Targeting H3N2 Influenza: Advancements in Treatment and Vaccine Strategies

1. Introduction

Zoonotic influenza remains a significant global public health threat [1,2,3]. Specially avian influenza, in different forms, represents a menace for a future pandemic [4]. In the case of H3N2, this is one of the emerging forms currently concerning in some areas of the world [5]. For some experts, the risk of spillover from this and other forms seems imminent for multiple epidemics [6,7]. This review examines the host's humoral immune response, primarily targeting primary epitopes carried by the hemagglutinin (HA) surface glycoprotein in influenza A viruses. The swift mutational evolution of HA leads to "antigenic drift," allowing viruses to evade the host's adaptive immune defence [8,9]. Notably, the H3N2 influenza virus crossed the species barrier, initiating human infection in 1968, and recent antigenic modifications have led to the emergence of antigenic clusters [9]. Influenza A virus causes acute respiratory illness, contributing to a global annual mortality estimate of 250,000 to 500,000 individuals, with notable pandemics including the 1918 H1N1, 1957 H2N2, 1968 H3N2, and 2009 H1N1 outbreaks [10,11,12]. Influenza A viruses are distinguished from types B and C by their nucleoprotein (NP) and matrix (MI) proteins belonging to the Orthomyxoviridae family. The influenza A virus genome comprises 11 protein segments on a single-stranded negative-sense RNA sequence [13]. There are 144 different HA-NA combinations in the influenza A virus family, with 16 HA and 9 NA subtypes identified by neuraminidase (NA or N) and hemagglutinin (HA or H) proteins on the virus surface [14,15,16].

The World Health Organization (WHO) recommends regular updates to the H3N2 component in influenza vaccines due to ongoing mutational dynamics within H3N2 viruses [17]. Amino acid alterations at residues 222 and 225 in the hemagglutinin chain have been identified, affecting receptor-binding characteristics [18,19]. Research in the Western Pacific region aims to assess the impact of H3N2 viruses on public health and mortality rates, emphasising the need for control strategies [20]. Predicting antigenic characteristics of A (H3N2) viruses outside E-SE Asia could improve vaccine strain selection and reduce morbidity and mortality [21]. H1N1 and H3N2 subtypes have circulated in humans since 1977, with age-specific variations likely due to childhood imprinting [22]. Fever is the most common symptom for both influenza A subtypes, with higher body temperatures during the A/H3N2 season, while myalgia, coughing, and sore throats were more common during the A/H1N1 season [23]. Antigenicity characterisation poses challenges for HA inhibitory antibodies, with differences in neutralising antibody titers between children and adults, suggesting the NI assay's potential in H3N2 infection testing and vaccine selection [24]. Figure 1 provides a visual overview of the Influenza A (H3N2) virus, emphasising recent occurrences and research methodologies for analysis.

2. Global Impact and Evolution of H3N2 Influenza: A Historical Perspective

The H3N2 influenza virus originated from avian influenza A and coupled the N2 neuraminidase from the 1957 H2N2 virus with the distinct H3 hemagglutinin to generate the 1968 pandemic. When this virus was first identified in September 1968, it was thought to have killed one million people worldwide, of whom 100,000 died in the United States of America (USA) [25]. The H3N2 seasonal influenza A virus continues its worldwide circulation. These seasonal H3N2 variants frequently undergo antigenic drift, mainly affecting older individuals and leading to severe illness [26]. Since 1977, human-adapted subtypes of influenza A viruses (H1N1, H1N2, H3N2) have posed a simultaneous infection risk. While these viruses infect various animals, their primary hosts are wild birds. Influenza outbreaks affect 10–30% of the world's population and result in 290,000–650,000 deaths yearly, mostly in adults 65 years of age and older. Sub-Saharan Africa and Southeast Asia have the highest death rates from influenza epidemics [20]. In 2011, 12 cases of human infection with the new A(H3N2) virus were identified in the US, with potential person-to-person transmission [27]. Between 2011 and 2018, A(H3N2) was the cause of 39.2% of confirmed influenza infections in 27 Asian countries. Epizootiology research confirmed the H3N2 pandemic strain resulted from avian-human virus reassortment. Various H3N2 variants have amino acid changes at five antigenic sites on the H3 head, and single amino acid substitutions can alter the virus. A 2021-2022 study investigated 161 A(H3N2) virus strains, showing high genetic linkage [28]. Table 1 simplifies the origin and impact of the 1968 H3N2 influenza pandemic by representing the year-wise occurrences of H3N2 human infections, aiding in a clearer understanding of its global impact.

