Streptococcus pneumoniae is naturally found in the human NP, and this nasopharyngeal carriage is important for active infection and transmission of the pathogen (McDaniel and Swiatlo, 2004). Its capsular polysaccharides (CPSs) are important virulence factors, especially for invasive disease that can be classified into 100 serotypes, based on chemical and immunological profiles (Ganaie et al., 2020). The most identified pathogen in OM following culture is S. pneumoniae (Tamir et al., 2023).

Currently, two types of pneumococcal vaccines are available. Both are based on CPS: (A) the 23-valent pneumococcal polysaccharide-based vaccine (PPV-23); and (B) the 7-, 10-, 13-, and 20-valent pneumococcal conjugate vaccines (PCV-7, −10, −13, −20).

PPV23 is based on the CPS antigens from 23 different serotypes of S. pneumoniae and is recommended for individuals aged 65 and older, as well as those aged 2 to 64 years who have multiple chronic conditions, such as diabetes and chronic cardiovascular disease (CDC, 2019). Most bacterial CPS are poorly immunogenic in young children, in part because they are T-cell independent antigens and induce little immune memory. Newborns and infants up to the age of 1.5–2 years of age are unable to produce antibodies to bacterial CPS due to less developed immunity (Rijkers et al., 1998), therefore, there is much debate surrounding the use of pneumococcal polysaccharide vaccines in young children (Borrow et al., 2012). In PCVs, the pneumococcal polysaccharide is coupled to a carrier protein to convert the antigens from T-cell independent to T-cell dependent antigens for enhanced immunogenicity (Pichichero, 2013). PCVs have been incorporated into the national immunization programs of 148 out of 194 World Health Organization (WHO) member states as of the end of 2020 (CDC, 2020). There has been a decline in OM and NP colonization by vaccine serotypes of S. pneumoniae with these vaccines, however, their use has been linked with replacement by non-vaccine serotypes of S. pneumoniae as well as NTHi and Mcat (Barenkamp et al., 2017; Kaur et al., 2017; Ben-Shimol et al., 2019; Pereira et al., 2023). Pneumococcal serotype replacement after vaccination has been noted for both childhood carriage (Lee et al., 2017) and in AOM (Gene et al., 2013), and in some cases the replacing strains are antibiotic resistant (Lo et al., 2019), so that vaccination may change the distribution of resistance and potential treatment failures not only for URTI/OM but also for pneumonia or invasive disease. A study in Israel evaluated the efficacy of PCV7 and PCV13 and reported that these vaccines reduced pneumococcal AOM by 77%, however, an increase in AOM cases due to non-vaccine serotypes was observed (Ben-Shimol et al., 2014). Similarly, a Costa Rican study of AOM in children reported that pneumococcus had a lower frequency and NTHi cultured at a higher frequency in vaccinated vs. unvaccinated children (Abdelnour et al., 2015). A 14-year observational study on the effects of PCV7, PCV10, and PCV13 in Swedish preschool children showed that Mcat and NTHi were proportionally more common than S. pneumoniae after PCV introduction in NP culture of PCV-immunized children with URTI (Littorin et al., 2021). A global surveillance study that used whole-genome sequencing for analysis reported that non-PCV serotypes15B and 15C (15B/C), 12F, and 35B/D were among the most prevalent pneumococcal serotypes in the post-PCV13 era (Lo et al., 2019). In Italy, non-vaccine serotype 24F has been reported as a prevalent serotype in pneumococcal otorrhea cases (Sings et al., 2019). Such epidemiological studies of pneumococcal serotypes from both carriage and OM inform future changes to polyvalent vaccines. Notably, PCV-20 includes several recently emergent serotypes (1, 3, 4, 5, 6A, 6B, 7F, 8, 9 V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F) and is now approved for use in all age groups in the United States (ACIP, 2023).

As there are at least 100 pneumococcus serotypes, increasing the number of vaccine serotypes in the PCV vaccines is more complex and expensive. In summary, both PPV and PCVs induce serotype-specific immunity and their usage has been linked with the emergence of replacement serotypes (Weinberger et al., 2011; McElligott et al., 2015; Kaur et al., 2016; Kawaguchiya et al., 2017). Nonetheless, the success of PCVs in reducing childhood mortality and morbidity due to pneumonia and invasive disease is significant, for example, see a recent review of pneumococcal disease in children in the United States (Walter and Smith, 2022).

Despite the success of PCVs, the challenge of regular re-formulation, and the theoretical and practical challenges of including more serotypes means the search for protein antigens has continued, aimed at developing pneumococcal vaccines targeting conserved and universally present pneumococcal proteins to complement or ultimately substitute PCVs. A protein-based vaccine would theoretically be simpler and cheaper to make than a conjugate vaccine. Several protein antigens have been investigated in human trials, individually, and in multivalent formulations. Table 1 summarizes antigens and vaccine candidates for pneumococcal diseases [Also recently reviewed by (Duke and Avci, 2023)].

Pneumolysin (Ply) is a cholesterol-dependent cytolysin that has haemolytic properties, and aids in bacterial pathogenesis and infection. Vaccination with a detoxified or non-toxic form of Ply (dPly) is known to confer protection against multiple serotypes in animal models (Alexander et al., 1994; Ogunniyi et al., 2001; Denoël et al., 2011). A genetically mutated pneumolysin derivative (PlyD1) was safe, robustly immunogenic, and induced neutralizing antibody responses in a phase 1 trial (NCT01444352) (Kamtchoua et al., 2013). More recently, a genetic toxoid of Ply with two amino acid mutations (G293S and L460D) termed Ply-D protected mice from the lethal challenge of various clinical pneumococcal isolates (Thanawastien et al., 2021).

