Universal flu vaccine test in pigs Dr. Paul Heinen Facultad de Ciencias Exactas y Naturales Universidad Nacional de Asunci贸n paulheinen@yahoo.com.ar
Vaccination of pigs with a DNA construct expressing an influenza M2-nucleoprotein fusion protein exacerbates disease after challenge with influenza A virus Heinen, P.P., Rijsewijk, F.A., De Boer-Luijtze, E.A., Bianchi, A.T.J. J Gen Virol 2002, 83, 1851-1859
Graduate School of Animal Health, Utrecht University, detached at the Institute for Animal Science and Health (IDLelystad), Department of Mammalian Virology, with Dr. AndrÊ Bianchi, Prof. Dr. Jan van Oirschot and Prof. Dr. Albert Osterhaus. June 1997 – September 2001
Influenza A virus.
Variability: - Reassortment - Antigenic shift - Antigenic drift
When a single cell is infected by two or more different influenza viruses, reassortment, gene exchange between parental viruses, gives rise to new progeny viruses. 8 segments → 254 combinations
Influenza A virus.
- Reassortment - Antigenic shift - Antigenic drift
Phylogenetic tree of influenza HA subtypes B H8 H12 H9 H1
16 subtypes of HA 9 subtypes of NA 144 possible combinations HxNy
H2
Seen in humans:
H5 H6 H11 H13 H16 H3 H4 H14 H10 H7 H15 10% of residues
H1, H2 and H3 Others sporadic N1 and N2
Reservoir ofinfluenza Influenza viruses Reservoir of A viruses
1993 The Netherlands, son of a pig farm worker with reassortant H3N2 from pigs 24 April 2009 Mexico, H1N1 first disease outbreak notice → pandemic
Hey, who said that?
He says icecream made him feel better, and Thank God has now recovered full health. But the rest of the planet has a quick – paced pandemic marching on‌.
'Patient Zero' in Swine Flu Outbreak Identified as 5-Year-Old Mexican Boy:
Edgar Hernandez
2009 H1N1 genome: Gene 1 PB2 polymerase 2 PB1 polymerase 3 PA polymerase 4 HA hemagglutinin 5 NP nucleoprotein 6 NA neuraminidase 7 NS nonstructural 8M M protein
. avian human avian swine swine swine swine swine
Origin North American H3N2 North American North American North American Eurasian North American Eurasian
.
40 million death 2 million, <0.1% 1 million, <0.1% Reservoir of Influenza viruses Reservoir of influenza A viruses worldwide, 2.5%
¼ - ½ million death a year worldwide, <0.1%
H1N1
PB1
? H1N1
¼ - ½ million death a year worldwide , 0.03%?
Reasons to study flu and universal vaccines in pigs: • Pigs are a good flu vaccine model. Pigs can be naturally infected by avian, swine and human flu viruses • Pigs are a potential intermediate host and ¨mixing vessel¨ for flu viruses. • Swine flu causes considerable economic loss to the pig farming industry
Design of potential universal vaccine A universal influenza A vaccine based on the extracellular domain of the M2 protein. Nat. Med. 1999;5(10):1157â&#x20AC;&#x201C;1163. Neirynck S, Deroo T, Saelens X, Vanlandschoot P, Jou WM, Fiers, Walter. M2e Protection in mice after vaccination with M2e-HBc protein M2e HBc Hepatitis B core protein Injected as protein
DNA construct expressing fusion protein of influenza extracellular part of M2 (M2e,) fused to Nucleoprotein A universal influenza A vaccine based on the extracellular domain of the M2 protein. Neirynck S, Deroo T, Saelens X, Vanlandschoot P, Jou WM, Fiers W. Nat. Med. 1999;5(10):1157â&#x20AC;&#x201C;1163.
Protection in mice after vaccination with M2e-HBc protein M2e HBc Hepatitis B core protein Injected as protein
NP Influenza Nucleoprotein Injected as DNA plasmid
Conserved target for antibody mediated immunity Conserved target for cell mediated immunity (T-helper and Cytotoxic T-cells)
Vaccination-challenge experimental design Day 0, 21, 42: 6 6 6 6
Pigs vaccinated with
non-vaccinated controls
M2eHBc fusion protein M2eHBc fusion protein + adjuvant M2e-NP DNA PBS
Day 70, 4 weeks later, challenge with 108 TCID50 A/Swine/Best/96 (H1N1)
Clinical signs Laboured breathing Abdominal breathing Anorexia Apathy Coughing M2eHBc fusion protein (▲,a) M2eHBc fusion protein + adjuvant (,b) M2eNP DNA
(○,c)
3 pigs died day 1/2
control pigs
( □)
Antibody titers
M2eHBc fusion protein (▲,a) M2eHBc fusion protein + adjuvant (,b) M2eNP DNA
(○,c)
3 pigs died day ½ p.i.
control pigs
( □)
Lymphoproliferation
Lymphoproliferation (cell-mediated immunity) M2eHBc fusion protein (▲,a) M2eHBc fusion protein + adjuvant (,b) M2eNP DNA
(○,c)
3 pigs died day ½ p.i.
control pigs
( □)
T-lymphocyte populations in the lung Significantly more T-helper cells (and cytototoxic T-cells) after challenge, in lung fluid of DNA vaccinated pigs.
M2eHBc fusion protein (▲,a) M2eHBc fusion protein + adjuvant (,b) M2eNP DNA
(○,c)
2 pigs died day ½ p.i.
control pigs
( □)
Hypothesis to explain severe clinical signs after challenge infection: •
extensive influenza virus infection
•
non-neutralising M2e antibodies
•
the NP-specific T-helper cells
Th cell NP!
NP
MHC II APC
FcR
M2e Ab
MHC II APC •
all act together to stimulate macrophages in the lung, leading to an overinduction of cytokines (TNF-), inflammation and severe clinical signs.
FcR
1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger* and David M. Morensâ&#x20AC; *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and â&#x20AC; National Institutes of Health, Bethesda, Maryland, USA
Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the virus to host and environmental factors, possibly including immunopathology (e.g., antibody-dependent infection enhancement associated with prior virus exposures (as in dengue)) and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents.
Death rate per 100,000 persons
40 million death worldwide Emerging Inf. Dis. Jan 2006
Conclusion • Non-neutralizing antibodies to M2e seem to cause exacerbation of disease during influenza infection • Especially in combination with cellular immunity • We should be very careful before using M2e in people. • Unfortunately this could not be investigated further
Interest in M2e and universal vaccines has increased, especially since the 2009 H1N1 outbreak. M2e flu vaccine trial Acambis , January 2008 Phase I clinical trial: 79 volunteers. Response measured in 90% of volunteers, and no adverse effects seen. Adverse effects upon infection??? Phase II clinical trial M2e vaccine criticism, Company does not really expect a usable vaccine but does research on universal vaccines only to attract attention and raise the value of stocks. WHO vaccine research, September 2009, results presented cast doubt on the feasibility of using safely M2e as an immunogen in humans.
Perhaps we will hear more about this vaccine in the future. Or perhaps notâ&#x20AC;Ś.
Universal flu vaccine test in pigs Dr. Paul Heinen Facultad de Ciencias Exactas y Naturales Universidad Nacional de Asunci贸n paulheinen@yahoo.com.ar