HA head comprises several immunodominant antigenic sites (33). Varma et al. conducted a study utilizing a nasal gel formulated with recombinant computationally optimized broadly reactive HA antigen and cyclic guanosine monophosphate-adenosine monophosphate for the administration of the influenza vaccine. The experimental findings indicated that the administration of the gel resulted in significantly elevated levels of IgG2c and IgA in the mice (34). This vaccine formulation elicited stronger serum humoral responses compared to the absence of the gel, while also inducing a mucosal IgA response.

HA stem possesses epitopes capable of eliciting cross-reactive antibody responses and exhibits a greater degree of conservation compared to the head domain (35). In a study conducted by Tsybalova et al., the bacterial flagellin gene was fused with a fragment of HA2 gene and subsequently expressed it in the Escherichia coli (E. coli), resulting in the creation of three recombinant proteins: Flag-4M2e, Flag-HA2-2-4M2e, and Flag-HA2-1-4M2e. Notably, a portion of the HA2 in Flag-HA2-1-4M2e is derived from the A/H1N1pdm09 strain. The study reported the presence of specific anti-M2e IgG and IgA antibodies in the serum of immunized mice, alongside the generation of anti-HA antibodies to targeting various subtypes of the influenza A virus (36).

H1N1 acts as a multihoming virus, evolving through antigenic drift and shift as it spreads across species. Antigenic drift occurs due to point mutations in surface antigens, such as HA and NA, during viral replication. HA, a primary target for influenza vaccines, is central to understanding influenza’s sequence evolution ( Figure 3 ). Recent advancements in H1N1 HA-based vaccine design have emphasized strategies like immune focusing, nanoparticle-based designs, and chimeric HA constructs to enhance cross-protection against diverse influenza strains (2). This focus is particularly pertinent in the context of viral antigenic drift and shift. Immune focusing redirects the immune response toward conserved regions, particularly the stem, which mutates less frequently (37). Techniques to achieve this include removing or masking the HA head to expose the conserved stem and developing HA mini-stem vaccines that use only the stem region. Preclinical studies show these approaches can induce broadly neutralizing antibodies (bnAbs) effective across multiple influenza subtypes (38). Nanoparticle vaccines also show promise by presenting conserved HA epitopes in structured arrays that better mimic the viral structure, leading to stronger immune responses. Chimeric HA vaccines replace the variable HA head with non-relevant sequences, guiding the immune system to focus on the stem and enhancing cross-reactivity among H1N1 strains (39). For instance, McCraw et al. created an HA stem-based chimeric nanoparticle vaccine using helix-A sequences on a hepatitis B virus capsid scaffold, producing cross-reactive antibodies that provided protection in mice (40). Mosaic HA vaccines further expand immune coverage by integrating epitopes from multiple strains. Together, these innovations offer potential pathways toward more universal and durable influenza vaccines (41).

Recent research has identified novel neutralizing antibodies that specifically target stem domain epitopes within the pre-fusion conformation of the HA protein, which are not accessible in the post-fusion conformation (42). In another study, McMillan et al. introduced a molecular clamp that effectively stabilized the formation of a stable HA trimer in its pre-fusion state. In the context of the H1N1 pmd09 challenge, C57BL/6 mice that received this treatment in conjunction with an adjuvant exhibited no significant weight loss or mortality, and cross-protection was successfully elicited in ferrets. Furthermore, vaccines stabilized by the HA clamp also generated neutralizing serum antibodies (43). These findings suggest that this innovative platform holds significant potential for the development of H1N1 vaccines, particularly in addressing the challenges posed by high mutation rates in the HA head and the limited efficacy of the HA stem.

NA has the activity of hydrolyzing sialic acid, which helps the virus to be released from the host cell. Numerous studies have indicated that the levels of both anti-NA and anti-HA antibodies diminish over time; however, experimental findings in murine models suggest that the decline of anti-NA antibodies occurs at a slower rate than that of anti-HA antibodies (44).