Pneumococcal histidine triad protein D (PhtD) is a highly conserved adhesin protein. The PhtD-based vaccines protect immunized mice against NP and lung colonization of pneumococcus (Khan and Pichichero, 2012; Kaur et al., 2014b; Brookes et al., 2015; Ochs et al., 2016). Combining PlyD1 and PhtD, and including PHiD-10 (conjugated to protein D of H. influenzae), Odutola et al. (2016, 2017, 2019) reported on phase 2 trials in African children (NCT01262872), while the same vaccine was assessed in toddlers in Czech republic for safety, immunogenicity, and non-inferiority to PCV-10 (Urbancikova et al., 2017). None of these trials assessed protection against OM. Pneumococcal surface protein A (PspA) is a choline-binding protein that is located on the cell surface in approximately all pneumococcus strains and is involved in the complement-mediated clearance of bacteria. PspA was shown to be safe during phase I clinical trials (Briles et al., 2000b). However, PspA shows homology to the human cardiac myosin and must be modified to prevent possible autoimmune disease or cardiac inflammation (Ginsburg et al., 2012). PspA recently underwent phase 1a clinical trial as part of a multi-element vaccine alongside PlyD (NCT04087460, 2022). Pneumococcal choline-binding protein A (PcpA) is a surface-exposed protein that is conserved among different pneumococcal strains (Glover et al., 2008). A bivalent PcpA/PhtD vaccine was immunogenic and safe in humans during a phase 1 clinical study (NCT01444339) (Bologa et al., 2012), and induced functionally relevant antibodies (Ochs et al., 2016). A trivalent vaccine containing recombinant PcpA, PhtD, and PlyD1 (designed from serotype 6B) protected infant mice from the serotype 6A challenge (Verhoeven et al., 2014). This vaccine formulation was considered safe and immunogenic in a phase 1 study including adults, toddlers, and infants (NCT01764126) (Brookes et al., 2015). Furthermore, serum IgG antibody responses against PhtD, PcpA, and PlyD1 were in synchrony, indicating they are similarly immunogenic and therefore compatible with combining in a trivalent protein vaccine (Ren et al., 2015).

Another serotype-independent pneumococcal protein vaccine candidate called PnuBioVax™ is expected to offer broader coverage at a low unit price. This vaccine is formulated by fermenting a genetically modified S. pneumoniae TIGR cell substrate. Following harvesting, the protein antigens are detergent-extracted and purified using ion exchange chromatography (Cecchini et al., 2015). During phase 1 human trials (NCT02572635), this vaccine demonstrated good safety and immunogenicity profile (Entwisle et al., 2017; Hill et al., 2018).

Other antigens have been investigated in animal models and show promise for inclusion in a multivalent protein vaccine. Kong et al. developed a PspA vaccine with an intranasal vaccine delivery system based on a nanometre-sized hydrogel (nanogel) composed of a cationic cholesteryl group-bearing pullulan (cCHP). Intranasal vaccination with the cCHP-PspA vaccine protected mice from a lethal S. pneumoniae Xen10 challenge by reducing the invasion and colonization of bacteria in the upper and lower respiratory tracts (Kong et al., 2013). Wang et al. utilized a bacterium-like particle (BLP) delivery system designed to express PspA on the BLP surface. Intranasal delivery of this vaccine-induced PcpA-specific IgG and IgA antibodies in mice and protected the animals from a lethal challenge with S. pneumoniae (Wang et al., 2018). A recombinant PspA-based protein vaccine (PspAB_1-5) consisting of the B region fragments from clades 1 to 5 of both families resulted in improved C3 complement component depositioning in immunized mice. The antibodies induced were cross-reactive against pneumococci from clades 1, 2, and 5. Consequently, the PspA vaccines based on the B region of all clades may be able to provide better protection against S. pneumoniae (Akbari et al., 2019).

Elongation factor Tu (EF-Tu) is a universally expressed surface protein that is highly conserved in different S. pneumoniae. EF-Tu has chaperone activity and mediates peptide biosynthesis, protein folding, and cellular stress response (Gregersen and Bross, 2010). A vaccine containing recombinant EF-Tu from S. pneumoniae strain D39 protected immunized mice against lethal challenges with serotype 2 and a multidrug-resistant serotype 15A, and increased the levels of cytokines including TNFα, IL-6, IL-17, and IFN-γ. The anti-EF-Tu serum demonstrated an enhanced phagocytic activity against S. pneumoniae, irrespective of its serotypes (Nagai et al., 2019). Hence, EF-Tu presents as a potential broad-spectrum vaccine candidate against common pneumococcal serotypes. LytB is an endo-β-N acetylglucosaminidase involved in the formation of biofilm, separation of daughter cells, and pathogenesis (Bai et al., 2014). An anti-LytB antiserum demonstrated significant protection in mice from a lethal pneumococcal challenge (Wizemann et al., 2001). In another study mice immunized with LytB showed enhanced complement-mediated immunity against various pneumococcal serotypes. Anti-LytB serum-stimulated neutrophil-mediated phagocytosis against S. pneumoniae (Corsini et al., 2016). Thus, LytB could be an effective antigen in a pneumococcus vaccine.

In an interesting approach, Mann et al. fused peptides of choline-binding protein A (CbpA) with a non-toxic Ply (L460D). In mice, this fusion was more effective than L460D at reducing nasal colonization, OM, pneumonia, OM, and meningitis (Mann et al., 2014).

Shi et al. developed three live recombinant Salmonella Typhi vectors expressing PspA pneumococcal protein. These live vectors were shown to be highly immunogenic in mice and highly susceptible to killing in human blood (Shi et al., 2010). In a phase I clinical trial, administration of these live attenuated Salmonella strains was safe and well-tolerated in healthy adult subjects. The immunogenicity profile of these live vectors was limited perhaps because of pre-existing cross-reactive antibodies, therefore further genetic manipulation is needed to develop a candidate with improved immunogenicity (Frey et al., 2013).

