Maimoona Ayyaz Ansari
Nishtar Medical University Multan
As already developed a safe and effective nasal vaccine delivery system using a self-assembled nanosized hydrogel (nanogel) made from a cationic cholesteryl pullulan are available. In this research three pneumococcal surface protein fusion antigens as a universal pneumococcal nasal vaccine and then encapsulated each PspA into a nanogel with Cu nano particles and mixed the three resulting monovalent formulations into a trivalent nanogel–copper formulation. First, to characterize the nanogel–PspA formulations, native polyacrylamide gel electrophoresis (PAGE) was used to determine the average number of PspA molecules encapsulated per nanogel molecule. Second, two methods were adopted—a densitometric method based on lithium dodecyl sulfate (LDS)–PAGE and a biologic method involving sandwich enzyme-linked immunosorbent assay (ELISA)—to determine the PspA content in the nanogel formulations. Third, treatment of nanogel–PspA formulations by adding copper nanoparticles released each PspA in its native form, as confirmed through circular dichroism (CD) spectroscopy. Results showed that trivalent nanogel–PspA formulation induced equivalent titers of PspA-specific serum IgG and mucosal IgA Abs in immunized mice. These results show that the specification methods we developed effectively characterized our nanogel-based trivalent copper nasal vaccine formulation.
Epidemic infections, such as cholera, malaria, and the sudden outbreak of COVID-19, cause global life threats and socioeconomic recession. One of the most devastating pandemics was the Black Death, an infection caused by Yersinia pestis, which killed over 75 million people in 1350. At present, the world is still in the midst of the COVID-19 pandemic, with 3,398,302 COVID-19-related deaths recorded as of May 19, 2021. Fur- thermore, antibiotic resistance leads to the ineffectiveness of tradi- tional antibacterial treatments, and nearly half a million new cases of multidrug-resistant tuberculosis (MDR-TB) occur worldwide .Among coping strategies for emerging infectious diseases, preventive vaccination is a crucial strategy  and the only one with wide and thorough effects on the public health. For example, since 2000, vaccination has reduced the reported incidence of measles by 83%, thereby preventing over 20 million deaths . Vaccines work by training and utilizing the body’s immune system to recog- nize and fight the pathogens. However, the high infection rates, widespread transmission, or high fatal ratio of certain epidemic germs raised huge challenges for the design of prophylactic
vaccines.Vaccines have evolved from the live-attenuated or inactivated vaccines to the subunit/peptide vaccines. Regardless of the safety issues, the live-attenuated and inactivated vaccines, which are nanoparticles themselves , are ready to be captured and pro- cessed by antigen-presenting cells (APCs), including dendritic cells (DCs), macrophages, and B cells, resulting in an efficient immune response. In contrast, subunit vaccines or recombinant vaccines are characterized by improved safety, but much weaker immuno- genicity. To break the bottleneck of previous vaccine types, advanced delivery systems (e.g., micro/nano delivery) exhibiting both high efficacy and safety are greatly required. Such systems will likely help to catalyze novel candidate vaccines toward clinical testing at an unprecedented speed. For example, lipid nanoparticles dictated the success of the mRNA vaccines in 2020, making an enormous contribution to control the spread of COVID-19 at a global level.
The advantages of vaccine delivery systems have been well documented in recent reviews. For example, Ding et al.  sum- marized the nanosystems with superior therapeutic or preven- tive effects, providing an important clue on maintenance of our well-being by exploiting the immunomodulatory property of nanomaterials. Other works also referred to using nanotechnol- ogy or materials science approaches with delivery systems [5,6]. In addition, vaccine delivery systems focusing on tackling a particular infectious disease (e.g., COVID-19)  or cancer  were reviewed. Nevertheless, there has been no systematic sum- mary of advanced delivery systems and design concepts for pro- phylactic vaccines for a wide range of epidemic infectious diseases. Taking into account the diverse species and pathogenic mechanisms of infection pathogens, the protection efficacy of different systems against the same infection has not been suffi- ciently compared. In addition, the requirements and priorities of the delivery systems for different infectious diseases or stages are also case by case. Obtaining such data would greatly support designing the optimal delivery vectors for specific infectious diseases.
In this review, we provide an overview of the major or epidemic infectious diseases (pneumonia, diarrhea, candidiasis, malaria, and others) caused by bacteria, viruses, fungi, and parasites. We also list advanced delivery systems against different infectious diseases and compare their protective efficacy from different aspects. More- over, we include the current vaccines and vaccine delivery systems that are either newly licensed (e.g., COVID-19 vaccines) or close to licensure. In particular, we highlight the advanced delivery systems with high efficiency, cross-protection, or long-term pro- tection against epidemic pathogens.
2. Bacterial infectious diseases and advanced delivery systems
2.1. Respiratory infectious diseases and advanced delivery systems
Respiratory infections represent a serious health problem worldwide that mainly affects children, older people, and immuno- compromised individuals. Pneumonia vaccines (e.g., 23-valent polysaccharide vaccines, 13-valent polysaccharide conjugate vacci- nes) have achieved great success worldwide. Nevertheless, the existence of a variety of serotypes (>95) of S. pneumoniae makes the capsular polysaccharide (CPS)-based vaccines unable to pro- vide broad protection , while the cost of preparing vaccines con- taining all serotypes is very high. In addition, there may still be undetected serotypes . In this case, effective delivery systems of proteinaceous antigens have been developed for the prevention of pneumonia. In addition to the delivery efficacy, a combined mucosal and systemic immune response is also appreciated for pneumonia vaccines , which not only requires novel delivery design but also a specific administration route (e.g., noninvasive mucosal routes). At present, there are a variety of delivery systems for pulmonary infectious diseases and they have shown good results (Table 1).
