRef]
  60. Temprana, C.F.; Argüelles, M.H.; Gutierrez, N.M.; Barril, P.A.; Esteban, L.E.; Silvestre, D.; Mandile, M.G.; Glikmann, G.; Castello, A.A. Rotavirus VP6 protein mucosally delivered by cell wall-derived particles from Lactococcus lactis induces protection against infection in a murine model. PLoS ONE 2018, 13, e0203700. [Google Scholar] [CrossRef] [PubMed]
  61. Joan, S.S.; Pui-Fong, J.; Song, A.A.; Chang, L.Y.; Yusoff, K.; AbuBakar, S.; Rahim, R.A. Oral vaccine of Lactococcus lactis harbouring pandemic H1N1 2009 haemagglutinin1 and nisP anchor fusion protein elevates anti-HA1 sIgA levels in mice. Biotechnol. Lett. 2016, 38, 793–799. [Google Scholar] [CrossRef]
  62. Tang, L.; Li, Y. Oral immunization of mice with recombinant Lactococcus lactis expressing porcine transmissible gastroenteritis virus spike glycoprotein. Virus Genes 2009, 39, 238–245. [Google Scholar] [CrossRef]
  63. Cui, L.C.; Guan, X.T.; Liu, Z.M.; Tian, C.Y.; Xu, Y.G. Recombinant lactobacillus expressing G protein of spring viremia of carp virus (SVCV) combined with ORF81 protein of koi herpesvirus (KHV): A promising way to induce protective immunity against SVCV and KHV infection in cyprinid fish via oral vaccination. Vaccine 2015, 33, 3092–3099. [Google Scholar] [CrossRef] [PubMed]
  64. Pant, N.; Hultberg, A.; Zhao, Y.; Svensson, L.; Pan-Hammarstrom, Q.; Johansen, K.; Pouwels, P.H.; Ruggeri, F.M.; Hermans, P.; Frenken, L.; et al. Lactobacilli expressing variable domain of llama heavy-chain antibody fragments (lactobodies) confer protection against rotavirus-induced diarrhea. J. Infect. Dis. 2006, 194, 1580–1588. [Google Scholar] [CrossRef] [Green Version]
  65. Li, Y.J.; Ma, G.P.; Li, G.W.; Qiao, X.Y.; Ge, J.W.; Tang, L.J.; Liu, M.; Liu, L.W. Oral vaccination with the porcine rotavirus VP4 outer capsid protein expressed by Lactococcus lactis induces specific antibody production. J. Biomed. Biotechnol. 2010, 6, 708460. [Google Scholar]
  66. Li, Y.; Li, X.; Liu, H.; Zhuang, S.; Yang, J.; Zhang, F. Intranasal immunization with recombinant Lactococci carrying human papillomavirus E7 protein and mouse interleukin-12 DNA induces E7-specific antitumor effects in C57BL/6 mice. Oncol. Lett. 2014, 7, 576–582. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  67. Reese, K.A.; Lupfer, C.; Johnson, R.C.; Mitev, G.M.; Mullen, V.M.; Geller, B.L.; Pastey, M. A Novel Lactococcal Vaccine Expressing a Peptide from the M2 Antigen of H5N2 Highly Pathogenic Avian Influenza A Virus Prolongs Survival of Vaccinated Chickens. Vet. Med. Int. 2013, 22, 316926. [Google Scholar] [CrossRef] [Green Version]
  68. Di-Qiu, L.; Xin-Yuan, Q.; Jun-Wei, G.; Li-Jie, T.; Yan-Ping, J.; Yi-Jing, L. Construction and characterization of Lactobacillus pentosus expressing the D antigenic site of the spike protein of Transmissible gastroenteritis virus. Can. J. Microbiol. 2011, 57, 392–397. [Google Scholar] [CrossRef]
  69. Asahi-Ozaki, Y.; Yoshikawa, T.; Iwakura, Y.; Suzuki, Y.; Tamura, S.; Kurata, T.; Sata, T. Secretory IgA antibodies provide cross-protection against infection with different strains of influenza B virus. J. Med. Virol. 2004, 74, 328–335. [Google Scholar] [CrossRef]
  70. Kikuchi, Y.; Kunitoh-Asari, A.; Hayakawa, K.; Imai, S.; Kasuya, K.; Abe, K.; Adachi, Y.; Fukudome, S.; Takahashi, Y.; Hachimura, S. Oral administration of Lactobacillus plantarum strain AYA enhances IgA secretion and provides survival protection against influenza virus infection in mice. PLoS ONE 2014, 9, e86416. [Google Scholar] [CrossRef] [Green Version]
  71. Renegar, K.B.; Small, P.A., Jr.; Boykins, L.G.; Wright, P.F. Role of IgA versus IgG in the control of influenza viral infection in the murine respiratory tract. J. Immunol. 2004, 173, 1978–1986. [Google Scholar] [CrossRef]