Whole-cell vaccines are an attractive alternative to polysaccharide-based vaccines because they take advantage of whole-cells expressing various protein antigens without involving the purification of individual antigens. Live-attenuated or killed whole-cell vaccines from unencapsulated S. pneumoniae can provide serotype-independent protection in animal models. One caveat is that use of live attenuated strains must be used cautiously in populations with a risk of impaired or sub-optimal immune status. The pneumococcal whole-cell vaccine (wSp) consists of a killed, unencapsulated pneumococcal strain delivered with an alum-based adjuvant. The purpose was to provide a cost-effective vaccine with broader coverage. In phase 1 studies in healthy adults in the United States (NCT01537185), the vaccine demonstrated a good safety, tolerability, and immunogenicity profile (Keech et al., 2020). It has also been tested in phase 1 and 2 trials in healthy adults and toddlers in Kenya to evaluate its safety and immunogenicity which was reported to be satisfactory (NCT02543892). Although it has not been assessed for its efficacy in the human paediatric OM population, a subcutaneous dose of wSp decreased the density of pneumococcus in the ME of mice, however, it could not protect from S. pneumoniae-induced OM (Manning et al., 2019). A whole-cell vaccine consisting of an ethanol-killed capsule-deficient S. pneumoniae mutant prevented the colonization of serotype 19F and 4 strains in mice (Xu et al., 2014; Jang et al., 2019). Chan et al. reported a multiple-antigen vaccine (MAV) based on bacterial lysates. In addition to having the advantages of a whole-cell vaccine, the preparation of this vaccine improved the amount of surface antigens as it had heat shock proteins (Hsps), PspA, and Ply proteins. In animal studies, MAV was able to elicit functional antibody production against multiple serotypes of S. pneumoniae, including non-vaccine serotypes (Chan et al., 2022). Based on these observations, it can be anticipated that the MAV approach may confer serotype-independent protection against S. pneumoniae.

Immunization of mice with a live attenuated whole-cell vaccine based on S. pneumoniae D39 (lacking pep27 and comD genes to eliminate the reversion of the bacterium to wild-type phenotype) showed significantly higher IgG titers against serotype D39. This immunization also reduced colonization regardless of the serotype. Furthermore, this strain depicted a good safety profile in both normal and immunocompromised mice. Thus, the Δpep27ΔcomD strain presents efficiency and safety in preventing pneumococcal infections (Kim et al., 2019). Another live attenuated vaccine strain has the pro-lipoprotein diacylglycerol transferase (lgt) gene deletion from the capsule of the pneumococcal strain TIGR4 (TIGR4Δlgt). This strain evokes a significantly reduced inflammatory response and is reduced in virulence. Intranasal immunization of mice resulted in protection against invasive pneumococcal infections (Jang et al., 2019). Thus, TIGR4Δlgt is an attractive broad-spectrum vaccine candidate.

Numerous NTHi vaccine candidates have been investigated in the past years. In this section, we review only those candidates who presented great potential to undergo further development.

Many NTHi proteins including H. influenzae adhesin protein (Hap), H. influenzae autotransporter (Hia), Protein D (PD), outer-membrane protein 6 (P6), and outer-membrane protein 26 (OMP26) have been studied by several groups for the prevention of NTHi disease (see review by (Khan et al., 2016)). Antisera raised in guinea pigs against high molecular weight (HMW) proteins HMW1/HMW2 or Hia proteins showed opsonophagocytic activity against a wide range of NTHi strains (Winter and Barenkamp, 2014). Given these observations, a vaccine formulated with HMW1/HMW2 and Hia proteins may protect against a broader range of NTHi strains. Pre-clinical studies with these proteins revealed that PD immunization with OMP26 produced lowered antibody responses against PD, however, PD immunization with P6 did not mask PD immune responses (Michel et al., 2022), emphasizing the necessity of compatibility studies when combining antigens. Mice immunized with a truncated adhesin protein F (PF) showed faster clearance of NTHi infections compared to mice immunized with a control peptide (Jalalvand et al., 2014). Type IV pili (Tfp) are crucial for NTHi biofilm development, adherence, competence, and twitching motility (Das et al., 2017). Antisera raised against recombinant soluble PilA (rsPilA) dispersed in vitro formed NTHi biofilms and blocked NTHi-induced OM in chinchilla (Novotny et al., 2015b, 2017).

A recombinant fusion protein consisting of immunologically important components of protein E (PE) and PilA was evaluated in pre-clinical trials. PE-PilA-induced anti-PilA antibodies halted NTHi biofilm development and disrupted in vitro established biofilms, as seen for rsPilA. After the intranasal NTHi challenge, NP colonization was significantly reduced in immunized mice, and in chinchillas, symptoms of experimental OM were notably hindered (Ysebaert et al., 2019). A multi-component NTHi vaccine consisting of a free recombinant PD and a recombinant fusion protein PE-PilA was tested in a phase 1 clinical trial in healthy adults (NCT01657526), with a good safety profile and acceptable reactogenicity (Leroux-Roels et al., 2016). A further development added the Mcat antigen UspA2 to this combination, to test a multi-component vaccine in two-dose and three-dose phase 1 and 2 clinical trials, showing promising safety and immunogenicity profiles in healthy adults and adults aged 50–71 with a history of smoking (De Smedt et al., 2021; Galgani et al., 2022).

Other vaccine candidates have not yet been tested in human trials but show promise in a range of animal studies. Intracellular elongation factor thermal-unstable (EF-Tu) was identified recently as a novel NTHi surface protein (Thofte et al., 2018); and it was reported that antibodies against EF-Tu can result in complement-dependent killing of NTHi (Thofte et al., 2019), indicating the potential of this protein as a NTHi vaccine antigen. To date, no further investigations in animal models have been reported.

