Center for Research and Assistance in Technology and Design of the State of Jalisco A.C. , Medical and Pharmaceutical Biotechnology, Guadalajara, Jalisco, Mexico
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Center for Research and Assistance in Technology and Design of the State of Jalisco A.C.
Medical and Pharmaceutical Biotechnology Av. Normalistas No. 800. Colinas de la Normal
Copyright: © 2019 López-González HE, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 international License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Influenza is a viral infection that causes annual epidemics, besides the latent risk of a possible
pandemic. Nowadays the best choice to control influenza is vaccination. However vaccine
effectiveness oscillates between 30 and 60%, it is inconvenient and must be reformulated
every year, because of the high rate of mutability of the influenza virus. On the other hand,
vaccinated population has caused the virus to undergo immunological pressure, causing
mutations to escape. The principal target of this immunological pressure is due to the
presence of glycoproteins on the viral surface, in where there are some well characterized
antigenic sites. In the present study, hemagglutinin sequences from Influenza A/H1N1 from
the last ten years were analyzed focusing on appearing and disappearing of N-glycosylation
sites. As per previous reports, conserved glycosylation sites were identified, mostly on the
stalk, other glycosites where found on the globular domain, such as the site 179, which in
less than 10 years went from a very low rate, to be present in almost all the sequences, but,
apparently this site is still undergoing through a stabilization phase, the main evidence, are
the recent mutations that have arisen in the sequon. We discuss the possible repercussions
that this could have on the choice of vaccine strains, and the impact of the substrate in
where vaccines are developed and how this could be fundamental in their effectiveness.
Influenza A H1N1; Hemagglutinin; N-glycosylation; Influenza evolution; Influenza
Influenza is a viral disease caused by influenza A or B virus. Every year, this virus infects and spread throughout the world population causing more than 300,000 respiratory cases. In 2018, a larger number of cases were reported from 2017 . The severity of the illness is variable from a mild auto limited infection to where the patient barely presents the symptoms up to death . Until now, the most effective prophylactic measure is vaccination. However vaccine effectiveness oscillates between 30 and 60% [3-6]. Every year in pursuit to improve protection over this illness, vaccination campaigns take place all over the world and there is continuous research and development of new vaccines. Some of which have already been applied to population . A very important consequence of this is, in general terms, the diversity of antibodies generated on world population in one way or the other have lead influenza virus to undergo a evaluative pressure, but for better understanding of this impact, it is necessary to highlight some of the virus characteristics and part of their biological cycle.
Influenza A virus belongs to Orthomyxoviridae family and has an average size of 100 nm, it is covered by a lipid bilayer taken from the host cell , where two important and antigenic glycoprotein’s are embedded: hemagglutinin (HA), from which 18 types are known (numbered from H1 to H18); and neuraminidase (NA), with 11 types known (numbered from N1 to N11). HA is the most abundant in the viral surface and has the ability to trigger a major immune system . These glycoproteins are fundamental for some biological process, HA participates in the recognition of the cell receptor, besides it has a participation on biological process, such as fusion of the viral membrane with the membrane of the target cell; on the other hand, NA is a necessary enzyme for the release of newly synthetized virions from the host cell .
The survival of the virus is determined by its capacity to transfer from one person to another. However, it is a very common and prevalent disease. Most of the people already have a certain level of protector antibodies acquired from previous infections causing a very drastic selection, among each virus having potential of new host. If we consider influenza virus from a very general point of view like any other species, in order to survive, it must adapt to the changing environment where it survives, it must have high mutation rate, capability of producing multiple variants which try to escape from immunological pressure and these will survive, infect and spread. For mutations to be effective, they must be translated to change on the external glycoproteins. One of the most studied changes is N-glycosylation [10-12].
N-glycosylation is a very common post-transductional modification, where a oligosaccharide is attached to a specific motif called sequon, composed by three aminoacids: N – X – S/T, where X could be any except proline . It is important to mention, that for N-glycosylation to occur, there are other factors to be considered, besides the sole presence of the sequon, factors such as the location of the sequon in the structure, and the process of the glycosyltransferases and its function .
Another important aspect of the process is regarding the nature of the glycan attached to the sequon which depends on the host or the system used for vaccine production, this means the same sequon from the same strain can bear different types of glycans, depending on the substrate or kind of cell where the virus was replicated .
Like mentioned before, HA is the most abundant glycoprotein on the viral surface, and because of this, it is the major target of the immune response; this molecule is formed from a precursor HA0, that is cleavage into two subunits: HA1 and HA2 . On the viral envelope, HA forms a homotrimer, with a globular head formed by HA1; and a stalk, formed by HA2. The globular head displays two important features: The first one is the high mutation rate, compared with the stalk ; and the other is the presence of five antigenic sites, Sa and Sb, located near the tip of the HA and the receptor binding site; Ca1 and Ca2 located among the subunits; and Cb, located on the head (S refers to strain-specific, and C to common); therefore it is important to keep up with the mutations on these sites, especially for those vaccine candidate strains [8,16].
