Influenza vaccine response: future perspectives

KEYWORDS:

• Influenza vaccine response
• host immune response
• influenza vaccine

1. Introduction: influenza vaccines, types and recommendations, and vaccine response

The World Health Organization (WHO) estimates that influenza annually is the cause of one billion infections, of which 3–5 million are severe diseases, with between 300,000 and 500,000 deaths worldwide. Among pediatric populations, influenza causes more than 800,000 hospitalizations in children of 5 years old and younger and with more than 300,000 hospitalizations in children of ages <1 year old. Vaccination is the primary strategy for the prevention and control of influenza. Indeed, annual influenza vaccination is currently recommended for groups at high risk of complications from influenza infection such as pregnant women, elderly people, young children, people with chronic diseases, and occupational groups [1].

The most widely used vaccines are either killed/inactivated or live attenuated influenza vaccines (LAIVs). The trivalent inactivated vaccines (TIVs) produced annually are currently targeted against the circulating H3N2, the pandemic H1N1/09, and an influenza B lineage strain as determined by the WHO. Three formulations are currently available: whole virus, detergent-split, and subunit vaccines, administered intramuscularly or subcutaneously. These vaccines rely on the induction of neutralizing antibodies targeting the globular head of viral hemagglutinin (HA) and neuraminidase (NA) antigens. Recently, quadrivalent inactivated vaccines, including a second influenza B virus in addition to the viruses in trivalent vaccines, have become available and are expected to provide wider protection against influenza B virus infections.

The LAIVs, which are administered intranasally, can stimulate humoral response, inducing both secretory IgA (S-IgA) and serum IgG, as well as cell-mediated immune response, similarly to natural influenza infection. However, they do have some limitations; for example, they are currently not approved for use in high-risk groups, due to the high incidence of allergic reactions as well as the risk of transmission following vaccination. Intranasal LAIVs are produced by reverse genetics using the HA and NA genes from circulating viruses on an attenuated, temperature-sensitive, cold-adapted virus backbone. LAIVs were found to be effective in influenza prevention in several controlled trials conducted in children before the A/H1N12009 pandemic [2]. However, the licensed LAIV in the United States has suffered a number of issues concerning its vaccine effectiveness (VE) over the past three seasons (2013–2014, 2014–2015, and 2015–2016) [3]. Consequently, in June 2016, the American Advisory Committee on Immunization Practices (ACIP) recommended that LAIV should not be used during the 2016–2017 influenza season, while the same decision was not accepted by several other health authorities such as Canada, UK, and Finland where LAIV VE was considered adequate [4]. Possible explanations for reduced LAIV VE has been a decreased immune response against A/H1N1pdm09 (thought to be the result of a more highly vaccinated population during seasons 2013–2014 and 2015–2016), antigenically drifted A/H3N2 viruses during the 2014–2015 season, potential interference of viruses included in the LAIV, heat susceptibility, and also methodological issues in studies (biases in the design and statistical limits) [5].

Several preclinical studies on adjuvant-combined nasal-inactivated vaccines have revealed that nasal S-IgA reacted with homologous virus HA and were cross-reactive with viral HA variants, resulting in protection and cross-protection against infection by both homologous and variant viruses. Data suggest that adjuvant-combined nasal-inactivated vaccines have advantages over the current injectable vaccines because they induce both S-IgA and serum IgG. Moreover, nasal-inactivated vaccines seem to be superior to the LAIV ones because noninfectious preparation could be used also in high-risk populations. Therefore, the development of intranasal-inactivated vaccines has to be recommended [6].

Recently, Protein Sciences have developed a vaccine, Flublok®, based on the HA protein; this is the first recombinant HA vaccine and data from the clinical studies were sufficiently compelling to support its FDA licensure for adults older than 18 years old (initially on 16 January 2013 and expanded on 29 October 2014) for the prevention of influenza (see next sections) [7].

The influenza vaccines available in the USA for 2016–2017 influenza season are summarized in Table 1.

Table 1. Influenza vaccines available in the USA for 2016–2017 influenza season.