Kim et al. conducted a study on the 2009 pandemic H1N1 NA influenza virus-like particles (VLPs) vaccine. Their research involved animal models, which revealed that mice immunized with the VLP vaccine exhibited a cross-protective immune response when compared to those immunized with inactivated split vaccine. Notably, this immune response was unaffected in mice deficient in the Fc receptor γ-chain (45). Deng et al. demonstrated the expression of NA tetramers and monomers using Expi293F cells, revealing that only the tetrameric form of NA exhibited enzymatic activity. This tetrameric NA provided enhanced protection to mice against viral infection and elicited a greater production of specific binding antibodies (46). The active site of the NA enzyme is situated within its head domain (47). Furthermore, there exists potential for the incorporation of additional subunits within the NA head domain to augment its immunogenicity, or for the combination of NA with other antigens to develop a multi-component vaccine targeting various infectious diseases.

The matrix protein is composed of two components, M1 and M2. The M2 serves as a proton channel, playing a crucial role in regulating pH levels during the processes of viral entry and replication within host cells (48). The M2e is distributed across the surface of the viral particles; however, it is obscured by the HA and NA antigens. This structural arrangement results in M2e being less accessible to antibodies specific to influenza, thereby reducing its susceptibility to antigenic variation and contributing to its greater conservation (49).

The immune response elicited against the M2e epitope is characterized by its relative weakness, and the resulting specific antibodies exhibit an inability to neutralize the virus effectively. Consequently, the incorporation of carriers or adjuvants is crucial for enhancing the efficacy of M2e-based vaccines (2). In a particular study, researchers successfully expressed a fusion protein designated Flg4M2eHA2-1, which comprises two copies of the human consensus M2e sequence (M2eh) and two copies of the M2e sequence from the A/H1N1pdm09 strain (M2es), all linked to the C-terminus of the S. typhimurium flagellin protein FljB. Animal studies demonstrated that mice immunized with the recombinant protein exhibited high titers of M2e-specific IgG antibodies in both serum and bronchoalveolar lavage fluid; however, the immune response to the HA2 component was notably weak (50). This diminished response may be attributed to the positioning of the HA2 sequence between the flagellin and the tandem M2e copies.

NP is a prevalent intracellular protein that plays a critical role in the transcription and replication of the viral genome, thereby affecting the host specificity and virulence of viruses (51). Upon infection with an influenza virus, NP serves as the primary antigen identified by host cytotoxic T lymphocytes, ultimately contributing to the eradication of the virus (52).

In a recent study, trimethyl chitosan was utilized both as a mucosal adjuvant and as a delivery system to create nanoparticles incorporating purified HA2 and NP proteins, with a focus on assessing their immunogenicity. In vitro analyses conducted on primary human nasal epithelial cells demonstrated that trimethyl chitosan nanoparticles (TMC nPs) enhance the uptake of HA2 and NP proteins. When compared to control TMC nPs devoid of active components, human nasal epithelial cells treated with various formulations of influenza nanoparticles (including HA2-TMC nPs, NP-TMC nPs, and HA2-NP-TMC nPs) exhibited a significantly elevated production of pro-inflammatory cytokines, suggesting a heightened immune response. Furthermore, in the subsequent experiments simulating viral infection, the influenza nanoparticle formulations were found to markedly inhibit the replication of the influenza virus (53).

The E. coli expression system is widely recognized as a prevalent platform for the expression of recombinant proteins (55). The expression system utilizing E. coli has gained widespread acceptance as a host for the production of recombinant heterologous proteins, owing to its advantages, which include high expression levels, rapid production cycles, and cost-effectiveness.

Two different HA fragments (rHA1_1-326 and rHA1_53-269) were expressed in E. coli and used to vaccinate mice, where rHA1_1-326 demonstrated a significant level of protection against a homologous H1N1 viral challenge. In contrast, the rHA1_53-269, which encompasses neutralizing epitopes, fails to form higher-order oligomers. Therefore, it does not elicit the same protective response as rHA_1-136, which contains both neutralizing epitopes and a trimerized domain (56). It is well established that E. coli, being a prokaryotic organism, may result in inadequate post-translational modifications, the formation of inclusion bodies, and potential protein degradation (57). Molecular chaperones play a crucial role in assisting newly synthesized, aggregated or misfolded proteins in achieving their native conformations, and their application has been extensively documented to enhance the expression of various proteins (58). One particular study highlighted the use of heterologous caveolin-1 as a chaperone, in conjunction with the Oct1 DNA-binding domain as a fusion partner, which significantly improved the solubility of influenza HA (59).