  72. LeCureux, J.S.; Dean, G.A. Lactobacillus Mucosal Vaccine Vectors: Immune Responses against Bacterial and Viral Antigens. mSphere 2018, 3, e00061-18. [Google Scholar] [CrossRef] [Green Version]
  73. Cao, Y.; Wang, X.; Yang, Q.; Deng, H.; Liu, Y.; Zhou, P.; Xu, H.; Chen, D.; Feng, D.; Zhang, H.; et al. Critical Role of Intestinal Microbiota in ATF3-Mediated Gut Immune Homeostasis. J. Immunol. 2020, 205, 842–852. [Google Scholar] [CrossRef]
  74. Bermúdez-Humarán, L.G.; Kharrat, P.; Chatel, J.M.; Langella, P. Lactococci and lactobacilli as mucosal delivery vectors for therapeutic proteins and DNA vaccines. Microb. Cell Fact. 2011, 1, 1475–2859. [Google Scholar] [CrossRef] [Green Version]
  75. Xu, R.; Jia, H.; Terkawi, M.A.; Xuan, X.; Zhang, H. Immunogenicity of orally administrated recombinant Lactobacillus casei Zhang expressing Cryptosporidium parvum surface adhesion protein P23 in mice. Curr. Microbiol. 2011, 62, 1573–1580. [Google Scholar] [CrossRef] [PubMed]
  76. Ohkouchi, K.; Kawamoto, S.; Tatsugawa, K.; Yoshikawa, N.; Takaoka, Y.; Miyauchi, S.; Aki, T.; Yamashita, M.; Murooka, Y.; Ono, K. Prophylactic effect of Lactobacillus oral vaccine expressing a Japanese cedar pollen allergen. J. Biosci. Bioeng. 2012, 113, 536–541. [Google Scholar] [CrossRef] [PubMed]
  77. Lei, H.; Peng, X.; Jiao, H.; Zhao, D.; Ouyang, J. Broadly protective immunity against divergent influenza viruses by oral co-administration of Lactococcus lactis expressing nucleoprotein adjuvanted with cholera toxin B subunit in mice. Microb. Cell. Fact. 2015, 14, 111. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  78. Lei, H.; Peng, X.; Zhao, D.; Ouyang, J.; Jiao, H.; Shu, H.; Ge, X. Lactococcus lactis displayed neuraminidase confers cross protective immunity against influenza A viruses in mice. Virology 2015, 476, 189–195. [Google Scholar] [CrossRef] [Green Version]
  79. Mohseni, A.H.; Taghinezhad, S.S.; Keyvani, H.; Razavilar, V. Extracellular overproduction of E7 oncoprotein of Iranian human papillomavirus type 16 by genetically engineered Lactococcus lactis. BMC Biotechnol. 2019, 19, 019–0499. [Google Scholar] [CrossRef] [PubMed]
  80. Taghinezhad-Saroukalaei, S.; Keyvani, H.; Bermúdez-Humarán, L.G.; Donders, G.G.; Fu, X.; Mohseni, A.H. Twenty years of research on HPV vaccines based on genetically modified lactic acid bacteria: An overview on the gut-vagina axis. Cell Mol. Life Sci. 2020, 78, 1191–1206. [Google Scholar] [CrossRef]
  81. Schroder, K.; Hertzog, P.J.; Ravasi, T.; Hume, D.A. Interferon-gamma: An overview of signals, mechanisms and functions. J. Leukoc. Biol. 2004, 75, 163–189. [Google Scholar] [CrossRef] [PubMed]
  82. Dong, H.; Rowland, I.; Yaqoob, P. Comparative effects of six probiotic strains on immune function in vitro. Br. J. Nutr. 2012, 108, 459–470. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  83. Sehgal, K.; Dhodapkar, K.M.; Dhodapkar, M.V. Targeting human dendritic cells in situ to improve vaccines. Immunol. Lett. 2014, 162, 59–67. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  84. Jin, Y.B.; Yang, W.T.; Shi, C.W.; Feng, B.; Huang, K.Y.; Zhao, G.X.; Li, Q.Y.; Xie, J.; Huang, H.B.; Jiang, Y.L.; et al. Immune responses induced by recombinant Lactobacillus plantarum expressing the spike protein derived from transmissible gastroenteritis virus in piglets. Appl. Microbiol. Biotechnol. 2018, 102, 8403–8417. [Google Scholar] [CrossRef] [PubMed]
  85. Kathania, M.; Zadeh, M.; Lightfoot, Y.L.; Roman, R.M.; Sahay, B.; Abbott, J.R.; Mohamadzadeh, M. Colonic immune stimulation by targeted oral vaccine. PLoS ONE 2013, 8, e55143. [Google Scholar] [CrossRef]
  86. Mierau, I.; Kleerebezem, M. 10 years of the nisin-controlled gene expression system (NICE) in Lactococcus lactis. Appl. Microbiol. Biotechnol. 2005, 68, 705–717. [Google Scholar] [CrossRef]
  87. Rosales-Mendoza, S.; Angulo, C.; Meza, B. Food-Grade Organisms as Vaccine Biofactories and Oral Delivery Vehicles. Trends Biotechnol. 2016, 34, 124–136. [Google Scholar] [CrossRef]
