2017 Biology week lectures: Vaccine research overview from key virologists

 

Prologue

Having recently moved back to the UK I was not aware that there is such a thing as a biology week. Thankfully the Royal Society of Biology made sure to bring this celebration to my immediate attention by broadcasting it on its web-page so that I became aware of both its existence and some of the lectures and events on during this celebration of biology.

Scrolling down the list of events from the Royal Society of Biology webpage I identified two promising talks about vaccines and promptly booked myself a place in them. This blog is dedicated to sharing some of the insights from these lectures:

• “Around the world in 80 days” by Professor Wendy Barclay, Chair in Influenza virology at Imperial College London which would be hosted as part of the RSB AGM in Reading
• “Vaccines for a 21st century society” by Professor Rino Rappuoli, Chief Scientist & Head of External R&D at GSK Vaccines as part of the Environmental Microbiology Annual Lecture 2017 hosted by the sfam in London

Part 1: Professor Wendy Barclay -Influenza A virus overview

Wendy gave a lovely talk starting with a brief description of influenza virus components and with the importance and impact of influenza in society which included a description of yearly spikes of occurrence, epidemiological and pandemic occurrences and Prince Philip’s statement that he had “not had flu for 40 years”. She also mentioned the potential reasons for this such as:

• Acquired Immunity
• Exposure to Airborne Virus
  
• Reduced Stress Levels  

Wendy then went on to talk about the barriers viruses face when attempting to infect a different host type.

Influenza species are categorised as A (avian), B (human) and C. The natural hosts of influenza A virus are birds where typical infections with influenza A virus do not show any symptoms. Some strains however cause systemic infection with involvement of the central nervous system.

In wild ducks, influenza viruses replicate preferentially in the cells lining the intestinal tract, cause no disease signs, and are excreted in high concentrations in the faeces (Webster 1992 & 1978).

She pointed out while showing us a number of images of birds such as pigeons, ducks, swans and chickens in close proximity to humans how infrequently humans become infected with avian influenza despite our frequent exposure to the avian virus infested birds. Although, she mentioned, lethality is high in the known cases that humans have caught avian influenza the avian virus is usually incapable of becoming airborne and transmitted to further humans from a human host meaning that the zoonotic strain normally does not spread from the infected human. Indeed according to Wan et al approximately 60% of 385 human infections with avian Influenza in Vietnam have been fatal [3], but very few have been transmitted from person to person [4]. Infections are usually acquired by contact with H5N1 infected poultry or poultry products, and the H5N1 viruses isolated from human cases are often virtually identical to isolates from poultry [5].

  

She went on to explain that viruses have adapted over the centuries to infect specific hosts in a way which permits viral infection and replication in a manner which is less detrimental to their favourite host. This evolution includes adaption to have a preference for certain mammalian cell receptors, for the virions to be stable under different pH conditions, to be able to make use of the their viral RNA-dependent RNA polymerase in its host cells.

For example, avian influenza virus polymerase which is responsible for viral replication and spread has adapted to the temperature of the avian enteric tract (40°C) and restricts the virus infectivity  in humans at the lower temperature of the human proximal airways.

In order to understand these preferences we need to understand a bit about the nature of influenza virus structure and biology.

The influenza A virus comprises of an outer host derived lipid bilayer embedded with the virus-encoded glycoproteins hemagglutinin (HA) and neuraminidase (NA) and matrix protein 2 (M2), and within that an inner shell of matrix protein which protects the nucleocapsids of the viral genome at the centre of the virus particles. The lipid bilayer of Influenza B contains an addition NB component and Influenza C contains only an M2 protein and the hemagglutinin–exterase-fusion (HEF) protein that combines the functions of HA and NA.

HA mediates the viral binding to host cell receptors. Human influenza preferentially binds α2,6-linked sialyloligosaccharide receptors (mostly found in the upper respiratory tract) and avian influenza prefers α2,3-linked sialyloligosaccharide receptors which are more abundant in the lower respiratory tract. The M2 ion channel equilibrates the pH across the viral membrane and NA cleaves sialyloligosaccharide residues (oligosaccharides with one or more sialic acid residues) from host cell surfaces aiding in the release of the viral particles.

The influenza virus genome comprises of eight single-stranded, negative-sense RNA molecules (vRNA) as well as multiple copies of NP (Nucleoprotein) with which it forms a viral ribonucleoprotein (vRNP) complex with a single RNA polymerase which is a trimer or the three proteins; PA (Polymerase acidic protein), PB1 (RNA-directed RNA polymerase catalytic subunit)and PB2 (Polymerase basic protein 2).

Hundreds of mammalian cellular factors have been identified that affect virus replication or whose expression is modulated by the virus (Zhao M. 2017). As can be expected a large number of those interacting proteins interact with the viral polymerase.

A number of studies have found that the avian viral RNA polymerase functions weakly in human cells while certain mutations, particularly of its PB2 protein, are selected during adaption of the avian strains to mammalian cells (Mänz B 2013, Subbarao, E. 1993, Naffakh, N. 2008). Particularly residue 627 of PB2 which sports a lysine (rarely an arginine) in human Influenza and a glutamic acid in avian Influenza has been found to play a crucial role to host adaption.