The yeast expression system possesses several advantageous characteristics, including the capacity to elicit specific immune responses against the recombinant antigens, its adjuvant properties (60) and its role in the activation of the antigen presenting cells (APCs) (61). Notable yeasts utilized in this context include Saccharomyces cerevisiae and Pichia pastoris, among others. Saccharomyces cerevisiae is the most frequently employed yeast for the production of recombinant proteins (62). However, Pichia. Pastoris is increasingly favored over Saccharomyces cerevisiae due to its ability to achieve high cell density cultures, which is facilitated by its aerobic respiration and the expression of proteins under the tightly regulated methanol inducible AOX1 promoter (63).

Kopera et al. conducted a study examining the potential of the Pichia cells for the production of a soluble H1N1 HA antigen. Their immunization experiments revealed that the H1 antigen elicited a robust HI-immune response in murine models (64). Concurrently, research is underway to develop yeast-based oral influenza vaccines. Oral vaccination has the capacity to activate both the humoral and cellular immune responses at systemic and mucosal levels, thereby representing an effective strategy for inducing protective sIgA responses (65). Lei and colleagues utilized Saccharomyces cerevisiae EBY100 to successfully formulate oral vaccines for H7N9-HA (66) and H5N1-HA (67). Subsequent animal studies demonstrated significantly elevated levels of HA-specific antibodies and HI titers were detected, which are anticipated to confer protection against both homologous and heterologous strains of influenza viruses. These findings are promising for the development of an oral vaccine targeting H1N1 using yeast expression systems.

The baculovirus expression vector system has emerged as a critical platform for the production of recombinant proteins utilized in vaccines (68). The baculovirus expression system is characterized by several key features: 1) a high safety profile, as it is unable to complete a full replication within mammalian cells; 2) a substantial capacity for the incorporation of exogenous genes; and 3) an efficient expression of these genes.

In a research investigation, H1N1 HA gene was successfully cloned into baculovirus expression vector. The recombinant vector was subsequently introduced into E. coli for propagation, followed by transfection into SF9 insect cells for protein expression. Comparative analyses using hemagglutination inhibition and enzyme-linked immunosorbent assay demonstrated that the recombinant H1N1 protein, when formulated with adjuvant, elicited a greater antibody response against viral challenge than the commercially available H1N1 influenza vaccine. Furthermore, the results from animal studies indicated that the protective efficacy of the novel vaccine was 96%, surpassing that of existing alternatives (69).

Mammalian cell expression systems are increasingly favored for H1N1 antigen production in influenza vaccine development due to several advantages over traditional egg-based systems (70). Cell-based influenza vaccines are particularly advantageous as they help avoid the antigen mismatch issues that can arise with chick embryo-based production (71). Common mammalian cell lines for recombinant protein production include Chinese hamster ovary (CHO) cells and human embryonic kidney 293 (HEK293) cells (72, 73). CHO cells, which are non-secreting, rarely produce endogenous proteins, making it easier to separate and purify the target protein after expression. Additionally, CHO cells can yield high protein quantities in large-scale cultures, though their glycosylation patterns differ from those in humans (74). HEK293 cells, on the other hand, are easy to culture, have high transfection efficiency, and can produce recombinant proteins in substantial quantities, making them suitable for transient transfection. Derived from human cells, HEK293 cells can better simulate human glycosylation patterns, yielding proteins more closely matching human conformation (75). However, their longer doubling time in suspension culture can reduce production rates compared to CHO cells (76). Overall, these mammalian systems are widely used for producing recombinant subunit proteins in vaccine development.

