Live-attenuated vaccines

These vaccines include a type of the living virus that has been weakened, thus it does not cause severe disease in people with healthy immune systems. Attenuated viruses are not able to replicate enough to cause illness but can protect people against future infection [10, 11]. These vaccines, as the closest models of natural infections, are suitable for training the immune system response against particular viruses. Live attenuated vaccines for certain viruses are relatively easily produced. This type of vaccine could become virulent for immunocompromised individuals, such as those with cancer or other immune system diseases. Live attenuated vaccines commonly have to be protected from light and refrigerated. This makes it hard to ship the vaccines overseas, particularly to places with no refrigeration [12].

Inactivated vaccines

Another technique of vaccine manufacturing is the inactivation of the pathogen by chemical treatment or by heat. Keeping the epitope structure on the epitope antigen during the inactivation method is critical. Chemical inactivation with formaldehyde has been more successful. For instance, formaldehyde inactivation is used to produce the Salk polio vaccine. Killed or inactivated pathogens do not replicate; the probability of those becoming virulent and causing a disease is zero [13]. Nearly a century ago, researchers reduced the toxicity of diphtheria toxin by formalin [14]. Furthermore, Ramon et al. [15] demonstrated the possibility of inactivating the toxins while keeping their structure intact. The influenza inactivated vaccine was the first vaccine that had been inactivated [16].

Toxoid vaccines

Toxoid vaccines have been sufficiently attenuated and can trigger a humoral immune response. Toxoid vaccines don’t give the recipient long-term immunity; therefore, like other kinds of vaccines may need a booster to provide ongoing defense against diseases.

Biosynthetic vaccines

Biosynthetic vaccines like the hepatitis B vaccine include man-made substances that are identical to fragments of the bacteria or virus. Biosynthetic vaccines induce a strong immune response targeting the key part of the bacterial and/or virus. These types of vaccines can safely be administered to immunocompromised individuals and those with chronic conditions [17, 18].

DNA vaccines

The encoded protein in the DNA vaccine is in its native form and has no denaturation or alteration. Thus, the immune response is similar to the antigen expressed by the pathogen. There are human trials underway with various DNA vaccines, like those for malaria, herpesvirus, and influenza. Currently, there is no human vaccine in use for defending against parasites; however, investigators are hopeful for the immunity of DNA vaccines against parasitic diseases [19].

Recombinant vaccines

These types of vaccines use attenuated microorganisms as vectors. A gene encoding the main antigen of a virulent microorganism can be introduced into an attenuated virus or bacterium. Upon the introduction of the modified virus to body, the immunogen is expressed, making an immune response against the immunogen that is derived. Human adenoviruses have been recognized as possible carriers for recombinant vaccines, mostly against diseases like acquired immune deficiency syndrome [19].

OVs

Mechanism of action of OVs

Routes for delivered antigens through the intestine are illustrated in Figure 1. These vaccines can access three parts of the immune system; (1) the lamina propria [that dendritic cells (DCs) and lymphocytes are distributed through the lymphatic drainage channels], (2) the Peyer’s patches (extremely structured lymphoid follicles specific for inducing of immune system responses) [12]; and (3) mesenteric lymph nodes [23]; the same lymphatics can also feed into the peripheral systemic blood circulation through the thoracic duct [24]. Recently, studies have revealed that antigens can be taken up by enterocytes by a phagocytic process [25]. The physicochemical properties of the antigen can control the route taken in the body. Water-soluble tiny particles can diffuse across the semipermeable membranes of blood microcapillaries, while large and lipidic compounds will be retained in the lymphatics and drain into the mesenteric lymph nodes [20].

Display full size

Figure 1. Three primary pathways by which orally administered vaccine antigens are delivered through the intestine to stimulate the body’s immune system. (1) Mesenteric lymph nodes: Antigens or antigen-loaded immune cells migrate to the mesenteric lymph nodes, a key site for orchestrating adaptive immune responses and promoting systemic immunity. (2) Lamina propria: Antigens cross the intestinal epithelial barrier and enter the lamina propria, where they are captured by antigen-presenting cells (APCs), initiating local immune responses. (3) Peyer’s patches: Specialized lymphoid structures in the intestinal mucosa, Peyer’s patches directly sample antigens from the gut lumen via M cells, facilitating the induction of mucosal and systemic immune responses.

