1. Linda M. Stannard, University of Cape Town
Flu and you: What influenza virus does when it infects and lessons from past & present
S. Mark Tompkins, PhD
Center for Vaccines and Immunology
Department of Infectious Diseases
March 20, 2017

2. Classified as type A, B, or C, based upon their protein composition
Influenza viruses
Orthomyxoviridae
Classified as type A, B, or C, based upon their protein composition
Type A
Found in many kinds of animals
Can cause epidemics and pandemics
Type B
Widely circulates in humans
Can cause epidemics
Type C
Found in humans, pigs, and dogs
Causes mild respiratory infections
Does not spark epidemics or pandemics.
Family: Orthomyxoviridae
Genus: Influenavirus A
Species: Influenza A virus

3. Nature Reviews Microbiology 6, 143-155 (February 2008)
Influenza A virus
A human, agricultural & zoonotic pathogen
HA – 18 different serotypes*
NA – 11 different serotypes
e.g. H1N1, H3N2, H5N1
Segmented, (-) sense RNA virus
Currently, H1N1 and H3N2 subtypes are circulating in humans, although zoonotic infections with other subtypes occur.
Highly Pathogenic Avian Influenza H5N1
Avian H7N9
H3N2v (variant) swine viruses
H1N2v swine viruses
Nature Reviews Microbiology 6, (February 2008)
Scientific barriers to developing vaccines against avian influenza viruses
Kanta Subbarao & Tomy Joseph
Nature Reviews Immunology 7, (April 2007)
a | The influenza A virus particle has a lipid envelope that is derived from the host cell membrane. Three envelope proteins — haemagglutinin (HA), neuraminidase (NA) and an ion channel protein (matrix protein 2, M2) — are embedded in the lipid bilayer of the viral envelope. HA (rod shaped) and NA (mushroom shaped) are the main surface glycoproteins of influenza A viruses. The ratio of HA to NA molecules in the viral envelope usually ranges from 4:1 to 5:1. b | The HA glycoprotein is synthesized as an HA0 molecule that is post-translationally cleaved into HA1 and HA2 subunits; this cleavage is essential for virus infectivity. The HA glycoprotein is responsible for binding of the virus to sialic-acid residues on the host cell surface and for fusion of the viral envelope with the endosomal membrane during virus uncoating. The NA glycoprotein cleaves sialic-acid receptors from the cell membrane and thereby releases new virions from the cell surface. M2 functions as a pH-activated ion channel that enables acidification of the interior of the virion, leading to uncoating of the virion. Matrix protein 1 (M1), which is the most abundant protein in the virion, underlies the viral envelope and associates with the ribonucleoprotein (RNP) complex. Inside the M1 inner layer are eight single-stranded RNA molecules of negative sense that are encapsidated with nucleoprotein (NP) and associated with three RNA polymerase proteins — polymerase basic protein 1 (PB1), PB2 and polymerase acidic protein (PA) — to form the RNP complex. The PB1, PB2 and PA proteins are responsible for the transcription and replication of viral RNA. The virus also encodes a non-structural protein (NS) that is expressed in infected cells and a nuclear export protein (NEP). The location of NEP in the virion is not known.
*Bat influenza: H17N10, H18N11

4. What’s in a name? Human A/Brisbane/59/2007 (H1N1) B/Florida/04/2006
A/Viet Nam/1203/2004 (H5N1)
A/South Carolina/1/1918 (H1N1)
Non-human
A/Goose/Guangdong/1/96 (H5N1)
A/Mute Swan/MI/451072/2006 (H5N1)
A/Ck/PA/13609/1993 (H5N2)
A/Ck/TX/ /2002 (H5N3)

5. Influenza A virus Seasonal Avian Swine Equine Pandemic Canine Seal flu
Cow flu?
Equine
Pandemic
Canine

6. The Impact of Seasonal Influenza
Infects 5-15% of the world population
>200,000 hospitalizations
~36,000 deaths in the US
$37.5 billion in economic cost (influenza and pneumonia)
3-5 million cases of severe illness worldwide
Up to ½ million deaths worldwide

7. Avian Influenza
Avian influenza is everywhere and found in many aquatic bird species or their habitats
Low Pathogenic (LPAI) and Highly Pathogenic (HPAI) viruses are a major concern to poultry production in the United States
HPAI outbreak in Pennsylvania in 1983
17 million birds destroyed at a cost of $65 M
LPAI outbreak in Virginia in 1997
5 million birds destroyed at a cost of >$130 M
>18 outbreaks of potential HPAI since 1997
More on 2015 H5N2 to come