3. Pathophysiology of H3N2 Influenza: Complex Interactions and Inflammatory Response Mechanisms

The H3N2 influenza virus invades the upper respiratory tract, developing within respiratory epithelial cells and triggering an inflammatory response. This response, orchestrated by viral infection, produces inflammatory mediators like chemokines and cytokines. Cytokines recruit leukocytes and activate immune reactions, while chemokines attract leukocytes to infection sites, thus contributing to inflammation. However, this intricate cascade can lead to excessive inflammation, resulting in tissue damage and organ dysfunction in critical illnesses related to the virus [36]. This is comparable to the complex pathophysiology of the H3N2 influenza virus, which starts in the upper respiratory system and progresses to the lower tract. Influenza virus H3N2 damages respiratory epithelium, causing inflammation and airway obstruction [37]. Inside the body, the H3N2 influenza virus attaches to cell receptors, infiltrates host cells, and initiates replication, prompting immune responses that restrict viral replication via antibodies and cytokines.

The virus, however, can also directly damage tissues, leading to complications like pneumonia [38]. Influenza transmission studies using ferrets reveal a significant route through the soft palate, favoured by human influenza viruses' hemagglutinin proteins and similar receptors in humans [39,40]. Influenza's primary pathophysiology involves lung inflammation and dysfunction due to viral infection of the respiratory epithelium, compounded by immune responses. Chronic inflammation can lead to multiorgan failure, with predominant effects on the lungs, causing severe respiratory distress [41]. The influenza virus directly affects the respiratory tract or weakens the immune system. Lung obstruction, alveolar structural loss, and extracellular matrix deterioration are possible outcomes. Acute pneumonia is identified in 30–40% of hospitalised patients with laboratory-confirmed influenza; older patients (more than 65), Caucasians, younger than five, and inhabitants of nursing homes are at higher risk of developing pneumonia. Moreover, influenza can result in secondary bacterial infections and severe pneumonia, which increase the risk of bacterial sepsis and ARDS. Influenza A is the most frequent virus that causes adult acute respiratory distress syndrome (ARDS) [36]. H3N2 Influenza Viruses (IAVs) have evolved rapidly since 1968, incorporating N-linked glycans, increased HA molecule net charge, and altered receptor binding preferences. Researchers have modified antigenic characterisation assays to adapt to these changes. The HAI assay's use of guinea pig red blood cells and 20nM oseltamivir carboxylate enables a more precise evaluation of contemporary IAVs (Figure 2) [42].

4. Evolving Vaccination Strategies

The primary method used to create inactivated influenza vaccines is to develop viruses in chicken eggs, which have been regulated for over 70 years. The FDA distributes, tests, and adapts vaccine viruses following the WHO's strain selection process. The global manufacturing infrastructure can produce 1.5 billion doses annually. Influenza vaccination efficacy is assessed through randomised clinical trials and observational studies. Since the mid-2000s, however, nothing has been known about the effectiveness of vaccines targeted to a particular strain. A meta-analysis of influenza vaccine clinical trials conducted between 1967 and 2011 identified eight placebo-controlled trials, most showing no particular benefit against H3N2. With the development of molecular diagnostic tests, influenza infections may now be detected with high sensitivity and specificity. An inventive 'test-negative' observational study design was employed in Canada in 2005 to quantify vaccine effectiveness for the first time using RT-PCR. This methodology is used in annual VE surveys conducted in the US, Canada, Europe, and Australia [43]. The primary public health approach to curbing influenza, including the H3N2 influenza virus, revolves around vaccination. Annual vaccines are advised for individuals who are deemed to be at risk, such as the elderly and those with high rates of morbidity and death, according to the WHO [44]. Seasonal influenza, primarily H3N2, is a significant cause of respiratory disease and mortality, with increased rates of hospitalisation and excess mortality among the elderly [43].