Role of the microbiota

The gut microbiota is intricately associated with the immune system; so it is possible that immune responses to all types of vaccines are modified depending on microbial communities [30]. It has been revealed that a lack of mucosal bacteria is linked with a reduction of antiviral T-cell responses [31]. Moreover, skin-resident DCs respond to skin microbes [32]. Toll-like receptor (TLR) sensing of bacterial compounds can also contribute to antibody responses. For example, research on the influenza vaccine exposes that interactions between host TLR and bacterial flagellin can trigger macrophages to enhance the production of factors like interleukin-6 (IL-6), a proliferation-inducing ligand (APRIL), and tumor necrosis factor alpha (TNF-α), thereby resulting in increased plasma cell differentiation [33]. A study on oral typhoid vaccination has demonstrated that intestinal microbial diversity affects vaccine responses. Greater commensal richness and variety showed higher interferon gamma (IFNγ) induction by cluster of differentiation 8 (CD8⁺) T cells [34]. This results in greater antibody production, as T cell-derived IFN-γ induces IgG2a class [35]. In vaccinated Bengali infants, the frequency of Actinobacteria was positively related to strong T cell responses to OPV, while the presence of Pseudomonadales, Enterobacterales, and Clostridiales was negatively associated with the vaccine responses [36]. The probable effect of intestinal microbiota on OVs is not limited to enteric pathogens [37]. The ensuing cross-talk between the microbiota and mucosal immune cells is vital to the progress of the intestinal immune system [38]. In addition to these effects on the immune system, the microbiota appears to ease the uptake and replication of enteric viruses [39]. Several studies have shown that augmenting the infant microbiota with probiotic supplements has the ability to enhance OV efficacy [40]. However, daily administration of supplements of Lactobacillus rhamnosus GG during a placebo-controlled trial provoked an improvement in Rotarix immunogenicity. The gut microbiota plays a critical role in shaping both innate and adaptive immune responses. These modulatory effects are particularly relevant for OVs, as a healthy and balanced microbiota can enhance antigen uptake, promote mucosal IgA production, and improve the efficacy of vaccine vectors delivered via the gastrointestinal tract [41].

Role of nutrition

Diet is perhaps the primary factor dictating the variety and composition/function of the gut microbiota [42]. A high fiber diet can help maintain a healthy gut microbiota [43]. Equally, disorders in the nutritional status of individuals are associated with impaired immune responses [44]. Malnutrition in children results in lower seroconversion rates of OVs [45]. Undoubtedly, nutrient deficits are related to the loss of OV efficacy. In the following sub-sections, we will discuss nutrition factors that influence the immune system and vaccine efficacy. Adequate nutrition is essential for the proper development and function of the immune system. In the context of OVs, optimal nutritional status can enhance antigen-specific immune responses, support mucosal barrier integrity, and improve the effectiveness of vaccine vectors, thereby increasing the likelihood of successful immunization [46].

Genetically modified plants

Genetically modified plants are formed by introducing the anticipated gene into a plant to make the encoded protein. OVs against several diseases like cholera, measles, and hepatitis are formed in plants, namely tobacco, banana, potato, etc. [64]. Tobacco, formerly used as a model plant, is perennial and has many benefits, such as fast growth, and has been widely used as an edible vaccine candidate [65]. A main drawback of using the potato as a vector is that it needs to be cooked before use, denaturing the antigen [66]. Maize or rice are staple foods in most of the countries. The main reason why they are appealing as a candidate edible vaccine vector is that they can be kept without chilling for a longer period [67].