  88. Mohseni, A.H.; Razavilar, V.; Keyvani, H.; Razavi, M.R.; Khavari-Nejad, R.A. Efficient production and optimization of E7 oncoprotein from Iranian human papillomavirus type 16 in Lactococcus lactis using nisin-controlled gene expression (NICE) system. Microb. Pathog. 2017, 110, 554–560. [Google Scholar] [CrossRef]
  89. Taghinezhad-Saroukalaei, S.; Razavilar, V.; Keyvani, H.; Razavi, M.R.; Nejadsattari, T. Extracellular overproduction of recombinant Iranian HPV-16 E6 oncoprotein in Lactococcus lactis using the NICE system. Future Virol. 2018, 13, 697–710. [Google Scholar] [CrossRef]
  90. Mohseni, A.H.; Razavilar, V.; Keyvani, H.; Razavi, M.R.; Khavari Nejad, R.A. Codon Usage Optimization and Construction of Plasmid Encoding Iranian Human Papillomavirus Type 16 E7 Oncogene for Lactococcus Lactis Subsp. Cremoris MG1363. Asian. Pac. J. Cancer Prev. 2017, 18, 783–788. [Google Scholar]
  91. Suebwongsa, N.; Panya, M.; Namwat, W.; Sookprasert, S.; Redruello, B.; Mayo, B.; Alvarez, M.A.; Lulitanond, V. Cloning and expression of a codon-optimized gene encoding the influenza A virus nucleocapsid protein in Lactobacillus casei. Int. Microbiol. 2013, 16, 93–101. [Google Scholar]
  92. Taghinezhad-Saroukalaei, S.; Razavilar, V.; Keyvani, H.; Razavi, M.R.; Nejadsattari, T. Codon optimization of Iranian human papillomavirus Type 16 E6 oncogene for Lactococcus lactis subsp. cremoris MG1363. Future Virol. 2017, 12, 499–511. [Google Scholar] [CrossRef]
  93. Fuglsang, A. Lactic acid bacteria as prime candidates for codon optimization. Biochem. Biophys. Res. Commun. 2003, 312, 285–291. [Google Scholar] [CrossRef] [PubMed]
  94. Taguchi, A.; Kawana, K.; Yokoyama, T.; Adachi, K.; Yamashita, A.; Tomio, K.; Kojima, S.; Oda, K.; Fujii, T.; Kozuma, S. Adjuvant effect of Japanese herbal medicines on the mucosal type 1 immune responses to human papillomavirus (HPV) E7 in mice immunized orally with Lactobacillus-based therapeutic HPV vaccine in a synergistic manner. Vaccine 2012, 30, 5368–5372. [Google Scholar] [CrossRef]
  95. van Roosmalen, M.L.; Kanninga, R.; El Khattabi, M.; Neef, J.; Audouy, S.; Bosma, T.; Kuipers, A.; Post, E.; Steen, A.; Kok, J.; et al. Mucosal vaccine delivery of antigens tightly bound to an adjuvant particle made from food-grade bacteria. Methods 2006, 38, 144–149. [Google Scholar] [CrossRef] [Green Version]
  96. Ramirez, K.; Ditamo, Y.; Rodriguez, L.; Picking, W.L.; van Roosmalen, M.L.; Leenhouts, K.; Pasetti, M.F. Neonatal mucosal immunization with a non-living, non-genetically modified Lactococcus lactis vaccine carrier induces systemic and local Th1-type immunity and protects against lethal bacterial infection. Mucosal. Immunol. 2010, 3, 159–171. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  97. Mohamadzadeh, M.; Duong, T.; Sandwick, S.J.; Hoover, T.; Klaenhammer, T.R. Dendritic cell targeting of Bacillus anthracis protective antigen expressed by Lactobacillus acidophilus protects mice from lethal challenge. Proc. Natl. Acad. Sci. USA 2009, 106, 4331–4336. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  98. Zhen-guo, W.; Ning-yi, J.; Ming-xiao, M.; Dong-liang, F.; Min, Z.; Ge-fen, Y.; Lei-li, J.; Kuo-shi, J.; Zhi-ping, X.; Ming-lan, J. Immunogenicity of Recombinant Fowl-pox Vaccines coexpressing HA of AIV H5N1, H7N1 and chicken IL-18. Virol. Sin. 2005, 20, 607–612. [Google Scholar]
  99. Seegers, J.F. Lactobacilli as live vaccine delivery vectors: Progress and prospects. Trends Biotechnol. 2002, 20, 508–515. [Google Scholar] [CrossRef]
  100. Cai, R.; Jiang, Y.; Yang, W.; Shi, S.; Shi, C.; Hu, J.; Gu, W.; Ye, L.; Zhou, F.; Gong, Q.; et al. Surface-Displayed IL-10 by Recombinant Lactobacillus plantarum Reduces Th1 Responses of RAW264.7 Cells Stimulated with Poly(I:C) or LPS. J. Microbiol. Biotechnol. 2016, 26, 421–431. [Google Scholar] [CrossRef]