Nevertheless, compared to other subtypes, the efficiency of the existing influenza vaccinations against H3N2 viruses is lacking [20,45]. This discrepancy is partially attributed to the H3N2 virus's rapid and unpredictable evolutionary pace in contrast to other seasonal flu viruses [46]. Further contributing to the reduced vaccine effectiveness is the composition of trivalent inactivated vaccine (TIV) influenza vaccines, which incorporate antigens from two A subtypes (A H3N2 and A H1N1) and only one B lineage. This setup frequently leads to mismatches between circulating and vaccine B strains. Consequently, the Quadrivalent influenza vaccine (QIV) has demonstrated enhanced immunogenicity compared to TIV across various age groups [44]. Furthermore, a prime-boost vaccination strategy involving the Ad5-HA + Ad5-NP vaccine followed by an inactivated H3N2 vaccine has proven effective in inducing cross-reactive immunity against H3N2 viruses in swine[47]. To improve vaccinations, yearly assessments of vaccine efficacy and genetic analysis of circulating influenza viruses are essential. The evidence base for choosing influenza vaccine viruses may be strengthened by combining clinical protection with virologic data [48]. The efficacy of influenza vaccines against influenza A(H3N2) viruses was lower than that of influenza B viruses, necessitating improved effectiveness. However, the 2016–2017 influenza vaccination trials demonstrated modest protection against outpatient influenza [49]. The antigenic distance hypothesis states that harmful interference from the previous season's immunisation may have an adverse effect on this season's protection from influenza. During three outbreaks (2010-2011, 2012-2013, 2014-2015), a study conducted in Canada assessed the effectiveness of vaccines against influenza A(H3N2) disease that required medical attention and laboratory confirmation. Consistent with the ADH, the results revealed considerable variations in the preceding vaccination effects by season. In 2014–2015, adverse effects were evident and statistically significant, indicating that low vaccine effectiveness in subsequent epidemics since 2010 could be attributed to influenza vaccinations administered more than once [50]. The discussion on H3N2 vaccine effectiveness involves summarising and presenting data from studies or clinical trials, providing a general outline to structure the discussion in Table 2. Vaccination remains the chief strategy to contain the H3N2 influenza virus. However, formulating effective vaccines encounters challenges due to the virus's rapid evolutionary rate.

4.1. Current Influenza Vaccines

The influenza vaccination now in use, which targets the H3N2 strain, has been updated for the flu season of 2022–2023 [56,57]. A component of the virus similar to A/Darwin/9/2021 (H3N2) is included in this vaccination [56,57]. However, it's crucial to understand that the flu vaccine's ability to fend off H3N2 viruses changes according to the season. Vaccinations against influenza A(H3N2) are often less effective against influenza B viruses and more effective against influenza A(H1N1) viruses [58]. The H3N2 strain in the flu season of 2021–2022 showed antigenic dissimilarity from the vaccine virus, which led to a decrease in the efficacy of the vaccine against H3N2 viruses [59]. The World Health Organization (WHO) has recommended the A/Darwin/6/2023 (H3N2) component of influenza vaccinations for the 2023–2024 influenza season in the northern hemisphere [60]. Maintaining a yearly vaccination regimen is pivotal in guarding against the flu, given the ongoing variations in the viruses [56].

4.2. Effectiveness and Limitations of Vaccines

H3N2 vaccines' effectiveness varies annually due to the virus's continuous evolution and adaptation. The potential deterioration in the effectiveness of the H3N2 vaccination can be attributed to several factors, including antigenic mismatch, vaccine component egg-adaptive alterations, and age-related effects, which allow older individuals to be less protected against A(H3N2) viruses due to previous exposure to non-A(H3N2) influenza viruses. According to recent studies, immunisation lowers the risk of influenza hospitalisation in younger, immunocompetent adults during seasons when the vaccine virus and influenza A(H3N2) are antigenically different. In 2020–2021, the COVID-19 pandemic saw a low level of influenza circulation; in 2021–2022, the circulation level increased. Immunisations decreased the likelihood of hospitalisation for younger persons but not for those over 65. Antivirals, vaccinations, and preventative measures all require advancements [59]. Among participants <50 years old, influenza vaccines displayed a 36% effectiveness against A(H3N2)-related illnesses [61].