	Presentation	Age indication	Mercury (from thimerosal), µg/0.5 mL	Latex	Route of administration
Inactivated influenza vaccine, quadrivalent standard dose	0.5 mL single-dose prefilled syringe	≥3 years	Not relevant	No	IM
	0.25 mL single-dose prefilled syringe	6 through 35 months	Not relevant	No	IM
	0.1 mL single-dose prefilled microinjection system	18 through 64 years	Not relevant	No	ID
Inactivated influenza vaccine, quadrivalent, cell culture-based standard dose	0.5 mL single-dose prefilled syringe	≥4 years	Not relevant	No	IM
Inactivated influenza vaccine, trivalent standard dose	0.5 mL single-dose prefilled syringe	≥4 years	≤1	Yes^a	IM
Adjuvanted inactivated influenza vaccine, trivalent standard dose	0.5 mL single-dose prefilled syringe	≥65 years	Not relevant	Yes^a	IM
Inactivated influenza vaccine, trivalent, high dose	0.5 mL single-dose prefilled syringe	≥65 years	Not relevant	No	IM
Recombinant influenza vaccine, trivalent (RIV3)	0.5 mL single-dose vial	≥18 years	Not relevant	No	IM
Live attenuated influenza vaccine, quadrivalent (LAIV4)	0.2 mL single-dose prefilled intranasal sprayer	2 through 49 years	Not relevant	No	NAS

ID: intradermal; IM: intramuscular; NAS: intranasal.

^aSyringe tip cap might contain natural rubber latex.

Modified from Grohskopf et al. [5]

The immune response to influenza vaccine is influenced by several host factors, such as age, genetic differences in immune responsiveness, history of infection and previous vaccination against influenza, gender, medical history, and health status.

2. Antigenic drift and antigenic shift

The capacity of influenza A and B viruses to undergo gradual antigenic change in their two surface antigens (HA and NA) complicated vaccination against seasonal influenza. The high variability of the influenza genome is due primarily to two mechanisms, known as antigenic drift and antigenic shift. The first one is caused by errors during the transcription of the viral genome as viral RNA polymerase lacks proof-reading activity, leading to accumulation of point mutations which, when affecting the antigenic domain, determine the emergence of new variant viruses. As the antibody prevalence to older variants increases in the population, the circulation of older previously dominant variants is suppressed, allowing new antigenic variants to become predominant; the virus is consequently able to escape immune recognition and cause repetitive influenza outbreaks. The second mechanism is a more dramatic change in virus antigenicity and happens when different subtypes of viruses enter a single cell. The replicated segments of various subtypes can reassemble with the exchange of genes, leading to new strains causative of pandemics, such as the last pandemic that occurred in 2009, caused by the A(H1N1)pdm09 infection [8]. The efficacy of current seasonal influenza vaccines is reliable when vaccine strains are matched with circulating influenza A and B strains; therefore, they need to be reformulated frequently. The low VE, as reported during 2014–2015 influenza season (33% related illness, confidence interval [CI], 26–39), was primarily due to antigenic mismatch between H3N2 strains and the A/Texas/50/2012 H3N2 vaccine strain [9]. Moreover, A (H3N2) viruses are shown to undergo changes in their receptor-binding specificity, which might result in genetic changes during their growth in eggs. These egg-adapted changes alter the antigenic properties of candidate vaccine viruses and contribute to a lower VE [10].

3. Vaccine manufacturing

The WHO officials together with National Health Agencies cooperate to identify dominant circulating strains that are likely to cause flu during the following winter in the northern and southern hemispheres. Current influenza vaccines are produced using the technology developed in the 1940s that relies on replicating the virus in embryonated hens’ eggs; gradually this production process may be transferred to cell cultures. The virions harvested from eggs are chemically inactivated, the viral envelope is disrupted with detergents, and the HA and NA proteins are then purified. HA is the main immunogen in inactivated influenza vaccines, and the levels of HA are used to standardize vaccine doses. The master virus strains (MSVs) for each of the three influenza virus components in LAIV are created through genetic reassortment. LAIV stimulates a strong cross-reactive antibody response. Adjuvants have been used to augment the immune response to vaccine antigens. Both their benefits and site effects are associated with activation of components of the innate immune system; for example, they permit antigen-sparing regimes, provide flexibility in the time interval between vaccinations, improve antibody avidity, and increase vaccine response in elderly people [11].

Flublok contains HA protein antigens derived from influenza virus strains. These proteins are produced in a proprietary non-transformed, non-tumorigenic continuous cell line grown in a serum-free medium, derived from Sf9 cells of the fall armyworm, Spodoptera frugiperda. The HAs are expressed in this insect cell line using the baculovirus Autographa californica nuclear polyhedrosis virus. Flublok contains 45 μg of each HA, whereas the standard vaccine contains 15 μg of each HA; the higher HA content offers the potential to provide cross-protection and the possibility for longer-lasting and improved immunogenicity [12,13]. The technology used to produce Flublok offers multiple advantages. For example, the vaccine will be an exact genetic match to the influenza virus and the manufacturing time is shortened. Furthermore, the manufacturing process does not require the biocontainment, the endotoxin content is carefully controlled, and no chemicals like formaldehyde are used in the process [7].