The aim of this study was to determinate the changes on
influenza virus that has provoked the acquisition of N-glycosylation,
and relate them to the possible effect produced by the continuous
use of vaccines on the population in the last 10 years.
For this study, protein sequences of influenza A H1N1 hemagglutinin were used. All sequences were downloaded from Influenza Virus Database. The criteria for the selection of the sequences were human origin pandemic complete sequences (554 to 566 amino acids), and, not more than two adjacent amino acid deletions. All the different sequences where selected from April 2009 to August 2018. The total sequences used for each year are indicated in Table 1.
Influenza database also contains seasonal influenza sequences,
and porcine-like sequences; to avoid the selection of these
sequences, multiple alignments were made on Clustal X, followed by
phylogenetic trees, using Neighbor-Joining method made on MEGA
X; and the reference sequences used for the phylogentic trees were:
Vaccinal strains A/Michigan/45/2015 (No. Access: AMA11475.1),
A/California/07/2009, (No. Access: YP_009118626.1) and A/
Brisbane/59/2007 (No Access: AET50439.1); reference strains A/
Puerto Rico/8/1934 (No. Access: ABD77675), A/WSN/1933 (No.
Access: ACF54598.1) and A/South Carolina/1/1918 (No. Access:
AAD17229); this way, we assure that only pandemic strains were
used for this study.
All the results showed in this study consider the numbering of each residue present in hemagglutinin, taking as a reference the numbering H1, starting with signal peptide.
The prediction on N-glycosylation sites was performed in all sequences that previously showed to be derived from the 2009 pandemic, and was made on NetNGlyc 1.0 server , taking positive results with potential of 0.5 or higher, probability of a real glycosylation on that site is estimated.
To determine differences in the sequons for site 170, from
2009 to 2018 progressive alignments were made on Clustal X, and
the percentage of each sequon was determinated, noting whether
glycosylated or not.
Each sequence was analyzed on NetNGlyc 1.0 Server, for the prediction of every N glycosylation site. The results are shown on Figure 1. Eight N-glycosylation sites where predicted: six sites very conserved Asn28, Asn40, Asn104, Asn304, Asn498 and Asn557; one site that disappeared Asn136, and two sites that increased trough this decade Asn179 y Asn293.
The glycosylated sequons are shown on Table 2. All sequons are conserved from 2009 to 2018 except Asn179, that has several changes through mutations.
Site 104, also reported on 1918 influenza virus , is located on the side head of HA, under the antigenic site Ca2, and has been stable for a very long time, as part of the HA structure , and its importance in the receptor binding union has already been described . The sites 28, 40, 304 and 498 located on the stalk, had already been reported as conserved trough influenza A H1N1 history [12,18,21,22], and has been found that these sites are fundamental for correct protein folding and hemagglutinin stability during synthesis [23-25].
Even though site 557 (with NGS sequon) has a very high incidence in N-glycosylation predictions, some research groups have analyzed the H1N1 variant ; the reference virus Caledonia/20/1999 ; and even A/Puerto Rico/8/1934 ; they all agree that sequon NGS does not consists of any kind of glycan, besides, this residue is part of the transmembranal domain that anchors to the membrane of the host cells .
On the other hand, site 293 (with NTT sequon) appears to be a relatively new site, because it started to appear steadily from 2009 to date, and has been a much conserved site, even on the sequences it is not predicted with any glycosylation. However, some researchers have analyzed this site through glycan characterization, and they agree that this site has a place where glycosylation process takes place [12,26].
She YM in 2017  described two more glycosylations detected on candidates for a vaccine derived from A/California/07/2009 virus. The first one is site 136, with the sequon NTS; however, this glycosylation just appeared from 2009 to 2013 in less than 0.4% of the sequences, to finally be substituted by mutation N136K, which already had been dominant in the sequences. The other site mentioned is 490, but this site does not present a formal N-glycosylation sequon, just the motif NTC, for this reason was not predicted as a N-glycosylation site.
The appearing or disappearing of N-glycosylation, and their influence over immunologic and physiochemical parameter on hemagglutinin trimer has been widely described. It has been demonstrated that the removal or addition of a glycosylation in 142 and 177 sites, change the resistance or susceptibility of antibodies. When this glycosylations where induced on Influenza A strains of 1918 and 2009, these did not affect the trimer structure ; also, it has been demonstrated that the addition or removal of these glycosylation sites have a different effect, depending of the site, this was seen on seasonal strains, when the site 142 was removed, the virus presented a minor neutralization rate, than when site 71 was removed .
Site 179 is relatively new, starting to appear constant in 2009 to 2014, with an oscillating rate of 0.28% to 5.86%, until 2015, where more of half of the analyzed sequences presented this site, and to keep growing in future years up to be dominant in more than the 99% of the sequences.
This glycosylation is ubicated in the antigenic site Sa and is
speculated to be some kind of “shield” to this site from immune
response. However, is interesting that this sequon has been in
constant change: the few sequences with glycosylation on this site
found in 2009 presented the NKS sequon. On the other hand, the
vaccinal strain A/Michigan/45/2015, a representative sequence
from that year presented NQS sequon, while the sequences from
2018 almost all present the NQT sequon.