Studies show that repeated annual influenza vaccine shots are less effective against influenza. In a trial, ferrets were given a prime-boost vaccination regimen twice and once, and they were then challenged with A/Hong Kong/4801/2014 (H3N2). The RV group lost weight more slowly and shed more virus, indicating that variations in the quality of the immune response could influence protection following recurrent immunisation [62]. Despite the continued widespread use of egg-based influenza vaccinations, emerging vaccine platforms promise to resolve drawbacks [20]. The investigation compared the H3N2-specific antibody responses of mice immunised with mRNA-LNP vaccines encoding wild-type and egg-adapted H3 antigens. The results showed that mRNA-LNP encoding wild-type H3 was superior to egg-adapted H3 or the egg-based Fluzone vaccination in neutralising the wild-type 3c.2A H3N2 virus. Both mRNA-LNP vaccinations produced significant levels of group 2 HA stalk-reactive antibodies, suggesting that mRNA-LNP-based vaccines modified with nucleosides can avoid problems associated with egg adaptation in the most recent 3c.2A H3N2 viruses. Because of their distinct glycosylation site, 3c.2A H3N2 viruses, a distinct offshoot of the 1968 H3N2 strain, came to light during the 2014–2015 influenza season and are still circulating worldwide [63,64]. Those with moderate diseases, such as mild upper respiratory tract infections, fever, or diarrhoea, can benefit from the nasal spray flu vaccine. Even if the vaccine does not precisely match the strain that is now circulating, immunisation remains the most effective means of protection against the flu overall [65].

4.3. Novel Approaches to Vaccine Design

Influenza H3N2 undergoes continuous mutations, presenting a challenge for vaccine creation. Nonetheless, researchers have explored innovative avenues to enhance vaccine efficacy. These strategies include the accurate administration of antigenic material, control over release patterns, and well-informed design based on a more profound comprehension of immune system mechanisms and pathogen-host interactions [66]. One study employed an H3N2 microneedle vaccine, which produced a cross-protective immune response against many H3N2 antigenic variants [67]. Alternate approaches encompass employing conserved antigens like HA, NA, matrix, and internal proteins, coupled with diverse vaccine platforms such as recombinant antigen/protein-based, virus-vectored, nanoparticle-based, DNA/RNA-based, virus-like particle (VLP), and multiplex vaccines [68,69,70]. How to expose highly cross-protective epitopes to the immune system has been investigated using glycan engineering of HA and NA proteins [68]. Researchers have also devised chimeric HAs by transferring unique HA globular head domains from exotic novel strains to the HA stalk domains of presently circulating human influenza viruses [71]. Moreover, adenoviruses have been transformed into vaccine vectors by disabling genes responsible for their replication, exhibiting the potential for developing a "universal" flu vaccine. Overall, pandemic-focused vaccine development aims to mitigate public health repercussions and societal disruption [72].

5. Antiviral Therapies of H3N2 Influenza Virus

Several antiviral medications are accessible for treating influenza, including H3N2. The four antiviral medications recommended for influenza treatment are oseltamivir, peramivir, zanamivir, and baloxavir [73,74]. These medications function by stopping the surfaces of infected host cells from releasing influenza virions [75]. Antiviral therapy should be started as soon as possible for hospitalised patients with suspected or confirmed influenza who have severe, complicated, or worsening symptoms or are at higher risk of developing influenza-related complications. It is not necessary to wait for laboratory proof of influenza virus infection before starting antiviral therapy in suspected influenza cases [73]. Vaccination is the most reliable method of preventing H3N2 influenza; it is recommended for all individuals six months of age and older and is typically available in the fall. Keeping up with proper sanitary practices is also crucial. These include avoiding direct contact with sick people, frequently washing hands with soap and water, and covering the mouth and nose when coughing or sneezing [75].