In a parallel approach to the recombinant PE-PilA, Novotny et al. found that ChimV4, a fusion of P5 peptide and rsPilA resolved NTHi-induced OM when delivered transcutaneously (Novotny et al., 2015a). The formulation also prevented OM in a viral-bacterial model of experimental disease. Transcutaneous immunization with chimV4 + dmLT significantly increased mature B-cell phenotypes and antibody-secreting cells within nasal-associated lymphoid tissues (Novotny et al., 2017; Novotny and Bakaletz, 2020). A bioinformatics approach to identify candidate peptides could result in more rapid development of polyvalent protein antigens (see section 2.2.5 on recombinant polypeptide antigens).

One challenge for mucosal pathogens is eliciting a response to target bacteria at the surfaces. Building on previous studies that showed NTHi P6 as a promising candidate (DeMaria et al., 1996; Bakaletz et al., 1999; Kodama et al., 2000;), Kodama et al. found that prior treatment with CCL20 enhanced murine responses and bacterial clearance following intranasal P6 immunization (Kodama et al., 2011a). Similarly, P6 was tested with FMS-like tyrosine kinase receptor 3 ligand as a mucosal adjuvant and this combination also improved nasopharyngeal clearance of NTHi and dendritic cell recruitment (Kodama et al., 2011b; Kodama and Suzuki, 2011). Nasal immunization with P6 protein alone produced minimal or no antigen-specific immune responses and hence no effective protection against NTHi (Bertot et al., 2004; Abe et al., 2006; Noda et al., 2011). More recently, when combined with cCHP, a cationic cholesteryl pullulan nanogel, cCHP-P6 nanogel nasal vaccine induced P6-specific mucosal IgA and serum IgG responses without additional biologically active adjuvant. The P6-specific IgG titers were equivalent to those generated by the intramuscularly administered vaccine containing alum adjuvant (Nakahashi-Ouchida et al., 2022). This highlights that route of delivery, and adjuvant choice are crucial to assess vaccines to reduce disease at mucosal surfaces.

LOS are a major component of the Gram-negative bacterial outer membrane and thus, in theory, they are an attractive target for vaccine development. In NTHi, five different antigenically heterogeneous LOS serotypes I-V are produced (Campagnari et al., 1987), but these glycans are phase-variable within a strain and occur during infection (Fox et al., 2014). Despite this, several studies examined LOS as a candidate vaccine. In a mouse model of NP colonization, intranasal immunization with detoxified LOS-tetanus toxoid (dLOS-TT) demonstrated a substantial reduction of the same LOS type III NTHi strains as well as types IV and V, but only 50% reduction of type I and 29% reduction of type II NTHi strains (Hirano et al., 2003). Parenteral immunization with dLOS conjugates in chinchillas produced anti-LOS antibodies in serum or ME which were bactericidal and opsonophagocytic and lowered homologous NTHi-induced OM (Sun et al., 2000). Notably, dLOS was tested in a phase I study of healthy adults vaccinated intramuscularly with dLOS-TT resulting in increases in serum antibodies, with acceptable safety. However, no further human trials have proceeded. An alternate approach is to target a glycan present in bacteria but absent from host tissues. Ketodeoxyoctonic acid (KDO) is present in most lipo-polysaccharides (LPS)/LOS, as well as in some CPS. A monoclonal antibody, 6E4, raised against KDO is bactericidal against 12 out of 33 tested strains of NTHi in vitro (Apicella et al., 2018). Using a glycoconjugate composed of either multiple KD residues or a KDO-N-acetyl-lactosamine conjugated to an immunogenic protein may be an approach to develop a component of a vaccine that would target many bacterial species.

OMVs produced by Gram-negative bacteria are enriched in outer membrane components, including major and minor outer membrane proteins and LOS. The functional activity of NTHi OMV-specific antisera and the protective ability of NTHi OMVs as vaccine antigens in the chinchilla OM model were tested. Immunization of chinchillas with OMVs isolated from HMW1/HMW2- and Hia- Hia-expressing NTHi prevented experimental OM (Winter and Barenkamp, 2017). In another study, immunization of NTHi-derived OMVs along with CpG-MPLA adjuvant induced the production of protective antibodies and cytokines in mice (Behrouzi et al., 2020). Several licensed and effective vaccines targeting meningococcal disease are based on OMVs. The presence of immunodominant proteins can lead to strain-specific responses, but either the removal of strain-specific antigens or the addition of conserved antigens can address this problem (Pizza et al., 2020). This approach may be feasible for both NTHi and Mcat but requires significant development.

Adding a lipid moiety to a recombinant protein is expected to increase the immunogenicity through Toll-Like Receptor 2 (TLR2) signaling of antigen-presenting cells and T-helper 17 cells (Th17) to evoke or enhance cellular responses in the nasal-associated lymphoid tissue (NALT). Recently, the effects of lipidation vs. non-lipidation of recombinant P6 and OMP26 were compared in a mouse model. Lipidated P6 and OMP26 elicited nearly 10- to 100-fold higher IgG antibody levels, and lipidated antigens also reduced NP colonization and ME bullae NTHi density more than non-lipidated formulation (Kaur and Pichichero, 2022). Therefore, lipidation of NTHi antigens represents a promising approach in the development of novel NTHi vaccine formulations (Table 2).