Genetically modified lactic acid bacteria

There has been interest in developing recombinant lactic acid bacteria (LAB) as the next class of OV vectors due to their stability at room temperature, natural acid and bile resistance, and endogenous activation of immune responses. LABs are generally recognized as safe (GRAS) and have long been used in food fermentation. Furthermore, they can improve the health of consumers when consumed in an adequate amount [68]. It is important to distinguish between LAB engineered as vaccine vectors and LAB or postbiotics functioning as immunostimulants. LAB vaccine vectors are genetically modified strains that express or deliver specific antigens and therefore act as part of the antigen-presentation pathway to induce targeted immune responses. In contrast, non-engineered LAB and postbiotics do not provide antigen specificity; instead, they enhance immunity through indirect mechanisms such as epithelial barrier reinforcement, cytokine modulation, and activation of mucosal immune cells. Thus, LAB vectors contribute directly to antigen presentation, whereas LAB and postbiotics primarily serve as immunostimulatory adjuvants that amplify or shape the host response to an external antigen [69]. General effects of LAB in the intestinal tract are recognized to include alteration of the intestinal microbiota, enhanced barrier function, and the modulation of the host immune system [70]. For decades, the use of probiotics as an OV vector has been developed against different viral and bacterial pathogens and toxins [71]. The typical LABs’ interactions with the mucosal immune system are shown in Figure 2. LABs can stimulate innate immune response via peptidoglycan and lipoteichoic acid that activate TLR2, nucleotide-binding oligomerization domain NOD-like receptor (NLR) family, and C-type lectin receptors [72, 73].

Display full size

Figure 2. Mechanism of action of probiotics in modulating the immune system. (1) Mucus binding: Probiotics adhere to the intestinal mucus layer, promoting colonization and competitive exclusion of pathogens. (2) Induction of epithelial defenses: Probiotics enhance the integrity of the intestinal barrier and stimulate the production of antimicrobial peptides. (3) Uptake through microfold (M) cells: Probiotics and their components are sampled by M cells in the gut-associated lymphoid tissue, facilitating antigen presentation. (4) Stimulation of humoral responses: Probiotics trigger B cells to produce immunoglobulins, such as secretory IgA, contributing to mucosal immunity. (5) Transport to lymphoid tissues: Antigens and probiotic components are delivered to lymphoid organs, promoting immune cell activation. (6) Activation of macrophages and dendritic cells: These antigen-presenting cells are stimulated to initiate adaptive immune responses. (7) Induction of effector immune responses: Probiotics enhance T cell-mediated responses and the secretion of pro-inflammatory cytokines, supporting pathogen clearance and immune regulation. LAB: lactic acid bacteria; DCs: dendritic cells; Tregs: regulatory T cells.

The immunomodulatory effects of LABs are noted in Table 1. In addition, some LABs can bind to the mucosal epithelium or microfold (M) cells, resulting in mucosal colonization. LABs can interact with APCs like DCs and induce IgG. The mechanism of immune cell activation and the cause of immune responses are extremely dependent on the LAB strain. For instance, murine DCs can have different responses reliant on the strain of LAB [73–77].