5.1. Neuraminidase Inhibitors (Oseltamivir, Zanamivir)

Neuraminidase inhibitors, such as oseltamivir and zanamivir, belong to a drug class that obstructs the neuraminidase enzyme, a vital component for influenza replication. The FDA has approved using oseltamivir, with a twice-daily dosing schedule, for treating acute, uncomplicated influenza within two days of the onset of illness. These antiviral drugs are commonly used to battle influenza viruses A and B [73]. Even in severe influenza, where rhabdomyolysis is present, zanamivir has proven beneficial when treatment is initiated more than 48 hours after the onset of symptoms. When used early in influenza treatment, Neuraminidase inhibitors effectively lower the incidence of severe cases and fatality [76]. However, individuals with severe immunosuppression face the highest risk of developing oseltamivir- and peramivir-resistant influenza virus infections during or after treatment with these drugs [73]. Ongoing observation of oseltamivir resistance is necessary to further protect public health due to the evolution of antiviral medication resistance among influenza viruses [77]. Public health organisations advise using neuroliminase inhibitors to treat and prevent seasonal and pandemic influenza infections (Figure 3) [78]. Sialic acid receptors are crucial for virus attachment and entry into host cells. Blocking these receptors and inhibiting virus-host cell interactions is the best way to control and prevent infection. Neuraminidase inhibitors, including oseltamivir and zanamivir, are the most efficient drugs for treating influenza A and B virus infections [79].

5.2. Polymerase Inhibitors (Baloxavir Marboxil)

Baloxavir marboxil, a polymerase inhibitor, serves as a treatment for uncomplicated influenza [80,81]. It prevents the viral polymerase complex's cap-dependent endonuclease activity, which is essential for viral replication [82]. About its use against H3N2 influenza, here are key insights: Clinical trials have established the efficacy of baloxavir marboxil against H3N2 influenza [81]. However, because to changes in the polymerase acidic protein, certain H3N2 viruses have shown reduced susceptibility to baloxavir marboxil [80,83]. Unlike neuraminidase inhibitors (NAIs) like zanamivir and oseltamivir, which are usually advised twice daily for five days, baloxavir marboxil is only given orally once [81]. In vitro, investigations have revealed that baloxavir acid, the active metabolite of baloxavir marboxil, can be combined with other inhibitors, such as NAIs and favipiravir, to augment antiviral efficacy against seasonal influenza A viruses [84]. Baloxavir marboxil exhibits potential as a treatment choice for uncomplicated influenza, including H3N2 strains. Nevertheless, the emergence of resistant strains underscores the ongoing necessity for research and the development of novel antiviral therapies.

6. Advancements in Molecular Detection Methods

6.1. Rapid Diagnostic Methods

Serological testing, antigen detection tests, and molecular assays are quick diagnostic techniques for H3N2 influenza. In ten to fifteen minutes, antigen detection tests known as Rapid Influenza Diagnostic Tests (RIDTs) can identify influenza viral antigens in respiratory specimens with low sensitivity. The antigen-detection assays known as RIDTs have moderate sensitivity and may detect influenza virus antigens in respiratory specimens in 10 to 15 minutes. These immunoassays detect influenza A and B viral nucleoprotein antigens in respiratory samples and yield a qualitative result (positive vs. negative). RIDTs have excellent specificity; however, they only offer modest sensitivity. On the other hand, outstanding sensitivity and specificity are offered by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and other molecular techniques for identifying influenza virus RNA or nucleic acids in respiratory materials [85,86]. Among the techniques used in serological research are the Hemagglutination Inhibition Assay (HAI), Single Radial Hemolysis (SRH), Virus Neutralization Assay (VN) or Microneutralization, and Enzyme-Linked Immunosorbent Assay (ELISA). These tests detect influenza virus antibodies in serum or other bodily fluids. However, their applicability for early influenza diagnosis is limited due to the need for paired serum samples collected at least 2 weeks apart. For H3N2 influenza, molecular assays and antigen detection tests stand out as the most common quick diagnostic techniques [87].

6.2. Molecular Detection Techniques

There are numerous molecular detection methods available for identifying the H3N2 influenza virus. Reverse transcription-polymerase chain reaction (RT-PCR) and other nucleic acid amplification tests are the most widely used methods for detecting influenza virus RNA or nucleic acids in respiratory samples because of their exceptional sensitivity and specificity. Several molecular techniques can distinguish between influenza A and B infections as well as identify seasonal influenza A virus subtypes, such as A(H1N1) pdm09 or A (H3N2) [87,88,89,90]. Loop-mediated isothermal amplification (LAMP), an alternative molecular-based influenza diagnostic, provides a rapid, accurate, and dependable molecular detection technique. This technique provides a stable foundation for influenza and COVID-19 testing [91,92,93]. The Multiplex One-Step Real-Time RT-PCR technology increases efficiency and simultaneously identifies the human H3N2 virus, the pandemic (H1N1) 2009 virus, and the reassortant avian H7N9 virus [94]. Recommended by the WHO, these molecular assays offer high accuracy and efficacy for diagnosing the H3N2 influenza virus [87,88,89,90].

