Abstract
The recurrence of influenza outbreaks and isolated cases of epidemics is a source of global concern and is a significant cause of human morbidity and mortality. The influenza viruses can easily evolve through antigen mutation which enables these viruses to overcome the barriers of human body immunity that lead to host adaptation and transmission.
However, mechanisms responsible for the viral evolution are systematically being elucidated. Influenza infection can be effectively prevented through vaccination; yet the recent developments in the emergence of new influenza virus as demonstrated by 2009, pandemic influenza H1N1, influenza A virus (H7N9) and the deadly avian influenza A virus (HPA1 H5N1) which have lately revealed the numerous challenges to the vaccine strategy.
The emergence of these virus strains stresses the need to have a vaccine that is effective in combating the influenza viruses on a wide spectrum. This paper provides a review of evolution among the important influenza proteins and the impact of these changes on viral on viral antigens, host adaptation and the pathogenicity of these viruses. Most importantly the paper discusses the development of a universal influenza vaccine founded on this knowledge.
Introduction
The A, B and C viruses of influenza represent the three out of the five genera of the family Orthomyxoviridae and are distinguished by the segmentation in the negative strand RNA genomes. Through sequencing a relationship in the ancestry of these viruses has been established; however, they have genetically diverged, and reassortment has been evident in each genus as opposed to such occurrence across the genus. Viruses of influenza type A are distinguished by the subtype of their surface glycoproteins, the hemagglutinin (HA) besides the neuraminidase (NA). Influenza type A virus is known to attack a variety of organisms apart from the human beings including pigs, birds, dogs horses among many other animal species (Zhu et al., 3).
Influenza type B is predominantly found in humans although they possess the same structure as the type A virus. The virus of influenza type A and B have a discrete segment as compared to the C and D influenza viruses which contain seven genomic parts only. Influenza type C virus is known to predominantly affect humans however cases of infection in other animal species have also been reported mainly in pigs, horses, birds and cattle. The type D virus has been reported in goats and sheep after being recently isolated from the swine and cattle. The type A influenza virus have been recognized to be the most severe in zoonotic infection and human influenza pandemics.
Type A and B influenza viruses are responsible for several epidemics across the globe annually with causality record of three to five million infections resulting in approximately 250000 to 500000 deaths annually. In the United States, the seasonal epidemics are responsible for over 200000 hospitalizations and between 30000 and 50 000 deaths annually. The elderly, children under five years, pregnant women, infants and individuals suffering from chronic ailments are groups most vulnerable to influenza infection (4).
Moreover, the seasonal epidemics are sometimes supplemented by the occasional pandemics with statistics revealing that in the past 200 years there have been five major pandemics. These pandemics include the 1918 H1N1 Spanish flu pandemic, the 1957 H2N2 Asian flu pandemic, the 1968 H3N2 Hong Kong flu pandemic, the H1N1 pandemic of 1977 and the H1N1 flu pandemic of 2009. The influenza virus strand of H5N1 has rapidly evolved and is feared to be the next causal agent of another flu pandemic in the world. Antigenic shift and drift are the major elements responsible for the rapid evolution of the influenza virus and a source of critical challenges encountered in developing new antiviral drugs and novel vaccines.
Evolution and vaccination of the Influenza virus
Technology has played a critical role in developing of the influenza virus vaccine. Sequencing technology has provided researchers with advanced data concerning the genetic composition of the influenza virus. The development in the sequencing technology has allowed for advanced sequence analysis of the influenza virus (Lam, Ching, et al. 1). The complexity involved in trying to make sense of the massive data available concerning the influenza virus necessitates technology to employ advanced and new methodologies that are capable of breaking down the data into simple, understandable bits.