  101. Mbow, M.L.; De Gregorio, E.; Valiante, N.M.; Rappuoli, R. New adjuvants for human vaccines. Curr. Opin. Immunol. 2010, 22, 411–416. [Google Scholar] [CrossRef]
  102. Davidson, L.E.; Fiorino, A.M.; Snydman, D.R.; Hibberd, P.L. Lactobacillus GG as an immune adjuvant for live-attenuated influenza vaccine in healthy adults: A randomized double-blind placebo-controlled trial. Eur. J. Clin. Nutr. 2011, 65, 501–507. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  103. Plant, L.J.; Conway, P.L. Adjuvant properties and colonization potential of adhering and non-adhering Lactobacillus spp following oral administration to mice. FEMS Immunol. Med. Microbiol. 2002, 34, 105–111. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  104. Petrarca, C.; Carpiniello, F.; DiGioacchino, M. Recombinant Probiotics for Allergen Immunotherapy. J. Vaccines Vaccin. 2015, 1. [Google Scholar] [CrossRef]
  105. Enomoto, M.; Noguchi, S.; Hattori, M.; Sugiyama, H.; Suzuki, Y.; Hanaoka, A.; Okada, S.; Yoshida, T. Oral administration of Lactobacillus plantarum NRIC0380 suppresses IgE production and induces CD4(+)CD25(+)Foxp3(+) cells in vivo. Biosci. Biotechnol. Biochem. 2009, 73, 457–460. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  106. Mitragotri, S. Immunization without Needles. Nat. Rev. Immunol. 2005, 5, 905–916. [Google Scholar] [CrossRef]
  107. Holmgren, J.; Czerkinsky, C. Mucosal immunity and vaccines. Nat. Med. 2005, 11, S45–S53. [Google Scholar] [CrossRef] [PubMed]
  108. Neutra, M.R.; Kozlowski, P.A. Mucosal vaccines: The promise and the challenge. Nat. Rev. Immunol. 2006, 6, 148–158. [Google Scholar] [CrossRef]
  109. Ellebedy, A.H.; Ducatez, M.F.; Duan, S.; Stigger-Rosser, E.; Rubrum, A.M.; Govorkova, E.A.; Webster, R.G.; Webby, R.J. Impact of prior seasonal influenza vaccination and infection on pandemic A (H1N1) influenza virus replication in ferrets. Vaccine 2011, 29, 3335–3339. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  110. Rose, M.A.; Zielen, S.; Baumann, U. Mucosal immunity and nasal influenza vaccination. Expert Rev. Vaccines 2012, 11, 595–607. [Google Scholar] [CrossRef]
  111. Lycke, N. Recent progress in mucosal vaccine development: Potential and limitations. Nat. Rev. Immunol. 2012, 12, 592–605. [Google Scholar] [CrossRef]
  112. Sundararaman, A.; Ray, M.; Ravindra, P.V.; Halami, P.M. Role of probiotics to combat viral infections with emphasis on COVID-19. Appl. Microbiol. Biotechnol. 2020, 104, 8089–8104. [Google Scholar] [CrossRef]
  113. Wypych, T.P.; Wickramasinghe, L.C.; Marsland, B.J. The influence of the microbiome on respiratory health. Nat. Immunol. 2019, 20, 1279–1290. [Google Scholar] [CrossRef] [PubMed]
  114. Vitiñi, E.; Alvarez, S.; Medina, M.; Medici, M.; de Budeguer, M.V.; Perdigón, G. Gut mucosal immunostimulation by lactic acid bacteria. Biocell 2000, 24, 223–232. [Google Scholar] [PubMed]
  115. Dieye, Y.; Usai, S.; Clier, F.; Gruss, A.; Piard, J.C. Design of a protein-targeting system for lactic acid bacteria. J. Bacteriol. 2001, 183, 4157–4166. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  116. Michon, C.; Langella, P.; Eijsink, V.G.; Mathiesen, G.; Chatel, J.M. Display of recombinant proteins at the surface of lactic acid bacteria: Strategies and applications. Microb. Cell Fact. 2016, 15, 70. [Google Scholar] [CrossRef]
  117. Bermúdez-Humarán, L.G.; Cortes-Perez, N.G.; Lefèvre, F.; Guimarães, V.; Rabot, S.; Alcocer-Gonzalez, J.M.; Gratadoux, J.J.; Rodriguez-Padilla, C.; Tamez-Guerra, R.S.; Corthier, G.; et al. A novel mucosal vaccine based on live Lactococci expressing E7 antigen and IL-12 induces systemic and mucosal immune responses and protects mice against human papillomavirus type 16-induced tumors. J. Immunol. 2005, 175, 7297–7302. [Google Scholar] [CrossRef] [PubMed]