As noted above, combining protein antigens can be achieved by identifying immunologically relevant epitopes, and generating fusion proteins (see PE-PilA and ChimV4). Since bacterial genomes have been available, bioinformatics analysis has identified putative surface-exposed proteins, in an approach described as “reverse vaccinology,” an approach that contributed to antigens in the GlaxoSmithKline vaccine against serogroup B meningococcus (Masignani et al., 2019). Significant improvements in bioinformatic analysis tools, availability of increasing numbers of genome sequences for each bacterial species, and increased understanding of immunologically relevant sequences and structure allow more sophisticated and rapid screening for candidate proteins, domains, or short peptide sequences. Recently Whitby et al. identified 56 putative surface proteins conserved in 26 genomes (selected to represent diverse strains). Potential surface exposed regions were identified using molecular modeling. Antisera were raised in rats against 10 synthetic peptides corresponding to surface-exposed regions highly conserved in strains. Five of the antisera were protective in the infant rat model of invasive NTHi infection establishing their in vivo efficiency (Whitby et al., 2015). A different bioinformatics platform identified two similar proteins as being future NTHi vaccine candidates (D’Mello et al., 2019).

The same group extended their strategy to produce a single polypeptide comprising 9 unique peptides from 6 different surface proteins. Immunization of chinchillas with this polypeptide resulted in faster clearance of NTHi-induced OM (Whitby et al., 2020). This approach has the potential to be applied to other pathogens that have complex diversity in surface antigens, including Mcat. In the coming years, it can be anticipated that high throughput bacterial genomics, computational-based vaccine candidate identification approaches, and experimental validation will identify additional vaccine candidates for NTHi.

As discussed earlier, presently only one Mcat protein antigen has been tested in clinical trials (UspA2). UspA2 interacts with host structures and extracellular matrix proteins and induces bacterial adherence and serum resistance (Singh et al., 2010; Su et al., 2013; Singh et al., 2015). Vaccination with UspA2 induced protective antibodies in mice (Chen et al., 1996). UspA2 has been reported to have variations in sequence and structure (Su et al., 2013), which may affect antibody recognition and binding. Despite this, anti-UspA2 antibodies raised in mice, rabbits, and guinea pigs were able to induce complement-mediated killing of many, but not all Mcat strains of different origins (Ysebaert et al., 2021). The NTHi-Mcat vaccine containing UspA2 was immunogenic in the phase 1 clinical trial, with a satisfactory safety profile both in healthy individuals and adults with a smoking history (Van Damme et al., 2019). A 4-year follow-up (NCT03201211) found that immune responses to NTHi antigens persisted over an extended period, whereas UspA2 did not (De Smedt et al., 2021). However, in all human trials to date, baseline titers of serum antibodies against UspA2 are relatively high, suggesting either natural immune responses to UspA2 or similar proteins in Mcat, or cross-reactivity with an antigen from another source. This observation complicates the interpretation of data in clinical trials on the persistence of serum antibody responses. In a randomized, placebo-controlled, phase 2b trial (NCT03281876) of adults with a history of acute exacerbations of COPD, this vaccine showed good safety, however, did not demonstrate efficacy in lowering the yearly rate of severe or moderate exacerbations of COPD (Andreas et al., 2022; Arora et al., 2022).

Several antigens have shown promise in early animal trials but have few recent reports. This includes Hemagglutinin Moraxella IgD binding protein (Hag/MID), PilA2 (the major protein subunit of Tfp), and outer-membrane protein CD (OMP CD). However, they have been identified as targets of natural antibody production in COPD patients, or children (LaFontaine et al., 2009; Ren et al., 2019). OMP CD is an adhesin and a porin protein (Murphy et al., 2003) with surface-exposed epitopes and is considered conserved among different Mcat strains (Sarwar et al., 1992; Murphy et al., 1993). Murine immunization with OMP CD increased Mcat clearance following pulmonary challenge (Liu et al., 2007). Hag/MID is an OMP involved in human erythrocyte agglutination (Pearson et al., 2002; Forsgren et al., 2003; LaFontaine et al., 2009). Hag/MID shows sequence diversity among different strains, however, the adhesive domain region is conserved. A truncated Hag/MID induced a protective response in mice (Forsgren et al., 2004). PilA2 was present in 57.5% of 106 tested clinical Mcat isolates (Luke-Marshall et al., 2011). It was required for the adherence of Mcat to in vitro cultured human epithelial cells and NP colonization in a chinchilla model (Luke et al., 2004, 2007). At present, no data is available on measuring the immune responses to the PilA2-based vaccine in animal models or humans. Ren et al. studied natural antibody production against OMP CD, Hag, and PilA2 in stringently defined otitis-prone (sOP) children compared to non-otitis-prone children (NOP). All proteins elicited antibodies, but the sOP population showed reduced serum antibody responses (Ren et al., 2019). Serum and sputum sample analysis from COPD patients revealed that a Hag/MID domain encompassing amino acids 706 to 863 was a target of serum IgG and sputum IgA (LaFontaine et al., 2009).

Moraxella catarrhalis-adherence protein (McaP) is an adhesin and an autotransporter having esterase and phospholipase B activities (Timpe et al., 2003). Moreover, this protein is highly conserved as 98–100% amino acid sequence similarity was observed among 8 studied strains (Timpe et al., 2003; Lipski et al., 2007). Mouse anti-Mcap serum hindered the binding of Mcat and recombinant E. coli expressing McaP to human respiratory epithelial cells (Timpe et al., 2003; Lipski et al., 2007). Outer-membrane protein E (OMP E) is a porin and is involved in the binding and transport of fatty acids. Gene sequence analysis showed that the ompE gene stayed stable during the colonization process (Martin et al., 1993). OMP E also expressed a highly conserved surface epitope (Bhushan et al., 1997). Further investigations are needed to gain a clear understanding of McaP and OMP E immunogenicity, its different antigenic domains, and the protective immune responses they elicit. M35 is an OMP that acts as a general porin and is essential for energy source uptake for Mcat (Easton et al., 2005). M35 is suggested to be essential for in vivo colonization and resistance mechanisms (Easton et al., 2008). M35 gene sequence showed high conservation (99.6–100%) among 18 tested isolates. In immunoblot analysis, mouse anti-M35 serum displayed binding to whole-cell protein preparations from all the tested isolates (Easton et al., 2005). Anti-sera from M35 immunized mice did not show bactericidal activity, however, it improved opsonic activity. In another study, mucosal immunization of mice with recombinant M35 through intra Peyer’s patch increased clearance of bacteria from the lungs of Mcat-challenged mice (Easton et al., 2011).