4. RNA structure and polymerase fidelity

RNA viruses exhibit high mutation rates due to the high error rates of viral RNA-dependent RNA polymerase. Viral replication during the egg adaptation process frequently results in gene mutations, which may alter the antigenic properties of the HA and NA proteins derived from the wild-type viruses. Frequent amino acid substitutions in HA have been reported during the propagation of the viruses in eggs. Furthermore, the growth of the virus in eggs can change the virus’s receptor-binding specificity through the alteration around the HA’s receptor-binding sites. Such mutations in vaccine viruses may reduce the efficacy of the influenza vaccines. Optimal vaccine production requires that seed viruses exhibit high growth in eggs, without substantial changes in their antigenic properties. Therefore, the development of a master virus with high genetic stability may be crucial for the preparation of vaccines [14]. In a recent work, Naito et al. described the generation of a high-fidelity and high-growth influenza vaccine MSV with a single V43I amino acid change in the polymerase basic 1 (PB1) of the high-growth A/Puerto Rico/8/1934 (PR8) master virus [15]. This can lead to the generation of high-growth vaccine viruses with high polymerase fidelity, low error rates of gene replication, and reduced antigenic diversity during virus propagation in eggs for vaccine production.

5. Role of host immunity on influenza vaccine response

Protection elicited by current seasonal influenza vaccines is antibody mediated. These antibodies bind to viral surface proteins that mediated virus entry or budding from host cells, leading to neutralization, opsonization, and/or complement fixation. CD4⁺ T helper cells are required for antibody responses during the induction, expansion, differentiation, and maturation of viral surface protein-specific B cells [16]. Vaccine efficacy (and effectiveness) is affected by various host factors, including age, genetic differences in immune responsiveness, history of infection and previous vaccination against influenza, gender, medical history, and health status. The most important factor among these is age. Additionally, a decline in immune response in the innate and adaptive immune system in the elderly leads to greater susceptibility to infection and lower responsiveness to the vaccine. Critical characteristics of immunosenescence include, for example, decreased number and function of dendritic cells, an alteration in the number of natural killer cells, and a decreased number of naive T and B cells [6,17,18]. The major factors known to determine influenza vaccine response are summarized in Table 2.

Table 2. Major factors known to determine influenza vaccine response.

Major factors known to determine influenza vaccine response
– Age
– Gender
– Genetic differences in immune responsiveness
– History of infection and previous vaccination
– Medical history
– Health status

The work of Khurana et al. demonstrated a strong correlation between activation-induced cytidine deaminase (AID) induction and in vivo antibody affinity maturation in human B cells following H1N1pdm09 vaccination. Results showed an age-related decrease in AID induction in B cells following H1N1pdm09 vaccination, suggesting that levels of AID mRNA before vaccination and fold-increase of AID mRNA expression after H1N1pdm09 vaccination directly correlate with an increase in polyclonal antibody affinity [19]. A study conducted on twins to evaluate the relative contribution of heritable versus nonheritable factors, by analyzing more than 200 different parameters, showed that most of these are determined by nonheritable factors, suggesting a large environmental influence on immune response [20].

Several studies support the observation of a reduced serological response with repeated vaccinations, suggesting that VE may be negatively influenced by prior vaccination, particularly when the antigenic distance between strains in consecutive vaccinations is small, as reported in a recent Japanese study [21]. In this context, the confounding effects of age on the immune response may also play a role. However, these findings are not confirmed by a recent study that showed how a single administration of the adjuvanted H1N1pdm09 vaccine is able to induce durable persistence of protective antibody titers and cross-reactive IFN-γ+CD4⁺ and CD8⁺ T cells, up to 4 years after vaccination. This is probably due to the AS03 adjuvant that is shown to increase vaccine immunogenicity. Per this finding, repeated annual vaccination can maintain strain-specific antibodies and T cells and important cross-reactive IFN-γ+CD4⁺ as well as CD8⁺ T cells, hence providing evidence to continue annual influenza vaccination [22].

6. Expert opinion

6.1. Current limits of the research

The use of LAIV has gained increased interest in recent years, thanks to the opportunity for simple intranasal administration that avoids intramuscular injection. However, it is licensed only for children ≥2 years of age and its real efficacy is currently debated. The decision of the ACIP to no longer support its use in the United States was not supported by several other health authorities where LAIV VE was considered adequate. Given the lack of clarity behind these differences, there is an urgent need for its clarification since LAIV can significantly improve vaccination rates in all ages, especially in children.