  118. Daniel, C.; Roussel, Y.; Kleerebezem, M.; Pot, B. Recombinant lactic acid bacteria as mucosal biotherapeutic agents. Trends Biotechnol. 2011, 29, 499–508. [Google Scholar] [CrossRef]
  119. Dicks, L.M.; Botes, M. Probiotic lactic acid bacteria in the gastro-intestinal tract: Health benefits, safety and mode of action. Benef. Microbes. 2010, 1, 11–29. [Google Scholar] [CrossRef] [PubMed]
  120. Hugentobler, F.; Yam, K.K.; Gillard, J.; Mahbuba, R.; Olivier, M.; Cousineau, B. Immunization against Leishmania major infection using LACK- and IL-12-expressing Lactococcus lactis induces delay in footpad swelling. PLoS ONE 2012, 7, e30945. [Google Scholar] [CrossRef]
  121. Bahey-El-Din, M. Lactococcus lactis-based vaccines from laboratory bench to human use: An overview. Vaccine 2012, 30, 685–690. [Google Scholar] [CrossRef] [PubMed]
  122. del Rio, B.; Dattwyler, R.J.; Aroso, M.; Neves, V.; Meirelles, L.; Seegers, J.F.; Gomes-Solecki, M. Oral immunization with recombinant lactobacillus plantarum induces a protective immune response in mice with Lyme disease. Clin. Vaccine Immunol. 2008, 15, 1429–1435. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  123. Adachi, K.; Kawana, K.; Yokoyama, T.; Fujii, T.; Tomio, A.; Miura, S.; Tomio, K.; Kojima, S.; Oda, K.; Sewaki, T.; et al. Oral immunization with a Lactobacillus casei vaccine expressing human papillomavirus (HPV) type 16 E7 is an effective strategy to induce mucosal cytotoxic lymphocytes against HPV16 E7. Vaccine 2010, 28, 2810–2817. [Google Scholar] [CrossRef] [PubMed]
  124. Lee, Y.-N.; Youn, H.-N.; Kwon, J.-H.; Lee, D.-H.; Park, J.-K.; Yuk, S.-S.; Erdene-Ochir, T.-O.; Kim, K.-T.; Lee, J.-B.; Park, S.-Y.; et al. Sublingual administration of Lactobacillus rhamnosus affects respiratory immune responses and facilitates protection against influenza virus infection in mice. Antivir. Res. 2013, 98, 284–290. [Google Scholar] [CrossRef] [PubMed]
  125. Azegami, T.; Yuki, Y.; Hayashi, K.; Hishikawa, A.; Sawada, S.I.; Ishige, K.; Akiyoshi, K.; Kiyono, H.; Itoh, H. Intranasal vaccination against angiotensin II type 1 receptor and pneumococcal surface protein A attenuates hypertension and pneumococcal infection in rodents. J. Hypertens. 2018, 36, 387–394. [Google Scholar] [CrossRef] [PubMed]
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Figure 1. Schematic representation of the structure of important SARS-CoV-2 proteins, targeting the ACE2 receptors and promoting viral entry in infected cells. The SARS-CoV-2 spike (S) protein mediates membrane fusion by binding to these cellular receptors (retrieved from https://app.biorender.com/biorender-templates (access date: 6 March 2021)): “SARS-CoV-2 Targeting of ACE2 Receptor and Entry in Infected Cell”).
Figure 1. Schematic representation of the structure of important SARS-CoV-2 proteins, targeting the ACE2 receptors and promoting viral entry in infected cells. The SARS-CoV-2 spike (S) protein mediates membrane fusion by binding to these cellular receptors (retrieved from https://app.biorender.com/biorender-templates (access date: 6 March 2021)): “SARS-CoV-2 Targeting of ACE2 Receptor and Entry in Infected Cell”).
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Figure 2. Diagram illustrating the development of the first oral COVID-19 vaccine candidate based on recombinant Bifidobacterium longum which has been engineered to deliver plasmids containing synthetic DNA encoding spike protein from SARS-CoV-2 (adapted from https://app.biorender.com/biorender-templates (access date: 6 March 2021)).
Figure 2. Diagram illustrating the development of the first oral COVID-19 vaccine candidate based on recombinant Bifidobacterium longum which has been engineered to deliver plasmids containing synthetic DNA encoding spike protein from SARS-CoV-2 (adapted from https://app.biorender.com/biorender-templates (access date: 6 March 2021)).