Moraxella catarrhalis filamentous hemagglutinin adhesin-like protein (Mha)B1 and MhaB2 (exoproteins), and MhaC (transporter) (Balder et al., 2007), which are also named M. catarrhalis hemagglutinin-like protein (Mch)A1 and MchA2 for the secreted proteins and MchB for the transporter are potential vaccine candidates (Plamondon et al., 2007). A mutant Mcat lacking MhaB1 and MhaB2 could not colonize the chinchilla NP, emphasizing the importance of these proteins. Immunization of chinchillas with a polypeptide shared by MhaB1 and MhaB2 induced antibodies that interfered with the colonization of Mcat and promoted bacterial clearance (Balder et al., 2011; Shaffer et al., 2013).

Murphy et al. screened the genome of Mcat strain ATCC 43617 for potential surface proteins. This identified several substrate-binding proteins (SBPs) of the ABC transporter family. The group evaluated CysP, AfeA, OppA, and Sbp2 for their immunogenic potential which are studied by other groups as well (Yang et al., 2011; Otsuka et al., 2014; Murphy et al., 2016; Otsuka et al., 2016; Murphy et al., 2017). CysP is involved in the uptake of sulfate, AfeA binds ferrous, ferric, zinc, and manganese ions, OppA is likely an oligopeptide binding protein of the oligopeptide permease ABC transport system, and Sbp2 mediates the uptake of arginine, a strict growth requirement of Mcat. All were found to express accessible surface epitopes, to be conserved within Mcat strains, and to provide enhanced clearance in a lung challenge model in mice (Yang et al., 2011; Otsuka et al., 2014; Murphy et al., 2016, 2017).

Moraxella surface proteins (Msp) including Msp22, Msp75, and Msp78 showed 97–99% homology in amino acid sequence among 10 tested strains (Ruckdeschel et al., 2008). Mucosal and systemic immunizations of mice with recombinant Msp22 and Msp75 induced IgG and IgA antibodies. Mouse and rabbit anti-sera to recombinant Msp22 and Msp75 were able to recognize corresponding proteins in numerous Mcat strains. In addition, intranasal immunization of mice with recombinant Msp22 substantially lowered the bacterial load in the lungs of Mcat challenged mice (Ruckdeschel et al., 2009; Smidt et al., 2013). Therefore, these proteins are attractive Mcat vaccine antigens, nonetheless, more studies are required to uncover their detailed functions and immunogenicity in humans.

Peptide-based vaccines offer numerous advantages, such as the exclusion of deleterious parts from full-length antigens, ease of chemical modification, absence of infectious material, and ease of production and storage. Although they generally have poor immunogenicity, this can however be compensated for by using modified formulations of the vaccine (Purcell et al., 2007). Lactoferrin-binding protein A (LbpA) is a receptor for human lactoferrin and is involved in iron transport. Whole LbpA, both native and recombinant was reported to be non-immunogenic (Du et al., 1998; Yu et al., 1999). Yassin et al. used immune-informatics analysis to identify a peptide from LbpA named peptide A which showed high immunogenicity in mice and successfully cleared Mcat from mouse lungs. Furthermore, anti-peptide LbpA antibodies were bactericidal against heterogeneous Mcat strains (Yassin et al., 2016). Peptide A is the first promising peptide-based vaccine against Mcat, and it warrants further investigation.

LOS comprises a major virulence factor of Mcat and performs a vital role in eliciting inflammatory immune responses (Hassan et al., 2012). It also mediates serum resistance (Zaleski et al., 2000) and adherence to human epithelial cells (Peng et al., 2005). LOS from Mcat is relatively conserved, with three major serotypes (Holme et al., 1999; Verduin et al., 2002; Ren et al., 2011b), and no phase variation within a strain (unlike NTHi). In common with CPS, LOS glycans are poorly immunogenic unless conjugated to a carrier protein. Removal of toxic Lipid A moieties is also necessary. Detoxified LOS (dLOS) is conjugated to a carrier protein. Immunization of rabbits and mice with the conjugates derived from all three serotypes induces significant levels of antigens-specific mucosal and serum antibodies. Bactericidal antibodies were also identified in immunized animals (Jiao et al., 2002; Yu and Gu, 2005, 2007). Ren et al. generated two mutant LOS from Mcat strain O35E to produce conserved LOS antigens and exclude a potential autoimmune response in humans. These mutant LOS had one or two terminal galactopyranose (Galp) residues deleted and conjugated to tetanus toxoid (TT). These conjugates exhibited broad-spectrum protection and induced high levels of serum IgG with bactericidal activity against all three serotypes (Ren et al., 2011b). In another study, dLOS-protein conjugates from all three serotypes were combined and immunized mice developed humoral and cell-mediated immunity which enhanced pulmonary clearance of six strains of all three serotypes of Mcat (Ren et al., 2011a). In a similar approach, truncated (common) LOS was conjugated to recombinant OMP26 of NTHi. This elicited complement-mediated bactericidal activity against tested strains of serotype A and Mcat and one NTHi strain (Singh et al., 2020).