Genetically modified LAB, particularly Lactococcus lactis and various Lactobacillus species, have become attractive platforms for mucosal vaccine delivery due to their GRAS status, safety, and intrinsic immunomodulatory properties [93]. Modern vaccine engineering strategies allow LAB to express, secrete, or display antigens in ways that enhance uptake by mucosal immune tissues. Plasmid-based expression remains one of the most widely used approaches, employing strong promoters such as P170, PnisA (used in the nisin-controlled expression system), or constitutive promoters like Phelp to achieve robust intracellular antigen production. The NICE system, in particular, enables precise induction of antigen expression during fermentation, improving protein yields while minimizing the metabolic burden on the host strain [94]. Chromosomal integration strategies provide an alternative with greater genetic stability and improved biosafety, eliminating the need for antibiotic selection markers. Stable integration into loci such as upp, thyA, or neutral chromosomal regions in L. lactis has been shown to maintain antigen expression during gastrointestinal transit, which is essential for consistent mucosal delivery [95, 96]. Another key advancement is the surface display of antigenic proteins using anchoring domains such as LPXTG (for sortase-mediated covalent attachment), or LysM repeats (for non-covalent binding). Surface-anchored antigens are more efficiently recognized by intestinal DCs and M cells, leading to improved stimulation of the GALT and enhanced mucosal IgA responses [97]. Secretion-based systems also represent a critical component of LAB-based vaccine technology. Signal peptides such as Usp45 from L. lactis have been extensively employed to direct heterologous proteins into the extracellular environment [98]. Usp45-mediated secretion is one of the most efficient systems currently available for LAB and has been utilized to deliver cytokines, viral antigens, and bacterial toxins in mucosal immunization models [99]. Other secretion leaders, including SlpA from L. acidophilus, further expand the repertoire of antigens and enzymes that can be released into the culture medium or intestinal lumen [100]. Although whole-cell vaccine vectors such as probiotics and plant-based systems offer powerful platforms for oral antigen delivery, increasing evidence shows that many of their immunological benefits are mediated by the bioactive molecules they produce rather than by the living organisms themselves. This provides a natural transition toward postbiotics, non-viable microbial components and metabolites that retain these immunomodulatory properties while offering improved safety, stability, and mechanistic precision [101]. In the following section, we discuss these postbiotic components in greater detail and highlight their relevance as emerging OV adjuvants.

Definition and safety aspect

The term postbiotics refers to all products obtained from non-viable probiotic microorganisms, lysates, cell walls, fractions, secretions, and metabolites that, when received in adequate amounts, show health benefits [102–109]. They exhibit strong antibiofilm activity, helping to disrupt and prevent the formation of harmful microbial biofilms that contribute to chronic infections [110–113]. Certain postbiotic compounds, including organic acids and specific enzymatic metabolites, can also aid in the detoxification and removal of mycotoxins and pesticide residues [114, 115], thereby enhancing food safety and reducing toxic exposure. Postbiotics also could be synthesized in a laboratory through different kinds of methods, such as high pressure, ionizing radiation, thermal treatment, and sonication [116, 117]. Due to the related safety aspect, numerous clinical studies have been done to validate the metabolism and absorption of postbiotics. A handful of randomized controlled trials (RCTs) have assessed the side effects of probiotics metabolite (postbiotics), including vomiting, diarrhea, and nausea in individuals consuming Lactobacillus acidophilus, but no side effects were revealed in the remaining RCTs [118, 119].

Mechanism of action of postbiotics

A key step in generating resistance to viral disease is to modulate the immune system [120, 121]. Studies have demonstrated that probiotic metabolites regulate the immune system in a variety of ways (Figure 3), particularly when it comes to seasonal and highly pathogenic influenza virus infections.

Display full size

Figure 3. Postbiotics could induce activation of dendritic cells, afterward, its mediated differentiation of naïve T cells into Tregs, which would control the extreme T cell response. Furthermore, postbiotics could stimulate the production of cytokines via macrophages that might induce homeostasis through the reduction of pro-inflammatory cytokines.

The mechanism of immunomodulation by healthy gut microbiota is extremely associated with their derived postbiotics [122–124]. Some immunomodulatory effects of postbiotics are shown in Table 2.