8. 2015 H5N2 HPAI outbreak in North America
Update on the Highly-Pathogenic Avian
Influenza Outbreak of
223
Detections Reported
48,091,293
Birds Affected
Dec ‘14 – Jun ’15
Length of Outbreak
$1.6 billion
Turkey and Laying Hen Cost

9. Avian influenza virus in humans
Infection of humans with avian influenza does not mean a pandemic will occur
Avian influenza has spilled over into humans repeatedly
Influenza virus requires host adaptation at a variety of levels before it can establish itself in a population

10. Human AI Virus infections: Non-H5N1 (excluding H7N9)
Year
Country
Subtype
Cases
Deaths
1959
USA
H7N7 LPAI 1
1978-9 ?
1996
United Kingdom
1999
China
H9N2 LPAI 5
1999, 2003, 2007
Hong Kong 4
2002-3
H7N2 LPAI 2
2003
Netherlands
H7N7 HPAI 89
2004
Canada
H7N3 LPAI / HPAI
2006
H7N3 LPAI
2007
Total
109
Adapted from slide by Dr. David Swayne, USDA

12. Avian Influenza A Virus Transmission to Humans in North America
H7N2 in Virginia, 2002
A low pathogenic avian influenza A (H7N2) outbreak occurred among turkeys and chickens at commercial farms in Virginia
A person involved with culling activities developed influenza-like illness
H7N2 in New York, 2003
A case of avian influenza A virus infection was detected in an adult male from New York, who was hospitalized for respiratory tract illness
A LPAI H7N2 virus was isolated from a respiratory specimen from the patient
The source of this person's infection is unknown
H7N3 in Canada, 2004
LPAI outbreak that evolved into an HPAI outbreak.
Culling operations and other measures were performed in an effort to control the spread of virus
Health Canada reported 2 cases of H7N3: one in culling operations and one in a poultry worker
Both patients developed conjunctivitis (eye infection) and mild illness
H7N2 In New York, 2016
Outbreak of avian lineage H7N2 influenza virus infection among cats in an animal shelter in NY City
A shelter worker having prolonged contact w/ cats was infected, had mild illness, and recovered

13. H7N9
2013

14. Pandemic Influenza in the 20th Century
Spanish Flu (1918)
20-50% serious illness
675,000 deaths in the US
>50 million deaths worldwide
2-5% of the world population died
Asian Flu (1957)
70,000 deaths in the US
1-4 million deaths worldwide
Hong Kong Flu (1968)
34,000 deaths in the US
1-2 million deaths worldwide
SOURCE: The Threat of Pandemic Influenza: Are We Ready? A Workshop Summary, pp
Graphic from nih.gov

15. 2009 H1N1 swine influenza pandemic
From April 12, 2009 to April 10, 2010
60.8 million cases (range: M)
274,304 hospitalizations (195, ,719)
12,469 deaths ( ,306) (all US numbers)
Vaccine in arms
Apr
Jun
Aug
May
Jul
Shrestha SS, et al., Clin Infect Dis Jan 1;52 Suppl 1:S75-82.

16. Let’s get back to seasonal flu
What is the impact?
What are we doing about it?
What is driving the seasonal reoccurrence, outbreaks, infections across species, and the rare pandemics?

17. Periodicity of the flu season
AN overlay of multiple years

18. Association of seasonal febrile respiratory illness and influenza infection
Positive cultures
Febrile respiratory illness
1975
1976
1977
1978
1979
1980
1981
1982
Surveillance in Houston, Texas,
Number
positive
Patients
A/Port
Chalmers
A/Victoria
B/Hong Kong
A/Texas
A/Brazil
B/Singa-
pore
A/Bangkok-
A/England
B/Singapore
A/Bangkok
1974
1983
Edwin Kilbourne, Influenza

19. Snapshot of the 2016 – 2017 season

20. While the incidence of complications is not very high, numbers add up…
≥65
50 – 64
0 – 4

21. Focus on the individual and the virus

22. The first steps of influenza infection
You inhale droplets containing influenza
These small particles enter airways and are trapped
The virus adheres to epithelial cells
The virus binds to specific receptors and initiates the replicative cycle
Your immune response is initiated
Innate responses hold the virus at bay
Adaptive responses clear the infection and establish immune memory
Bacterial co-infections and co-morbidities can result in hospitalization or death