This is where the unsupervised machine learning approach comes into play. It is applied to the highest levels of influenza genetic sequences to visualize the vaccine controlled, and non-vaccine prevented influenza viruses. While applying the unsupervised machine learning processes, two objectives are sort and achieved. One is the visualization of the evolutionary trajectories in influenza virus under the vaccine pressure in the wild in the absence of any previous information on the viruses (6). The second is objective is to bring out statistical evidence in support of the visualization results. The origin of the influenza virus is thought to be from the reservoir consisting of wild aquatic birds.
Hemagglutinin
The relationship between influenza virus proteins and vaccine development is best explained through a study on hemagglutinin and neuraminidase. The influenza hemagglutinin is the integral type 1 protein membrane. Hemagglutinin is best described by the ectodomain that consists of the outward globular HA1 domain and the HA2 stem domain (Bouvier, Nicole, Palese 1). The HA2 stem domain allows the ectodomain to anchor the entire protein to the virus membrane through spanning. The life cycle of the influenza virus begins with the recognition of the sialic acid in the host cell glycoprotein by HA which is then followed by endocytosis.
The process of fusion of the viral and endosomal membrane requires an acidic environment which is triggered by the Ha conformational change (7). HA is the primary component of influenza antigens and performs the crucial role of targeting the host humoral immune system. The domineering antibodies produced by the host are usually against the immune dominant head domain. It is worth noting, during viral infection the most changes of the vital amino acids happen at the domain head. This is mainly because the receptor domain head contains receptor sites that are influential in receptor binding affinity, transmission and specificity.
The results demonstrate Minuit antibodies that are against the hemagglutinin and as a result there is a broad binding effect at the globular head domain. Although there is high divergence at the head domain, the stalk domain manages to retain its sequence conservation. The development of a universal influenza virus vaccines dramatically depends on the approach of induction of a broader HA stalk related antibodies. There is evidence that some stalk antibodies have extensive binding capabilities with the one or both group 1 and 2 hemagglutinin as well as influenza B hemagglutinin.
Studies on the methods that underline the antibodies efficacy have revealed that antibodies can stop membrane fusion in viruses and endosome by creating a barrier between HA1 and HA2 sub-units which inhibits viral budding as a result of the interaction of hemagglutinin on the cell surface. Interaction with antibody-dependent cell mediated cytotoxicity. The use of chimeric hemagglutinins which have different head domains is influential in the induction of higher titers consisting of stalk-specific antibodies through a sequential immunization strategy.
Some new methods have been developed focusing on exposing the stalk part without any interference with its native structures to initiate neutralizing of antibodies are available. These methods include stitched HA systems that consist of conserved fragments, mimicry of the HA mini stem profusion conformation, and the manufacturing of an H3 subtype HA which exposes the HA subtype peptides at the head domain. Evidence points out that nanoparticles that are based on HA conserved stalk epitopes are in regard to heterologous prime-boost is a positive lead to the development of a widely protective and effective immune response.
It is also a possibility that the amino acid remains replaced at the glycosylation sites of the HA molecules are responsible for receptor binding, antigen change and transition in clusters. For instance for H5N1 viruses to effectively bind with the receptors of the human and transmit among the mammalian species the glycosylation loss at 158-160 is essential to this process. The lack of glycosylation at 158 was vital for the binding of H5N1 to occur at the α2 6-linked receptors (Rocha et al 5).
Although evidence from several studies points to glycosylation of the hemagglutinin having an impact of the virulence of the virus, the glycosylation modulator receptor binding properties have to be verified. Evidence form a recent study reveals that glycosylation changes that occurred in the H3N1 head domain are responsible for changes in receptor binding properties of the virus but did not have an effect on the virulence of the virus. It, therefore, implies that modifying the glycosylation sites at the in the hemagglutinin head domain is key to the development of another strategy to the regulation of the immunogenicity of the HA and thereby presenting a chance for the development of a new vaccine.