6.3. Point-of-Care Testing Advances

Point-of-care testing (POCT) for influenza is experiencing rapid growth, and multiple studies have demonstrated its benefits in diagnosing influenza among patients with acute respiratory tract infections [95]. POCTs represent swift diagnostic assessments performed at the point of care, whether in a doctor's office or an emergency department, delivering results within minutes [95,96]. Initially criticised for limited sensitivity and result variability, recent research has highlighted the high specificity and sensitivity of POCTs, establishing them as reliable and practical tools for early influenza identification [96,97,98]. POCTs have the potential to enhance patient flow, reduce hospitalisations, and facilitate targeted treatments [99,100]. Predominant POCT options for influenza encompass antigen detection tests and molecular assays like RT-PCR [95,96,98]. Additionally, the molecular-based Loop-mediated isothermal amplification (LAMP) assay introduces an innovative avenue for molecular diagnosis [99,101]. The introduction of POCTs offers fresh prospects for managing healthcare patients, as illustrated in Table 3.

7. Future Directions

In moving forward, this review has illuminated crucial paths for future research and development in effectively combatting the challenges of Influenza A (H3N2). These essential directions encompass strengthening global surveillance through advanced sequencing technologies and real-time data sharing, enabling rapid detection of new strains and swift public health responses. Predictive modelling holds promise in guiding the formulation of more effective vaccines by anticipating antigenic shifts. Universal vaccines targeting conserved viral regions could offer enduring protection against diverse strains. Investigating host immune responses can provide insights into disease severity and susceptibility, informing targeted interventions. Exploring combination antiviral therapies and integrating advanced diagnostic tools into public health strategies can enhance treatment and outbreak management. For comprehensive solutions, promoting multidisciplinary collaboration among professionals is essential. Investment in worldwide education and pandemic preparedness will improve the ability to control possible epidemics. Influenza A (H3N2) 's dynamic complexity must be anticipated, understood, and efficiently managed. Proactive collaboration, cutting-edge technology, and a multidisciplinary approach are essential.

8. Conclusion

This review delves into the humoral immune response to influenza A viruses, mainly focusing on recognising hemagglutinin surface glycoprotein epitopes. It emphasises the significant impact of the H3N2 virus, which breached the species barrier in 1968, leading to acute respiratory illness and global mortality. The WHO advocates for regular updates to H3N2 vaccines due to mutational dynamics, with research in the Western Pacific region underscoring the importance of awareness and control strategies. The devastating 1968 H3N2 influenza pandemic claimed one million lives globally, with seasonal variants continuing to cause severe illness. Since 1977, human-adapted subtypes have posed a significant infection risk, affecting a substantial portion of the world's population annually. Influenza A (H3N2) spreads through various means and exhibits mutability, causing sporadic outbreaks and pandemics. Notably, H3N2 has caused fatal outcomes in India, and its molecular epidemiology indicates the possibility of reinfection by the same subtype in a short time frame. The review highlights the challenge posed by H1N1 and H3N2 mutations, affecting vaccination effectiveness. The WHO strongly recommends yearly influenza vaccination, particularly for the 2023-2024 flu season. However, H3N2 mutations present obstacles to vaccine development, prompting exploration into innovative strategies such as alternative antigen formulations and pandemic-focused vaccine development. Despite longstanding regulation of inactivated influenza vaccines, their effectiveness against H3N2 viruses has been limited due to the virus's rapid evolutionary pace. The review comprehensively explores the evolution, epidemiology, clinical manifestations, vaccination strategies, and Influenza A (H3N2) antiviral interventions, underscoring the importance of diagnostic advancements and multidisciplinary collaboration for pandemic preparedness. It is a valuable resource for healthcare professionals and policymakers combating H3N2.