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Figure 3. Schematic representation of the stimulation of immune responses with genetically modified probiotics expressing therapeutic factors in the gut and the lung. The crosstalk and the reciprocal interaction of the gut and lung mucosa (gut-lung axis) is mediated by immune cells moving between the two districts via the bloodstream and the lymphatic ducts, leading to modulation of the immune response in both sites. Delivery of antigen via recombinant probiotic to antigen-presenting cells in Peyer’s patches causes the stimulation of naive B and T cells and induction of several immune factors, such as Th1 and Th2 cytokines. As a result, cells and immune factors migrate to the thoracic duct and the BALT through circulation and enhance the production of secretory IgA and the activation of effector CD4+ and CD8+ T cells, preventing the onset and progression of respiratory viral infections. IgA, immunoglobulin A, IL-4: interleukin-4, IFN-γ: interferon-gamma, Th1: T-helper cell type 1, Th2: T-helper cell type 2, BALT: bronchi-associated lymphoid tissue (adapted from https://app.biorender.com/biorender-templates (access date: 24 November 2020).
Figure 3. Schematic representation of the stimulation of immune responses with genetically modified probiotics expressing therapeutic factors in the gut and the lung. The crosstalk and the reciprocal interaction of the gut and lung mucosa (gut-lung axis) is mediated by immune cells moving between the two districts via the bloodstream and the lymphatic ducts, leading to modulation of the immune response in both sites. Delivery of antigen via recombinant probiotic to antigen-presenting cells in Peyer’s patches causes the stimulation of naive B and T cells and induction of several immune factors, such as Th1 and Th2 cytokines. As a result, cells and immune factors migrate to the thoracic duct and the BALT through circulation and enhance the production of secretory IgA and the activation of effector CD4+ and CD8+ T cells, preventing the onset and progression of respiratory viral infections. IgA, immunoglobulin A, IL-4: interleukin-4, IFN-γ: interferon-gamma, Th1: T-helper cell type 1, Th2: T-helper cell type 2, BALT: bronchi-associated lymphoid tissue (adapted from https://app.biorender.com/biorender-templates (access date: 24 November 2020).
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Table
Table 1. Pre-clinical and clinical studies of probiotic-based vaccines against respiratory and non-respiratory viruses.
Table 1. Pre-clinical and clinical studies of probiotic-based vaccines against respiratory and non-respiratory viruses.
ProbioticVirusHost/Inoculation RoutePathways of Immune System InductionNumberDosageReference
L. acidophilusAvian
influenza virus H5N1		Mouse/OralInduction of anti-HA1 IgA antibody, anti-HA1 IgG, lymphocyte proliferative reaction, and IL-46 times1 × 1010 CFU/mL[22]
L. delbrueckiisubsp. lactisAvian
influenza virus H5N1		Mouse/OralInduction of anti-HA1 IgA antibody, anti-HA1 IgG, lymphocyte proliferative reaction, and IL-46 times1 × 1010 CFU/mL
L. caseiPorcine rotavirusMouse/OralInduction of serum IgG and mucosal IgA9 times1 × 109 CFU/mL[23]
Infectious pancreatic necrosis virus (IPNV)Rainbow trouts/OralInduction of specific IgM anti-pIPNV, and reduction of viral loads2 times5 × 108 pfu/200 µL[26]
L. lactisHuman papillomavirus type 16 (HPV-16)Healthy women/OralInduction of E7-specific IgG and SIgA antibody and, E7-specific IFN-γ-secreting CD8+ T cell immune response20 times1 × 109, 5 × 109, and 1 × 1010 CFU/mL[27]
L. plantarumInfluenza virus H9N2Mouse/OralInduction of IgG, sIgA, HI antibodies, and CD8+ T cell immune response7 times1 × 109 CFU/mouse[28]
L. lactisInfluenza virus H1N1Mouse/OralInduction of specific serum IgG and IgA, and sIgA9 times1 × 1010 and 5 × 1010 CFU/mL[29]
L. caseiSevere acute respiratory syndrome (SARS)Mouse/Oral and nasalInduction of serum IgG and mucosal IgAFor oral: 20 times
For nasal: 8 times		For oral: 5 × 109 cells/100 µL