23. Influenza A virus life cycle
Box 1: Influenza A virus life cycle
FromInfluenza virus RNA polymerase: insights into the mechanisms of viral RNA synthesis
Aartjan J. W. te Velthuis
& Ervin Fodor
Nature Reviews Microbiology 14, 479–493 (2016) doi: /nrmicro
Viral infection is initiated when a virion binds to cell surface receptors that contain sialic acid, followed by endocytosis of the virion (see the figure). After fusion of the viral and endosomal membranes, the viral ribonucleoproteins (vRNPs) are released into the cytoplasm and then transported into the nucleus. In the nucleus, the viral RNA-dependent RNA polymerase replicates the single-stranded negative-sense viral RNA (vRNA) genome segments by copying them into complementary RNAs (cRNAs), which are positive-sense RNAs that form complementary RNPs (cRNPs) and act as templates for the production of vRNAs. The viral RNA polymerase also carries out transcription of the vRNA segments into mRNAs, which are 5′ capped and 3′ polyadenylated. Viral mRNA is exported to the cytoplasm for translation by cellular mechanisms. Newly synthesized viral RNA polymerase subunits (polymerase basic 1 (PB1), PB2 and polymerase acidic (PA)) and nucleoprotein (NP) are imported into the nucleus and bind to vRNA genomic segments and cRNAs to assemble vRNPs and cRNPs, respectively. Following nuclear export, progeny vRNPs are transported across the cytoplasm on recycling endosomes in a RAB11-dependent manner to the cell membrane, where the assembly of progeny virions takes place. Mature virions incorporate a substantial amount of host proteins73 and are released by budding. Auxiliary viral proteins (PB1-F2, PB1-N40, PB2-S1, PA-X, PA-N155, PA-N182, M42 and NS3) have been detected in infected cells but are not known to be incorporated into progeny virions; these proteins are not shown130. HA, haemagglutinin; M, matrix protein; NA, neuraminidase; NEP, nuclear-export protein; NS1, non-structural protein 1.

24. Not all infections are equal
Viruses from different species can cause distinct disease
By spread, they mean transmit

25. What makes avian and human influenza viruses different?
Sialic acid linkage preference of avian versus human influenza viruses
Sialic acid expression in different species
Receptor specificity
Aerosol versus droplet versus contact
Tissue distribution of sialic acid receptors
Route of transmission
Polymerase activity
Optimal temperature for replication
Host adaptation
Other?

26. Sialic acid linkage preferences of influenza viruses
Avian influenza viruses preferentially bind to sialic acids with 2,3 linkages
Human influenza viruses preferentially bind to sialic acids with 2,6 linkages

27. Influenza virus receptor density
2,6
2,3
But, don’t make the mistake of over-simplification!!

28. Attachment of influenza viruses to human respiratory tissues
Figure 1. Attachment of human (H3N2 and H1N1) and avian (highly pathogenic H5N1 and low pathogenic H5N9 and H6N1) influenza viruses in human trachea, lower respiratory tract (bronchus, bronchiole, and alveoli), and alveolar macrophages.
American Journal of Pathology. 2007;171:

29. Sialic acid preference is not absolute
Affinity is in the millimolar range (low) and binding relies heavily on the avidity of multimeric interactions
There is weak binding of other sialic acids
As few as 2 amino acid changes in the virus HA can alter specificity
Specificity may not equate infection or transmission
a) Avian flu predominantly binds -2,3 sialic acid on glycan arrays, but may interact with -2,6 and other sialic acids (left panel). On the other hand, human influenza primarily binds -2,6 sialic acid (right panel). (b) Depiction of -2,3- and -2,6-sialic acid–galactose-protein conjugate. As -2,6 sialic acid is bulkier than -2,3 sialic acid, the hemagglutinin receptor expressed by avian flu needs to change sufficiently to accommodate the predominantly human -2,6-sialic acid glycoconjugates. (c) In human patients infected by nonadapted viruses, such as avian flu and 1918 human influenza, the innate immune response is uncoupled from viral replication (left panel). The immune response in humans infected by adapted viruses, such as human H1N1 influenza, correlates with viral replication and effectively eradicates the virus.