• The single amino acid substitution, from glutamic acid to lysine at position 627 of the PB2 protein, converts a nonlethal H5N1 influenza A virus isolated from a human to a lethal virus in mice (Shinya).
• In one study the acquisition of an avian PB2 in an otherwise human Influenza restricted the viruses replication in mammalian cells (Clements). After several passages through mammalian cells the viral chimera acquired the ability to replicate by acquisition of the PB2 E627K mutation (Subbarao).
• The 1918 pandemic strain had a lysine residue at position 627 although the PB2 gene was otherwise avian virus-like implying that it did not acquire host adaption by re-assortment (Taubenberger et al). A total of ten amino acid changes in the polymerase proteins consistently differentiate the 1918 and subsequent human influenza virus sequences from avian virus sequences, five of which were to the PB2 protein.  Out of 253 available PB2 sequences from human H1N1, H2N2 and H3N2 isolates, these five changes are almost completely preserved, with the exception that two recent H3N2 isolates have the avian Lys residue at position 702. Only a small number of avian influenza isolates show any of these five changes, and it is intriguing that almost all of these isolates are from HPAI H5N1 or H7N7 viruses, or from the H9N2 lineage that infected a small number of humans in China in the late 1990s (ref. 29). Notably, a number of the same changes have been found in recently circulating, highly pathogenic H5N1 viruses that have caused illness and death in humans and are feared to be the precursors of a new influenza pandemic.
• In highly pathogenic avian influenza (HPAI) H5N1 which arose in Vietnam in 2004 and 2005  lysine 627 and asparagine 701 were both correlated with fatal disease in infected patients  (de Jong )
• It is interesting that in avian cells virus polymerases containing PB2 627K are only two times more efficient than those with 627E
• E627K was present in the single fatal human infection during the HPAI H7N7 outbreak in the Netherlands in 2003 (Fouchier ). A highly pathogenic avian influenza A virus of subtype H7N7, closely related to low pathogenic virus isolates obtained from wild ducks, was isolated from chickens. The same virus was detected subsequently in 86 humans who handled affected poultry and in three of their family members. Of these 89 patients, 78 presented with conjunctivitis, 5 presented with conjunctivitis and influenza-like illness, 2 presented with influenza-like illness, and 4 did not fit the case definitions. Influenza-like illnesses were generally mild, but a fatal case of pneumonia in combination with acute respiratory distress syndrome occurred also.Most virus isolates obtained from humans, including probable secondary cases, had not accumulated significant mutations. However, the virus isolated from the fatal case displayed 14 amino acid substitutions, some of which may be associated with enhanced disease in this case.
• Six influenza isolates were obtained from four different provinces of Thailand during the avian influenza outbreak in Asia from late 2003 to May 2004 (Puthavathana et al).
• In Hong Kong in 1997 and 2003, highly pathogenic avian influenza (HPAI) of subtype H5N1 was transmitted from birds to humans, of whom at least seven died (8–13).
• Pandemics of 1918 (H1N1), 1957 (H2N2), and 1968 (H3N2) were caused by influenza viruses harboring HA and NA genes of avian or swine origin (1, 2).

In search of the mechanisms which control the Influenza restriction to different hosts Wendy’s team focused on the activity of influenza A viral polymerase in heterokaryons formed between avian (DF1) and human (293T) cells. In their work they found that there is no restriction factor for avian-derived polymerase present in human cells. Thus, the restriction in human cells is likely the consequence of the absence of an interaction or a low-affinity interaction with an essential cofactor. Finally, this work also gives an explanation for the natural selection of the adaptative mutation E627K in PB2 by implying the presence of a human-specific positive cell factor that enhances replication of polymerases only when the PB2 627K motif is present. (Moncorgé O. et al, 2010).

Following this discovery Wendy’s team went on to look for a potential interacting factor. They found that the mammalian protein ANP32A interacts with the viral RNA polymerase PB2 protein and that “species-specific difference in host protein ANP32A accounts for the sub-optimal function of avian virus polymerase in mammalian cells”.

Wendy showed us an alignment of ANP32A protein sequences from animal and bird species which made it obvious that this protein is highly conserved and that an insertion event added a 33 amino sequence to avian ANP32A . This insertion was not present in human, porcine or ostrich ANP32A. She went on to explain the hypothesis that avian influenza must have adapted to bird species after the evolutionary event which separated mammals and land bound birds such as Ostriches from birds of flight. She presented some of he teams work which showed that the short added sequence present in avian ANP32A made all the difference between the species that avian influenza could infect  because it determined whether the  avian virus polymerase was able to function or not permitting viral replication in the host cells.

Wendy went on to show some metrics on influenza vaccination. She showed some epidemiological data that suggested that occurrences reduced during school breaks making children the largest culprits of infection spikes.