In a research investigation, the modified HA was produced as a soluble trimeric protein within mammalian cell systems. Subsequent assessments indicated that the recombinant HA exhibited functional activities comparable to those of their native counterparts present on the surface of influenza viral particles (77). However, the researchers did not assess the immunogenicity of the expressed HA proteins. In a separate investigation, the researchers developed chimeric HA and employed chimpanzee adenoviral vectors (AdC68) vaccine platform to produce recombinant adenoviral plasmids. Then they expressed the chimeric HA protein in HEK293 cells. Analysis of the experimental outcomes revealed that the AdC68-cH1-H7 construct demonstrated significant immunogenicity in murine models and conferred protection against challenges with various strains of the influenza virus (39). The suboptimal efficacy observed in other chimeric HA vaccines may be attributed to the concealment of antigenic sites resulting from their conformational structures.

TLRs play a crucial role in the activation of innate immunity through their interaction with pathogen-associated molecular patterns (94). Additionally, TLR adaptor molecules serve as a link between innate and adaptive immune responses (95). The activation of intracellular TLRs generally triggers a robust immune response characterized by the production of type I interferons (IFNs) and inflammatory cytokines (96). Consequently, the incorporation of targeted TLR adjuvants into protein subunit influenza vaccine has the potential to enhance their immunogenicity ( Figure 4 ).

In the field of influenza research, mice serve as the predominant animal model, with the C57BL/6 (103, 153) and BALB/c (154–156) strains being the most frequently utilized among inbred mouse populations.

Ong et al. employed the NvC-M2ex3 construct to immunize mice subcutaneously following a prime-double boost regimen. Subsequently, the mice were subjected to intranasal infection with mouse-adapted strains of H1N1 or H3N2. The results indicated no significant weight loss or observable signs of illness in the vaccinated mice, who exhibited elevated levels of IFN-γ and IL-12 in the pulmonary tissue following challenges with H1N1 and H3N2 (157). Additionally, AdC68 was utilized to generate recombinant chimeric HA, which were engineered by amalgamating multiple head domains from various influenza virus subtypes onto a single stalk domain. The AdC68-cH1-H7 construct demonstrated a significant reduction in viral infection and conferred protection to mice against lethal challenges posed by both H1 and H7 subtypes (39). However, it is important to note that mice are not natural hosts for the influenza virus. Unlike humans, mice exhibit replication of influenza A virus primarily in the lower respiratory tract, and research has indicated that the pathogenesis of the influenza virus in murine models differs to some extent from that observed in humans (158).

Ferrets were the inaugural animal species subjected to experimental infection with the influenza virus (152) and are regarded as the ‘gold standard’ for evaluating the transmissibility of influenza viruses through respiratory droplets (3). They exhibit a higher susceptibility to human influenza viruses compared to mice, possess a distribution of sialic acid residues in the respiratory tract that closely resembles that of humans, and demonstrate a comparable progression of disease and immune responses (159).

In a research investigation, partial female Fitch ferrets underwent preimmunization followed by intramuscular administration of 15 ug of computationally optimized broadly reactive NA antigen within a prime-boost framework. Subsequent challenges with influenza viruses H1N1 and H5N1 demonstrated that the vaccine successfully induced the production of specific neutralizing antibodies and effectively curtailed viral transmission among the ferrets (160).

As research has advanced, nonhuman primates have increasingly been utilized in biomedical studies (161). Among these, the cynomolgus monkey and the rhesus monkey have emerged as the most frequently employed species in this field (162). The genetic and physiological similarities of primates to humans, coupled with their extended lifespan, facilitate long-term studies (163). Research indicates that rhesus monkeys exhibit a greater propensity to display clinical symptoms when infected with H1N1, while cynomolgus monkeys demonstrate elevated levels of viral replication and increased body temperature in both the upper respiratory tract and lungs (164).

In a study, Swart et al. developed a subunit vaccine targeting the H1 stem protein, incorporating two additional point mutations to enhance the protein stability. They subsequently characterized the vaccine’s formulation efficacy in conjunction with various adjuvants in both murine and cynomolgus monkey models. A sustained humoral immune response specific to HA was observed in cynomolgus monkeys that were administered different adjuvants containing the H1 stem protein (165).

Typically, murine models are predominantly utilized in the initial phases of influenza vaccine research, while ferret models are employed in the intermediate stages. Ultimately, non-human primate models are introduced for a more comprehensive evaluation, contingent upon specific laboratory conditions and other relevant factors.