As mentioned above, D’Mello et al. used a computational approach to identify potential candidate antigens against a range of criteria, from NTHi and Mcat (D’Mello et al., 2019). Top Mcat candidates identified included a porin protein, an iron transporter protein, and 2 unstudied conserved-hypothetical proteins (D’Mello et al., 2019). Soltan et al. applied a different reverse vaccinology approach to identify potential protein candidates, including criteria of being essential, outer membrane-localized, involved in virulence, antigenic with no human homologs, with appropriate molecular weight and less than two transmembrane helices. Only LPS assembly protein LptD and outer membrane protein assembly factor BamA met these criteria. From this, several peptides of each were combined to construct a theoretical multi-epitope candidate peptide (Soltan et al., 2021). It is likely that future publications will reveal which of these are validated and can be further developed, but as with NTHi, a multi-component vaccine is likely required.

In summary, numerous Mcat antigens have revealed outstanding immunogenicity, induced functional antibodies, and generated protective responses in animal models. Nonetheless, there has been no clinical testing on them. Future studies are needed on these antigens to make advances in Mcat vaccine development.

Several animal models have been developed that examine the pathological changes associated with OM, in the absence of pathogen challenge. The principal anatomical cause of OME is ET dysfunction. To mimic this, animal models of OME are generated through ligation or cauterization of ET utilizing a trans-oral or trans-neck approach (Piltcher et al., 2002), or by injecting chemical materials through the tympanic membrane (Aynali et al., 2011). For the first approach, the ET orifice is cauterized, and the cartilage portion of the ET is ligated with nylon or an electrical cautery (Piltcher et al., 2002). Cauterization or ligation of the ET is however an irreversible process. The trans-neck method is a precise approach for blocking the tube, but it is more laborious as compared to the trans-oral approach. On the other hand, the trans-oral approach is easy, however, it is difficult to get consistent results with this method (Huang et al., 2012). Injecting chemicals via the tympanic membrane is comparatively simpler to perform than the methods mentioned above. Injection of histamine solution has been used to induce OME in rats (Aynali et al., 2011).

Rats exhibit the highest compatibility in terms of ME anatomy and histology to human infants and children as compared to other rodents, therefore, they are preferred models of AOM (Cayé-Thomasen and Tos, 2002). In addition, most human pathogens readily infect rat ME (Chaney et al., 2011) and the opening pressure of the ET is equivalent to that in humans (Hellstorm and Stenfors, 1983). The rat AOM course bears a close resemblance to that of humans (Hermansson et al., 1988), and ciliary clearance tracts and histological cell types are also analogous to humans (Daniel III et al., 1982). As a result of the relatively large tympanic bulla in the rat, bacteria can easily be inoculated into ME via the tympanic membrane or the bulla. Once infection has occurred, it usually resolves within 10–12 days without ME effusion signs and affecting tympanic membrane preservation (Hermansson et al., 1988; Prellner et al., 1999). It is also possible to induce a persistent OME in rats which can last for more than 16 weeks upon using appropriate inoculum (Piltcher et al., 2002). Moreover, rats are not prone to develop general sepsis which further enhances their usefulness (Prellner et al., 1999). Rat models of AOM with S. pneumoniae and OME model with ET obstruction by dental material are reported (Hermansson et al., 1988; Piltcher et al., 2002). An allergen-induced OME rat model was established using an intraperitoneal injection of ovalbumin and a subsequent intratympanic injection of ovalbumin into the ME (Hardy et al., 2001; Zhang et al., 2022). Rat OM model with methicillin-resistant S. aureus and P. aeruginosa injected into the ME cavity via the tympanic membrane have been developed (Yadav et al., 2017). Magnuson et al. utilized Haemophilus influenzae type b and NTHi strain 3,655 to inoculate the ME of Sprague–Dawley rats and studied the course of AOM development. Rats developed AOM with both bacterial strains 4 days post-inoculation (Magnuson et al., 1997). Rat models of OM with cholesteatoma have also been developed utilizing different chemical compounds such as dimethyl-benzanthrancene (DMBA) or propylene glycol injections into the ME (Klein-Szanto et al., 1982; Huang et al., 1988). An acute secretory OM (SOM) rat model was developed by delivering endotoxin into the ME cavity through the eardrum (Li et al., 2021). Mcat can induce OM in rats, but only with high dose, trans-tympanic inoculation, and OM is shorter lived than that caused by NTHi or S. pneumoniae (Westman et al., 1999).

Another rodent that is favored for AOM research is the chinchilla which displays general susceptibility to human bacterial and viral pathogens of OM (See review by (Bakaletz, 2009)). An advantage of using the chinchilla is that its tympanic membrane is almost the same size as humans. In addition, its large bulla facilitates the inoculation of pathogens and collection of ME effusions (Watanabe et al., 1982). The temporal progression and natural history of the disease process resemble human OM (DeMaria et al., 1996; Bakaletz, 2009). However, this species has a multi-loculated bulla, which is easily prone to fibrosis, with the ability to seal a chamber of the bulla and prevent infection from spreading. The external auditory canal of chinchillas is elongated and S-shaped, making it difficult to inspect the tympanic membrane with an ordinary otoscope and making the trans-tympanic challenge more difficult. It is only widely available in North and South America; therefore, lack of easy availability hinders its application. The rate of inner ear complications is high in chinchillas, and they tend to develop general sepsis, especially the younger chinchillas, and have a high mortality rate. Furthermore, chinchillas are not as capable of withstanding adverse conditions in laboratory settings as rats. Chinchillas tend to shed their fur at the insignificant sign of distress (Murrah et al., 2015).

AOM models of chinchilla with S. pneumoniae, H. influenzae type b, NTHi strain 86-028NP, and other ME-associated aerobic and anaerobic microbes have been developed through trans-bullar inoculation into the ME (Fulghum et al., 1982; Morton et al., 2012; Guan et al., 2014). Cholesteatomatous chronic OM chinchilla models have also been developed through the use of propylene glycol injection (Masaki et al., 1988).