A major limitation in the current research efforts concerns the factors which affect the immune system and their viral targets. Factors like age, sex, and immunity inherited factors are complex phenomena that cause several changes to different components of the immune system. Indeed, there are still controversies about repeated vaccination. These debates have emerged through data from a study performed in a household cohort during the 2010–2011 season, where reduced VE was demonstrated among individuals vaccinated in the prior year, and other studies subsequently observed similar findings [23–25]. These reductions in VE have primarily been associated with outbreaks of A(H3N2) infection. Studying this phenomenon for A(H3N2) is particularly challenging, given extensive histories of prior exposure to various strains through vaccination and natural infection. Furthermore, a recent study by Martínez-Baz et al. observed higher VE against A(H1N1)pdm09 (66%; 95% confidence interval [CI], 49–78) in individuals who received one to two prior doses [26]. It is imperative to understand the immunological mechanisms underlying this effect because clearly these observations have implications in vaccination policies in terms of frequency of vaccination, vaccine strain selection, and the development of novel influenza vaccines. Universal vaccines could, in part, solve the problem, but this scenario is not immediate and, in the meantime, it is necessary to understand the impact of sequential vaccination, studying current vaccines in homogeneous populations to avoid any bias.

6.2. Alternative cell cultures for vaccine manufacturing

The 60-year-old egg-based influenza manufacturing process has served us well. However, newer technology will enable us to overcome recognized egg substrate disadvantages of current influenza vaccine production. Such problems include the multiple passages required for virus proliferation in eggs and which may result in variant vaccine viruses; the long time required for production; the current need for high-level biocontainment facilities; and the current need for chemicals of concern to inactivate the influenza virus [12]. In the case of pandemic outbreaks, there is the possibility that vaccine numbers could be limited by egg supply so that vaccines are not produced in a timely manner. To overcome this potential production issue which is based on replicating the virus in embryonated hens’ eggs and guarantee fast mass immunization, several new influenza vaccine cell culture-based production methods have been granted commercial license in recent years.

Flublok is another step forward in the development of vaccines based on cell culture technology and represents the world’s first trivalent protein-based influenza vaccine developed with modern recombinant DNA technology. Flublok is produced using the baculovirus–insect cell expression system, as previously described. The HA proteins are exact analogs of wild-type circulating influenza virus HAs, with a threefold higher protein content compared with the TIVs. Furthermore, the HA component of the vaccine can be changed rapidly as needed on a seasonal basis [7,27]. Trial data have demonstrated that the higher antigen content in Flublok results in improved immunogenicity, while further data also suggest an improved efficacy and a slightly lower local reactogenicity compared with standard inactivated influenza vaccines, despite the presence of more antigen (statistically significant) [7]. Moreover, in the event of an influenza pandemic or vaccine supply shortage, this type of vaccine manufacturing has the potential to start up vaccine production much faster than the traditional influenza vaccine process.

6.3. The future of influenza vaccination: toward a universal vaccine

Current influenza vaccines induce immunity to the influenza virus strain-specific HA antigen and are not very effective against new pandemic viruses. A universal vaccine may have the capacity to protect against most varieties of influenza strains and subtypes. Many strategies for developing a universal influenza vaccine are based on raising an immune response against influenza proteins that are highly conserved across all strains [11]. To overcome these drawbacks, attractive approaches are aiming to develop a universal influenza vaccine, based on conserved protein regions or peptides (both B- and T-cell epitopes in the same formulation), shared by all strains and which are able to induce cross-protective neutralizing immunity against conserved viral antigens [16]. The current challenge is to develop strategies that allow strong antibody responses to the HA stem region, the less immunogenic region of the HA molecule. Several approaches have already been tested including the use of headless HA molecules, HA molecules with hyperglycosylated heads, or sequential immunization with chimeric HA molecules [28,29]. Both humoral and cell-mediated arms of the immune system should be activated to confer broadly protective immunity. Encouraging results have been obtained in studies on animals with virus-like particles that can induce strong humoral and cellular immune responses.

An influenza vaccine embodying the concept of a universal vaccine is the Multimeric-001® vaccine, which contains nine conserved linear epitopes from HA, nucleoprotein, and matrix protein-1. The Phase III trial will be pivotal and will run in 2017 and 2018 and in multiple centers across Europe and the USA. Another promising influenza virus antigen is the matrix protein 2 (M2), a transmembrane protein that acts as a proton-selective ion channel and plays a crucial role in helping release the genetic material of the virus into the host cell. It consists of a 23 amino-acid peptide containing the B-cell linear epitope, highly conserved across influenza virus subtypes. Phase I/II clinical trials have so far been successfully completed.

However, while advances are going to change the vaccination policies in upcoming years, a pressing need for the traditional egg-based production remains and advances are necessary to respond to a pandemic outbreak of influenza virus, which is predicted in the upcoming years. In the meantime, the creation of a cost-effective and flexible system to supply influenza vaccine for the world’s population remains one of the major challenges of the influenza vaccine industry.

Declaration of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

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Funding

This manuscript has not been funded.