She then introduced us to the concept of nasal vaccination of four attenuated influenza species in natural lipid particles delivered through a nasal spray which has adapted to survival at lower temperatures such as those found in the nose cavity of humans confining the viral infection anatomically by temperature restriction and eliminating the need for injections. Other major benefits of this type of vaccination include activation of both mucosal and systemic immune response, an increased safety profile as the vaccine is not injected and does not contain in potentially harmful adjuvants and broad specificity antibody responses which could protect against viral/bacterial strains not included in the vaccine. Most notably it was interesting to see that after vaccination reported infections were reduced not only on the year of vaccination but also the following year. She voiced concerns over the availability of this powerful vaccination strategy which protects against the strains found in UK as data from the US showed that one of the four influenza species which caused the majority of infections in US appears not to be as successful as the other three strains in protecting against infection when used in the nasal vaccine.

Finally she introduced pH lowering nasal sprays as an example of how pH influences influenza virus stability and its ability to infect cells. Influenza virus enters its host cells via endosomes/lysosomes and needed the acidic pH of these endosomes to release its DNA from the viral capsid into the host cell cytoplasm. Lowering of the nasal cavity pH with an acidic spray is therefore be a logical methodology for degrading the virus coat and exposing its genetic material to mucosal and environmental enzymes which can degrade it and protect from infection.

The lecture was followed by a questions and answers session. Intrigued by the fact that avian influenza which infects humans working in close proximity with birds do not usually transmit the virus to others and having in mind methods of horizontal DNA transfer and interesting findings of DNA both becoming encapsulated in viral particles and binding to them in viral DNA aggregates I took this opportunity to ask Wendy her opinion on the ability of influenza viruses to evolve not only by mutation but also by horizontal transfer of pieces of DNA from other species. Wendy stressed that picked up DNA would really have to confer the virus a selective advantage for it to be kept and integrated into its genome but to my delight she confirmed my suspicions by going on to mention that they have frequently found fragments of host DNA (ribosomal and other types) when sequencing viral genomes.

See also:

How flu shot manufacturing forces influenza to mutate

https://www.cdc.gov/flu/

Further reading:

1. Webster, Robert G., et al. “Evolution and ecology of influenza A viruses.” Microbiological reviews 56.1 (1992): 152-179 (inlcudes electron micrographs of Influenza A).
2. Webster, Robert G., et al. “Intestinal influenza: replication and characterization of influenza viruses in ducks.” Virology 84.2 (1978): 268-278.
3. Zhao, Mengmeng, Lingyan Wang, and Shitao Li. “Influenza A Virus–Host Protein Interactions Control Viral Pathogenesis.” International journal of molecular sciences 18.8 (2017): 1673.
4. http://jvi.asm.org/content/65/1/232.short nuclear transport
5. https://virologyj.biomedcentral.com/articles/10.1186/1743-422X-4-49 nuclear localization sequences
6. Moncorgé O, Mura M, Barclay WS. Evidence for Avian and Human Host Cell Factors That Affect the Activity of Influenza Virus Polymerase  . Journal of Virology. 2010;84(19):9978-9986. doi:10.1128/JVI.01134-10.
7. Mänz, Benjamin, Martin Schwemmle, and Linda Brunotte. “Adaptation of avian influenza A virus polymerase in mammals to overcome the host species barrier.” Journal of virology87.13 (2013): 7200-7209.
8. Subbarao, E. Kanta, and B. R. Murphy. “A single amino acid in the PB2 gene of influenza A virus is a determinant of host range.” Journal of virology 67.4 (1993): 1761-1764.
9. Naffakh, Nadia, et al. “Host restriction of avian influenza viruses at the level of the ribonucleoproteins.” Annu. Rev. Microbiol. 62 (2008): 403-424.
10. Hatta, Masato, et al. “Growth of H5N1 influenza A viruses in the upper respiratory tracts of mice.” PLoS pathogens 3.10 (2007): e133.
11. Scull, Margaret A., et al. “Avian Influenza virus glycoproteins restrict virus replication and spread through human airway epithelium at temperatures of the proximal airways.” PLoS pathogens 5.5 (2009): e1000424
12. Taubenberger, Jeffery K., et al. “Characterization of the 1918 influenza virus polymerase genes.” Nature 437.7060 (2005): 889-893.
13. de Jong, Menno D., et al. “Fatal outcome of human influenza A (H5N1) is associated with high viral load and hypercytokinemia.” Nature medicine 12.10 (2006): 1203-1207.
14. Wan, Xiu-Feng, et al. “Evolution of highly pathogenic H5N1 avian influenza viruses in Vietnam between 2001 and 2007.” PloS one 3.10 (2008): e3462.
15. Fouchier, R. A. et al. Avian influenza A virus (H7N7) associated with human conjunctivitis and a fatal case of acute respiratory distress syndrome. Proc. Natl Acad. Sci. USA 101, 1356–1361 (2004)
16. Shinya, K. et al. PB2 amino acid at position 627 affects replicative efficiency, but not cell tropism, of Hong Kong H5N1 influenza A viruses in mice. Virology 320, 258–266 (2004)
17. Puthavathana, Pilaipan, et al. “Molecular characterization of the complete genome of human influenza H5N1 virus isolates from Thailand.” Journal of General Virology 86.2 (2005): 423-433.