This perception is based on a study that revealed that immunization with multiple glycosylation sites in recombinant HA increased HA stalk-specific antibodies which resulted in improved protection against viral infection. According to the finding of a recent study that a deglycosylated influenza virus vaccine increased cross-reactive neutralizing antibody responses which point to the possibility of an improved cross- protection. Folding of the HA, intracellular transportation and membrane fusion have been linked to the glycosylation at the stalk region.
The pathogenicity of the influenza virus has also been linked to the mutations in the proteolytic sites of the HA. This is demonstrated by how low pathogenic H5 and H7 viruses transited to the highly infectious influenza virus through acquiring the polybasic amino acids from insertions at the HA cleavage sites (Lam, Ching, et al. 7). The mutations are responsible for the efficacy of the HA cleavage and are also responsible for increased virulence in H5N1 viruses. This evidence then shows that a change to live but attenuated vaccine strategies is essential to the prevention of the influenza virus.
Redesigning of the HA at the cleavage sites is also an approach that can be pursued into developing new vaccine methods. It should be noted that not all viral mutations are geared towards virus replication; however, the influenza viruses are resilient in overcoming these obstacles. This is demonstrated by how poorly replicated clinical H3N2 influenza virus strains can easily and quickly acquire HA and NA mutations and change in vitro which is responsible for active virus binding capabilities. Moreover, the replication of a virus that consists of attenuated mutation at the HA receptor sites can be replaced by other mutations in the HA and NA which increases the receptor binding properties (6). This evidence, therefore, calls for extra caution to be applied while applying the attenuated live virus vaccine strains.
Neuraminidase
This is a type 2 integral protein membrane that possesses sialidase enzymatic activity which is a requirement for the cleavage of host cells and viral glycoproteins. The NA glycoprotein is a homotetramer that comprises four resembling subunits. These subunits consist of the cytoplasmic domain, a transmembrane domain, a stalk domain and a globular head domain. The role of NA in virus replication is to produce new progenies and the prevention of their aggregation. The NA has been linked to a role play in the penetration of viruses in the mucous membrane of the respiratory tract by cleaving the sialylated decoys.
Neuraminidase is immunodominant and therefore possesses lower antigenic drift rates if compared to the immunogenicity of the hemagglutinin which then implies that the introduction of the neuraminidase based immune response will lead to the development of a new universal influenza vaccine. Findings from recent studies have shown evidence that NA immunity can protect against deadly homologous influenza virus infections but has reduced protection against heterologous virus infection.
Moreover, findings from several studies indicate that NA has same immune properties as the HA and the immune- dominance of the HA could be a result of its ability to distribute of the viral surface (Rocha et al.8) The efficacy of the NA immune-induced protection has been accepted though gradually.
Approaches that are similar to those that are used to promote high levels HA in stalk-specific antibodies can be utilized to realize stronger humoral responses against NA, which might include vaccinations with chimeric virus that consists of genetically modified HA and NA, sequential immunization using identical NA with a small portion of immunogenic HA, the employment of Na based nanoparticles and the introduction of the NA vaccine to supplement in the seasonal influenza vaccines.
The difference between the HA stalk domain and the NA stalk domain is that the latter is hypervariable. Evidence from several studies shows that discrepancy in the NA stalk domain which includes sequence deletion and glycosylation has an impact on viral pathogenicity and transmission. Evidence from one study shows that amino acid conservation in the stalk area was essential for maintaining the tetrameric structure of the NA and enzyme activities in the NA.
The enzyme activity in the NA head domain are conserved within the major subtypes and the conserved epitopes in the enzyme activity can be a decisive lead to realizing a new universal influenza vaccine. This demonstrated by evidence that the eight conserved amino acids sequence present in the enzymatic sites are universally conserved in the influenza viruses and played a vital role in the replication processes. Mutations have been recorded to occur on the surface of the enzymatic sites which is usually the target of antiviral drugs zanamivir and oseltamivir against NA. These mutative actions can cause drug resistance in influenza virus.