Postbiotics have potent antiviral properties against a variety of infections, especially viruses that cause respiratory illnesses [141, 142]. These molecules are used to prevent influenza A and respiratory syncytial virus infections by decreasing infectious symptoms, diminishing the duration of infection, lowering viral levels in the lungs or nasal washings, and boosting immune activity [143–145]. Lactobacillus plantarum L137, Lactobacillus plantarum 06CC2, and Lactobacillus gasseri TMC0356 soluble postbiotics can be used as possible antiviral agents in pharmaceutical products to treat virus infections [146]. Recently, it was postulated that gut dysbiosis in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) patients could be defined as a cytokine storm, explaining the proinflammatory status of the gut. This could be due to a decrease in bacterial metabolite synthesis [147]. Postbiotics’ anti-inflammatory attributes can reduce the severity of SARS-CoV-2 via decreasing the gut inflammation, activating Tregs, and reducing systemic inflammation by enhancing the gut barrier, thus preventing SARS-CoV-2 from spreading to other organs. To reduce cytokine storms, postbiotics also block several proinflammatory signaling pathways [148]. Balzaretti et al. [149] reported the immunostimulant properties of L. paracasei DG-derived exopolysaccharide by improving the gene expression of the proinflammatory cytokines, TNF-α and IL-6, and particularly that of the chemokines IL-8 and CCL20, in the human monocytic cell line Tamm-Horsfall protein 1(THP-1). Postbiotics exert a broad spectrum of immunomodulatory and anti-inflammatory activities through well-characterized molecular and cellular mechanisms. These effects are largely mediated through interactions with pattern-recognition receptors (PRRs), including TLRs and NLRs, which sense microbe-associated molecular patterns (MAMPs) derived from bacterial cell walls, metabolites, or secreted peptides. Components such as peptidoglycans, lipoteichoic acids, EPS, and short-chain fatty acids (SCFA) can bind to TLR2, TLR4, and NOD2, triggering downstream signaling through MyD88, NF-κB, and MAPK pathways. Activation of these pathways leads to balanced cytokine responses, including increased production of IL-10, IL-6, TGF-β, and IL-22, alongside controlled suppression of pro-inflammatory cytokines such as TNF-α and IL-1β [150, 151]. These immunomodulatory effects support enhanced IgA class switching in Peyer’s patches, improved antigen presentation by intestinal DCs, and modulation of Th1/Th2/Th17 differentiation, which collectively enhance mucosal immune responsiveness to orally delivered vaccines. Several postbiotic-derived metabolites also regulate antiviral and antibacterial immunity through cellular mechanisms. SCFAs such as acetate and butyrate enhance epithelial barrier integrity by upregulating tight junction proteins (ZO-1, occludin, claudins) via GPR41 and GPR43 signaling [152]. Indole derivatives generated by tryptophan metabolism act on the aryl hydrocarbon receptor (AhR), stimulating IL-22 secretion and supporting mucosal epithelial repair [153]. Cell-wall fragments from Lactobacillus and Bifidobacterium have been shown to increase DC maturation and M-cell transcytosis efficiency, improving the uptake of co-administered antigens. Additionally, bacterial extracellular vesicles (BEVs) can trigger type-I interferon responses via cGAS–STING and RIG-I pathways, offering synergistic adjuvant activity in OV formulations [154]. Although postbiotics are not antigen-specific vaccines, several experimental studies have explored their use as oral adjuvants in vaccine formulations and have reported indicative dosage ranges. In murine models, orally administered postbiotic preparations typically range between 10–200 mg/kg/day, depending on molecular composition and concentration of bioactive metabolites. For instance, heat-killed L. rhamnosus GG or its cell-wall fractions have been administered at 10⁸–10⁹ cell equivalents/day to enhance mucosal IgA responses [155]. SCFA-rich postbiotic extracts are commonly tested in the range of 50–150 mg/kg and have demonstrated increased antigen-specific IgA and balanced Th1/Th2 responses when co-administered with oral viral antigens [156–158]. Determining an appropriate treatment dose for postbiotics in humans requires a structured and evidence-based translational approach that moves systematically from preclinical data to clinical application. The first essential step is to establish the effective dose range in animal models by evaluating dose–response curves, pharmacodynamic markers (e.g., mucosal IgA levels, cytokine profiles, DC activation), and safety outcomes. The NOAEL (No-Observed-Adverse-Effect Level) identified in animals can then be converted into a human equivalent dose (HED) using standard allometric scaling based on body surface area. This approach is widely recommended by regulatory agencies such as the FDA for biologically active compounds [159]. The HED is then typically reduced by a safety factor (commonly 10-fold for novel immunomodulatory agents) to calculate a conservative and ethically acceptable starting dose for early human trials. Currently, no standardized clinical dosing guidelines exist for oral postbiotic vaccine adjuvants, and dose-response evaluations remain one of the major research gaps that require future preclinical and human trials.