Mongolian gerbils have been widely used to establish AOM models for the study of AOM and analysis of efficacies of different antimicrobials. The small size and cost-effectiveness of Mongolian gerbils made them popular in OM studies. They have a reasonably large ME making it easy to inoculate through the overlying skin. It is also easy to sample ME fluid, however, they have narrow external ear canals. Mongolian gerbils normally have healthy ears and rarely experience OM, while cholesteatoma is common in elderly gerbils (Chole et al., 1981).

Compared to chinchillas and rats, fewer studies using gerbils as a model for bacterially induced AOM are reported. Direct bacterial ME inoculation has been used with S. pneumoniae type 3, Haemophilus influenzae type b, and NTHi strain 119 (Fulghum et al., 1982, 1985; Von Unge et al., 1997; Cenjor et al., 1998; Larsson et al., 2003; Unge et al., 2009). For non-bacterial ME disease, cholesteatoma development or other ME disease was reported in gerbils through ligation of the external ear canal, ET blocking by electrocauterization or glue, and chemical injections into the bulla (Larsson et al., 2005; Yamamoto-Fukuda et al., 2010, 2011).

The MEs of guinea pigs have structural similarities to those of mice and rats, but the size of bulla in this animal is smaller compared to gerbils and chinchillas (Hellström et al., 1982; Foxwell et al., 1998). Several studies have used the AOM guinea pig model developed by using non-typeable S. pneumoniae and S. pneumoniae type 3 (Sp3) via a trans-bullar approach (Parks et al., 1996; Dai et al., 2009; Guan and Gan, 2013). LPS from Klebsiella pneumoniae was injected into the ME of the guinea pig to develop an OME guinea pig model (Ohashi et al., 1991; Dai and Gan, 2008). Yu et al. developed a guinea pig model of OME by reversible ET obstruction (Yu et al., 2013). Cholesteatoma guinea pig models with chemical inoculants in the bulla have also been developed (Yamamoto-Fukuda et al., 2011).

The NP-ET-ME complex of primates has been employed to model the normal and pathological activities of the human ET. There have been very few studies on monkey models of OM. The Rhesus monkey model has been used to evaluate the impact of allergic rhinitis in the development of ET obstruction and OME (Friedman et al., 1981; Doyle et al., 1985).

Dohar et al. (1998, 2005) established a CSOM cynomolgus monkey model by infection of P. aeruginosa to investigate the safety and efficacy profile of topical ciprofloxacin hydrochloride for treating experimental CSOM. The tympanic membrane of cynomolgus monkeys was perforated and the ME was injected with a strain of P. aeruginosa which forms biofilms (Dohar et al., 1998, 2005). Although monkeys share more similarities to humans than rodents, their large size makes their handling in experimental settings very difficult. They are costly and not readily available, and modern ethical considerations have also reduced their use.

Since 2010, mouse OM models have significantly increased as compared to other models and now the mouse is more utilized in OM research because it offers many remarkable advantages over other animals [see review by (Bhutta, 2012)]. The wide availability of reagents allows complex analysis of immunological progression and host responses. Importantly, several genetic, transgenic, and gene deletion strains are available that can be used in studying various aspects of OM pathophysiology as well as host susceptibility to OM (Mitchell et al., 1997; Hardisty et al., 2003; MacArthur et al., 2006; Zheng et al., 2006).

Although mouse models offer many benefits as model systems for OM, yet there are some limitations to consider. The small size of the tympanic membrane in mice makes it difficult to access as compared to larger rodents. Even with the use of a microscope, surgical procedures on mice can be challenging. Inoculating and removing fluid from the mouse ME is difficult, and sampling large amounts of effusion is impractical because of the small size of the ME. The mouse is less hardy in tolerating general anaesthesia and surgical bleeding. Since major pathogens of OM such as NTHi, S. pneumoniae, and Mcat are not natural murine pathogens (Murphy, 2005; Doherty et al., 2006), variations can be observed in patterns of colonization and immune responses resulting from infecting mouse ME and NP than those observed in humans. This notable limitation is also associated with other rodent models. Nonetheless, it can be overcome via direct ME inoculation or multiple IN inoculations. Genetically modified mice which are more susceptible to human pathogens can be used for this purpose. Moreover, the use of mutant bacterial strains capable of adhering and invading the murine mucosa more effectively than wild-type bacteria offers another approach (Tu et al., 2007).

To study the natural course of OM in mice, Dewan et al. have described a novel pathogen capable of transmission between mice that causes OM (Dewan et al., 2019; Ma et al., 2022). Bordetella pseudohinzii efficiently replicates in the NP, quickly escalates the ET, and colonizes the ME. The resulting acute and chronic histopathological transformations with an increasing decline in hearing acuity closely represent OM in humans. Laboratory mice experimentally inoculated with a very small inoculum of B. pseudohinzii consistently had their MEs colonized and subsequently transferred it to cage mates (Dewan et al., 2019; Ma et al., 2022).

A major challenge to Mcat vaccine development is the unavailability of a suitable animal model and a dependable correlate of protection. Under experimental settings, Mcat does not readily survive and replicate in the rodent NP. Although, chinchilla has been used in evaluating the NTHi and S. pneumoniae vaccine antigens, however, it readily clears Mcat from the ME (Bakaletz, 2009). Therefore, either NTHi and/or viruses are used in chinchillas to study Mcat infections and vaccine antigens (Armbruster et al., 2010; Brockson et al., 2012; Shaffer et al., 2013; Perez et al., 2014), or direct intra-bullae inoculation (Blakeway et al., 2019). Nevertheless, the chinchilla NP colonization model, the mouse lung clearance model, or the in vitro functional assays are unable to provide a fixed correlate of protection for assessment in animal models. Therefore, the development of improved animal models for evaluating potential Mcat vaccines is highly desired.