For nasal: 2 × 109 cells/20 µL		[30]
L. plantarumNewcastle disease virus (NDV)Chicken/OralInduction of sIgA, CD3+CD4+T, T lymphocytes proliferation and increasing survival rates9 times109 CFU/0.2 mL[31]
L. lactisHuman papillomavirus type 16 (HPV-16)Mouse/OralInduction of E7-specific antibody and E7-specific CD4+ Th and CD8+ T cell precursors, specific IL-2- and IFN-γ-secreting T cells9 times1 × 108, 1 × 109, and 1 × 1010 CFU/mL[32]
L. plantarumInfluenza A virus H1N1Mouse/OralInduction of Peyer’s patch (PP) DC, PP B220+IgA+, sIgA, growth centers (GCs) in PPs, T immune response, CD8+IFN-γ+ cells, and reduction viral load6 times-[33]
Goose parvovirus (GPV)Mouse/OralInduction of CD11c+, CD3+CD4+, CD3+CD8+, IFN-γ+ and TNF-α, and sIgA14 times2 × 109 CFU/mL[34]
Avian influenza virus Chicks/OralInduction of specific humoral, mucosal, and T cell-mediated immune responses, and reduction viral load6 times2 × 109 CFU/300 μL[35]
Avian influenza virus H9N2Mouse/OralInduction of specific mucosal antibody responses and B and T cell responses, specific CD8 T cells, and antigen specific cytotoxicity6 times1 × 109 CFU/mouse[36]
L. casei Mouse/Oral and nasalInduction of serum IgG, mucosal IgA, and cell-mediated immune responseFor oral: 10 times
For nasal: 8 times		For oral: 1 × 1010 CFU/100 µL
For nasal: 1 × 109 CFU/20 µL		[37]
Influenza A virusesMouse/Oral and nasalInduction of serum IgG and their isotypes (IgG1 & IgG2a), mucosal IgA, sM2- or HA2-specific cell-mediated immunity, IFN-g, and IL-4For oral: 8 times
For nasal: 6 times		For oral: 1 × 1010 CFU/100 µL
For nasal: 1 × 109 CFU/20 µL		[38]
Transmissible gastroenteritis virus (TGEV)Mouse and pregnant sow/Oral and nasalInduction of IgG and sIgAFor oral :20 times
For nasal: 8 times		For oral:
5 × 109 CFU/mL
For nasal: 2 × 109 CFU/mL
[39]
Human papillomavirus type 16 (HPV-16)Mouse/OralInduction of L2-specific serum IgG and vaginal IgG, and IgA30 times5 × 109 cells/mL[40]
Transmissible gastroenteritis coronavirus (TGEV) Oral/PigletInduction of systemic and mucosal immune responses, cellular immunity, switching from Th1 to Th2-based immune responses1–48 h1 × 1010 CFU/mL[41]
Classical swine fever virus (CSFV) and porcine parvovirus (PPV)Pig/OralInduction of mucosal and systemic CSFV-specific CD8 CTL responses, anti-PPV-VP2 serum IgG, and mucosal IgA6 times1 × 1010 CFU/mL[42]
Infectious pancreatic necrosis virus (IPNV)Juvenile rainbow trouts/OralInduction of IgM and IgT, IL-1β, IL-8, CK6, MHC-II, β-defensin, TNF-1α, and reduction in viral load.2 times1 × 109 CFU/mL[43]
Human papillomavirus type 16 (HPV-16)Human/OralInduction of E7-specific humoral, cellular, and mucosal immune response20 times500, 1000, and 1500 mg/day[44]
L. lactisHuman papillomavirus type 16 (HPV-16)Healthy women/OralInduction of E6-specific IgG and SIgA antibody and, E6-specific IFN-γ-secreting CD8+ T cell immune response20 times1 × 109, 5 × 109, and 1 × 1010 CFU/mL[45]
L. acidophilusHuman immunodeficiency virus 1 (HIV-1)Mouse/OralTLR5-stimulating activity, maturation and cytokine responses of DCs, induction of gamma interferon-producing cells, and Gag-specific IgA-secreting cellsThree daily doses on weeks 0, 2, and 42 × 109 CFU/mL[46]
L. lactisStreptococcus pneumoniaeMouse/NasalInduction of PspA-specific IgG and IgA antibodies, and Th1-mediated immune response3 times1 × 109 CFU/mL[47]
L. casei Porcine epidemic diarrhea virus (PEDV) Mouse/OralInduction of mucosal and systemic immune responses,
IL-4, and IFN-γ		9 times2 × 109 cell/0.1 mL[48]
L. lactisAvian influenza virusMouse/OralInduction of specific anti-HA1 IgA and IgG antibodies, IL-4, and IFN-γ6 times1 × 1010 CFU/mL[49]
Avian Influenza
(HA1) Virus		Mouse/OralInduction of HA-specific serum IgG and fecal IgA, CD8+ T cell proliferation, and IFN-γ+13 times1 × 1010 CFU/mL[50]
L. plantarumInfluenza virus H9N2Mouse/OralInduction of CD3+CD4+IL-4+, CD3+CD4+IFN-γ+ and CD3+CD4+IL-17+ T cells, CD3+CD8+IFN-γ+ T cells, serum IFN-γ, IgA, sIgA, and increasing survival rate9 times109 CFU/0.1 mL[51]