2.1.1. S. pneumoniae
S. pneumoniae is a global endemic pathogen causing a wide range of clinical diseases, such as pneumonia, meningitis, and sep- sis, which frequently lead to death among children all over the world, especially in developing countries . Colonization with
S. pneumoniae in humans is universal ; however, it provides an opportunity for the remaining serotypes to establish residence and progress to virulence [48,49]. In addition to direct infection, the bacteria usually exist in the form of biofilm, and some destruc- tive events, such as viral infection, can prompt the release of a vir- ulent subpopulation of bacteria to the lungs, blood, middle ear, and other parts of the body, causing the aforementioned diseases [50,51]. Therefore, high levels of IgG antibodies produced by humoral immunity are very important for invasive infections, and antigen-specific sIgA antibodies are the key to prevention of
S. pneumoniae colonization of the upper respiratory tract.
Various delivery vehicles (e.g., polymers, virus-like particles (VLPs), L. lactis, liposomes) have been used to deliver S. pneumoniae protein or glycan antigens, which showed a strong ability to pre- vent bacterial invasive infections and inhibit the colonization of the respiratory tract (Table 1). Among them, some vaccine formu- lations offered universal pneumococcal disease prevention. For example, Jones et al. proposed a vaccine platform through the lipo- somal encapsulation of polysaccharides (LEPS) technology. The completed LEPS vehicle (about 300 nm in size) was coupled with the PncO and GlpO protein antigens (identified through an antigen discovery and validation model that selectively targeted pneumo- cocci virulence transition ) for the liposomal containment of polysaccharides (serotypes 19F, 11A, and 35C). Thus, this vaccine not only prevents the colonization of the most aggressive S. pneu- moniae serotypes, but it also restricts virulence transition . Since pneumonia vaccines are often used in children and older adults, the safety and immune activation ability of the delivery car- riers need to be greater. Thus, we further focused on the latest several delivery systems that can produce the best protective effect.
It has been reported that a self-assembled nanosized hydrogel (nanogel) containing a cationic type of cholesteryl group-bearing pullulan (cCHP) can be used as a vaccine delivery system [53–55]. This nanogel could effectively transfer antigen to nasal epithelial cells and dendritic cells (DC) under the basement membrane and induce antigen-specific immune response as a non-adjuvanted vaccine (Fig. 1). After loading with a single protein antigen PspA, both specific IgG levels in the serum and bronchial fluids and IgA levels in the nasal fluid were significantly elevated in nasally administered mice , and all of the responses were involved in establishing protective immunity against pneumococci [56–59]. Further, a study in rhesus macaques revealed that the cCHP-based pneumococcal vaccine induced significantly elevated PspA-specific IgG and IgA levels and kept them for a long time . Moreover, positron emission tomography (PET) analysis com- bined with magnetic resonance imaging (MRI) has confirmed that the cCHP nanogel vaccine is not deposited in the olfactory bulbs and brains in macaques , suggesting that it is also safe to use as a nasal vaccine in humans.
Hybrid biological-biomaterial vector
Yi et al. have reported a combined delivery device named hybrid biological-biomaterial vector, which consists of a bacterial core electrostatically coated with a cationic polymer (a mannosylated poly (b-amino ester) (PBAE)) . The biological portion of the vector is a bacterial cell (live or dead), containing natural adjuvant properties, and it is ben- eficial for passive targeting of phagocytic APCs (Fig. 2). The vector’s composition and the surface characteristics endowed by the man- nosylated PBAE engage APC receptors and enhance the uptake upon vector administration. After subcutaneous injection of the vector expressing pneumonia PspA protein antigen in mice, a strong specific immune response providing a wide range of protec- tion against 10 different clinical S. pneumoniae was induced. More- over, the localization of PspA in the cytoplasm could provide a stronger immune protection effect than that in the periplasm or on the surface of bacteria . As for the combination of biomate- rial and biological components, each has its own antigen delivery characteristics and plays a role in the immune process. Moreover, such synergy is conducive for obtaining the best immune effects. In addition, E. coli can be further modified by genetic engineering to be more suitable for antigen delivery. Because the antigen is loaded with bacterial vectors, it provides a variety of possibilities for using different antigen locations and different loading forms, such as proteins and nucleic acids.
K. pneumoniae belongs to gram-negative family Enterobacteri- aceae. It exists widely in the natural environment and acts as opportunistic pathogen, so serious infections may be induced in patients with severe infections and weakened immune system . Klebsiella species remain the world’s most common nosoco- mial pathogens  and the main cause of hospital-acquired pneu- monia resulting in high mortality rate. Currently, the biggest challenge of K. pneumoniae treatment is drug resistance, which makes the use of common antibiotics ineffective. Indeed, the extended-spectrum b-lactam-producing and carbapenem- resistant K. pneumoniae (CRKP) has been recognized by the World Health Organization as a critical public health threat .
Outer membrane vesicles (OMVs)
A large number of gram-negative bacteria can naturally produce extracellular OMVs, which have significant advantages in vaccine development. Compared with other lipid nanoparticles, OMVs contain toll-like receptor (TLR) agonists, such as outer membrane proteins, lipoproteins and lipopolysaccharides (LPS), and a variety of immunogenic endogenous antigens. Generally, the diameter range of OMVs is from 50 nm to 250 nm, which is suitable for targeting and being phagocytized by APCs . The use of OMVs has become a very promising strategy of vaccination. K. pneumoniae-derived OMVs can induce strong humoral and cellular immunity response, pre- venting bacteria-induced lethality in intraperitoneally immunized mice . Although natural OMVs are considered potential vaccine candidates, they have some shortcomings. One is a wide size range of OMVs naturally secreted by bacteria, which may complicate the vaccine dynamics in vivo, and the low stability of natural OMVs