30. Immune response to influenza virus infection

31. Influenza A virus antigens (not exhaustive)
Variable (classical Ag)
Hemagglutinin
Neuraminidase
Conserved
Nucleoprotein
Capsid (Matrix 1)
Polymerases
Pore (Matrix 2)
Non-structural (NS)
Nature Reviews Microbiology 6, (February 2008)

32. Life cycle of influenza virus and role of the adaptive immune response during infection
Immunity 24, 5–9, January 2006
Figure 1. Life Cycle of Influenza Virus and Role of the Adaptive Immune Response during Infection
Influenza virus attaches to the epithelial cell surface through binding of the viral hemagglutinin (HA) protein to cell surface sialic acid receptors (1, 2). The virion is internalized through endocytosis and fusion (3). Opening of the M2 channel allows proton flow across the viral membrane (4), triggering fusion of viral and endosomal membranes and release of viral genes into the cytoplasm, from where they travel to the nucleus. Viral proteins produced in cytoplasm assemble with viral genes and bud from the cell membrane as progeny virions (5). Release of new virus
particles (6) requires the viral neuraminidase (NA) protein, which cleaves sialic acid receptors from the cell membrane. Antibodies (Abs) to the HA protein block virus attachment (inset, upper left), thereby decreasing the number of cells infected. They can also function to prevent fusion (4). Abs to the NA protein (inset, upper right) bind virus to the cell, preventing release of new virions. Abs to theM2 protein bind virus to the cell and prevent release of viral particles into the extracellular fluid (inset, lower left). Cell-mediated immunity contributes to resistance when CD8+ T cells specific for viral proteins such as nucleoprotein (NP) or polymerase proteins (PB2 and PA) recognize viral peptides presented by MHC class I proteins, resulting in the release of cytokines with antiviral activity (IFN-g and TNF-a) and perforins that mediate cytolysis of the infected cell (inset, lower right). Lysis of the infected cell decreases the amount of virus released by the cell. The latter three mechanisms, NA Abs, M2 Abs, and CD8+ T cells, operate after a cell becomes infected. Only HA Abs prevent infection; this is likely to be why they are the most effective in vivo.
Cartoon from Immunity (2006) 24:5

33. Course of immune response during influenza infection
Serum antibody titers to 1918 Spanish Influenza were detected in survivors more than 90 years later!!
Influenza virus titers peak at approximately 3 days after infection, at which time antibodies (Abs) and T cell responses begin to appear. Activated T cell responses peak on days 6–9 during the primary infection and then subside into a memory or resting state, whereas serum and mucosal Ab concentrations are sustained. Abs present at the time of reinfection result in lower viral titers and a reduction in symptoms. Upper respiratory infection, URI, lower respiratory infection, LRI.
From Immunity (2006) 24:5

34. After infection we are left with:
Virus specific:
Serum and mucosal antibody responses, both virus neutralizing and non-neutralizing
Memory B cells
Memory CD4 and CD8 T cells
These responses do not guarantee immunity
Non-neutralizing antibodies may be protective
T cell responses can help control infection
These responses provide heterosubtypic immunity
Heterosubtypic immunity will likely not protect from infection
Het may be relevant to protection from disease

35. We take advantage of the potent anti-HA neutralizing antibody response for vaccines
Vaccines since the 1940’s target the serum neutralizing antibody response to HA
This specific response can be measured by a serum hemagglutination inhibition assay
This is an established correlate of protective immunity
A 4-fold increase in HAI titer (or >1:40) is indicative of protection from influenza virus infection by a matched strain.

36. Hemagglutination inhibition assay
When we combine virus with immune serum and RBCs, the absence of haze (a dot) means there are virus-specific antibodies
Negative control
Positive control
Virus-specific
antibodies

37. Flavors of licensed influenza vaccines
Type
Substrate
Fluzone (Sanofi)
Formalin inactivated, split, and purified
Embryonated chicken eggs
Flucelvax (Seqirus)
β-propiolactone (BPL) inactivated, split, and purified
Madin-Darby canine kidney (MDCK) cells
FluBlok (Protein Sciences)
rHA protein subunit, detergent extracted, purified
SF9 (insect) cells
Flumist (MedImmune)
Live-attenuated, purified, and filtered
Inactivated (subunit) vaccines (IIV)
Target the HA eliciting neutralizing serum antibody responses
Most have “contaminating” antigens, including NA, but these are not quantitated or considered in immunogenicity or efficacy
Live-attenuated influenza vaccine (LAIV)
Elicits mucosal and serum antibody, as well as cellular immune responses

38. To sum up
Multiple types and subtypes of influenza are circulating (and changing) in humans
Zoonotic influenzas are all around us posing potential threats
Not all influenzas are equal
Outcomes of infection are varied
Immunity to infection is potent, although potentially not long-lived

39. Questions?
This is a mystery, and I don't like mysteries. They give me a bellyache, and I've got a beauty right now. – Captain James T. Kirk