L. lactisHepatitis E virus (HEV)Mouse/OralInduction of ORF2-specific mucosal IgA and serum IgG, and cellular immunity6 times1 × 1010 CFU/mL[52]
Human papillomavirus type 16 (HPV-16)Mouse/OralInduction of specific IgA and IgG, specific IL-2- and IFN-γ-secreting lymphocytes, and increasing survival rate9 times1 × 109 CFU/mL[53]
L. caseiHuman papillomavirus type 16 (HPV-16)Human/OralInduction of cellular and mucosal immune response1, 2, 4, or 6 capsules/day at weeks 1, 2, 4, and 8250 mg/ capsule[54]
L. lactisDengue (DEN) virusMouse/Oral and nasalInduction of anti-EDIII antibody responses6 timesFor oral: 1 × 1010 CFU/mL
For nasal: 1 × 108 CFU/mL		[55]
Human immunodeficiency virus (HIV)Mouse/OralInduction of HIV-specific serum IgG, fecal IgA, and Cell-mediated immune responses5 times1 × 108 CFU/mL[56]
L. plantarum SARS-CoV-2 ----[57]
Avian influenza virus H9N2Mouse and chicken/OralInduction of HI antibodies and T cell immune responses6 timesFor mouse:1 × 108 CFU/200 μL
For chicken: 5 × 108 CFU/500 μL		[58]
L. caseiHuman papillomavirus type 16 (HPV-16)Oral/MouseInduction of E7-specific mucosal IFNγ-producing cells and mucosal Th1 immune response16 times1 × 105 cells/head[59]
L. lactisRotavirusMouse/Oral and nasalInduction of Anti-rotavirus IgG and IgA antibodies, and reduction viral loadFor oral: 27 times
For nasal: 3 times		30 μg/dose[60]
New influenza A H1N1Mouse/OralInduction of anti-HA1 sIgA antibodies and humoral response9 times1 × 1010 CFU/mL[61]
Porcine transmissible gastroenteritis virus (TGEV)Mouse/OralInduction of IgG and IgA antibodies and local mucosal immune responses.9 times1 × 109 CFU/mL[62]
L. plantarumSpring viremia of carp virus (SVCV)Craps/OralInduction of IgM and reduction of viral loads27 times1 × 109 CFU/gr[63]
L. paracaseiRotavirus-induced diarrheaMouse/OralReduction of infection in cell cultures, shortened disease duration, severity, and viral load4 times1 × 107, 1 × 108, and 1 × 109 CFU/mL[64]
L. lactisRotavirusMouse/oralInduction of sIgA and IgG9 times1 × 109 CFU/mL[65]
Human papillomavirus type 16 (HPV-16)Mouse/NasalInduction of E7-specific cytotoxic T-lymphocyte response, antigen-specific immune response, high survival rate3 times1 × 109 CFU/mL[66]
Avian influenza virusChicken/NasalInduction of specific serum IgG9 times4 × 1010 CFU/100 µL[67]
L. pentosus Transmissible gastroenteritis virus (TGEV) Mouse/OralInduction of serum IgG and mucosal IgA9 times2 × 109 CFU/100 µL[68]
B. longumSARS-CoV-2Human/OralOngoing project; the final results will be made available on 28 February 2022.Single dose1 × 109, 3 × 109, and 10 × 109 CFUNCT number: NCT04334980
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Taghinezhad-S, S.; Mohseni, A.H.; Bermúdez-Humarán, L.G.; Casolaro, V.; Cortes-Perez, N.G.; Keyvani, H.; Simal-Gandara, J. Probiotic-Based Vaccines May Provide Effective Protection against COVID-19 Acute Respiratory Disease. Vaccines 2021, 9, 466. https://doi.org/10.3390/vaccines9050466

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Taghinezhad-S S, Mohseni AH, Bermúdez-Humarán LG, Casolaro V, Cortes-Perez NG, Keyvani H, Simal-Gandara J. Probiotic-Based Vaccines May Provide Effective Protection against COVID-19 Acute Respiratory Disease. Vaccines. 2021; 9(5):466. https://doi.org/10.3390/vaccines9050466

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Taghinezhad-S, Sedigheh, Amir Hossein Mohseni, Luis G. Bermúdez-Humarán, Vincenzo Casolaro, Naima G. Cortes-Perez, Hossein Keyvani, and Jesus Simal-Gandara. 2021. "Probiotic-Based Vaccines May Provide Effective Protection against COVID-19 Acute Respiratory Disease" Vaccines 9, no. 5: 466. https://doi.org/10.3390/vaccines9050466

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Taghinezhad-S, S., Mohseni, A. H., Bermúdez-Humarán, L. G., Casolaro, V., Cortes-Perez, N. G., Keyvani, H., & Simal-Gandara, J. (2021). Probiotic-Based Vaccines May Provide Effective Protection against COVID-19 Acute Respiratory Disease. Vaccines, 9(5), 466. https://doi.org/10.3390/vaccines9050466

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