Strategy for Influenza Vaccine
Presently the influenza virus vaccine comprises of the attenuated live virus of the influenza A strains and the B strains which together form the trivalent influenza vaccine as recommended by the World Health Organization (WHO). Apart from the trivalent vaccine, the current vaccine market contains quadrivalent and pandemic influenza virus vaccines (Zhu et al.,7). The recommendation of a vaccine composition depends on the general considerations made pertaining to the vaccines’ antigenicity, immunogenicity, ability to be produced and predictions that are founded on the influenza surveillance information or data.
A new strategy on the influenza vaccine is being developed based on the advanced understanding of the influenza virus structure including the HA stalk domain, M2e ectodomain and the HA head computationally optimized broad reactive antigen better referred to as the COBRAS. The objective of these new strategies is to induce an improved humoral immune response to the influenza infection (9). The introduction of immune responses especially the memory oriented immune response will play a critical part in the prevention of influenza-related infections globally. Studies conducted have revealed that formulations consisting of influenza peptides from NP, PA or MI if combined with other adjuvants can trigger cytotoxic T lymphocyte response, reduce lung viral titer and combat influenza infection on a larger scale.
Multimeric-001 is a newly designed influenza virus vaccine which is based on epitopes from influenza type MI, HA and NP and has satisfactorily produced improved humoral and cellular responses to the influenza viruses. If the multimetric-001 and the conventional influenza vaccines are combined to use the possibility of developing a universal vaccination solution to the influenza virus is achievable. In the same way, other vaccines resulting from modification of conserved peptides such as the FP-01.1 and the M2e multiple peptides can be a very reliable source of a universal vaccine against the influenza virus.
Skin vaccination has recently gained popularity due to its unique advantages of containing enough blood vessels, lymphatic vessel and the availability of numerous immune cells that are vital for the regulation of innate and adaptive immunity (10). This type of immunization has the potential to trigger an effective innate immune response and an effective cellular immune response. There is a new technology that has been developed in skin vaccination known as the biodegradable microneedle dissolving patch (MNP).
The application of this technology has elicited excitement due to its ability to trigger improved immune responses and a better protective efficiency compared to the conventional intramuscular injections. The current study has demonstrated that improving skin vaccination with M2e encapsulated MNP broadens the immune responses and offers enhanced protection to the recipients who had initially received the conventional influenza vaccine. It is important to note that MNP encapsulated vaccines are nontoxic, painless, stable and are self-administered. In addition, the quick adaptability of the manufacturing process in MNPs makes this type of vaccination convenient compared to the soluble type vaccine types.
The two major challenges that have been a pediment to achieving the universal influenza vaccine development has been the ability to rapidly evolve in the influenza virus and the low immunity levels in the most vulnerable groups (12). Most notably though is the challenge of having antigenic shifts and drifts within the circulating virus strains. The other challenge relates to the most vulnerable groups such as the elderly, pregnant women, infants and young children as well as individuals suffering from chronic ailments; these groups have the lowest immune responses to the influenza virus vaccination. These challenges necessitate the need to have a vaccine that has equal effectiveness across all populations and a vaccine founded on the conserved regions of the influenza virus could provide a remedy across populations, trigger long term immunity and enhance vaccine production duration.
Conclusion
Vaccination is key to the prevention of influenza infection; however, it faced numerous emerging challenges. These challenges include the section of the most appropriate vaccine type needed to combat a dominant influenza strain during an outbreak. These vaccine choices are usually informed by the available influenza data and as such the antigenic drift and shift may render the vaccines useless especially in the case of new pandemic virus outbreaks.
The future of the influenza vaccine will be determined by a better understanding between the genomic sequences, structure and the functions of all influenza virus proteins. This knowledge is essential in deciding viral evolution, transmission, host changing and pandemic formations. The understanding of the aspects of influenza virus antigen protein shades the light for pre-determining seasonal and pandemic influenza types and guide the production of universal influenza vaccines or at least specific vaccines for each strain.
