INFLUENZA AND

PRODUCTION OF

VACCINES AGAINST

INFLUENZA VIRUSES

PRESENTED BY:RIMJHIM ROY CHOUDHURY

Characteristics

• Influenza virus belongs to the orthomyxoviridae family.• Influenza virus have segmented negative-sense single

stranded RNA molecules, which are eight in number.• It’s an enveloped virus which is made up of proteins,

glycoproteins, and lipid bilayer.• Helical in symmetry.

3

Orthomyxoviruses

M1 protein

helical nucleocapsid (RNA plus NP protein)

HA - hemagglutinin

polymerase complex

lipid bilayer membrane

NA - neuraminidase

type A, B, C : NP, M1 protein sub-types: HA or NA protein

Type A Type B Type C

Severity of illness ++++ ++ +

Animal reservoir Yes No No

Human pandemics Yes No No

Human epidemics Yes Yes No(sporadic)

Antigenic changes Shift, drift Drift Drift

Segmented genome Yes Yes Yes

Amantadine, rimantidine Sensitive No effect No effect

Zanamivir Sensitive Sensitive

Surface glycoproteins 2 2

Types of Influenza virus

Influenza Biology

M2

HA

HA attaches to the membrane of epithelial cells via sialic acid-galactose

After attachment endocytosis occurs

H+ ion passes through the viral M2 matrix ion protein channels

Acidification of the core leads to conformation changes in HA making it a fusogen

Inside the endosome pH gets lowered

Fusion of endosome membrane and viral membrane; release of RNA and NP

Influenza Biology

Mode of action of H1 N1

Virus infects upper respiratory system

Hydrolyzing the mucus membrane by neuraminidase

Virus adheres to epithelial cells (receptor mediated endocytosis)

Action of lysosome on endosome

When endosome pH decreases, haemagglutine molecule undergoes conformational change. The hydrophobic end of haemagglutine spring outward & extends towards the membrane of endosome.

Mode of action of H1 N1

Nucleocapsid is released into the cytoplasm

Virus replication in epithelium cell nucleus

Assembly of viron particles

Released through budding

Undigested virus particles engulf by macrophages

Ag presentation by class 1 MHC molecule on macrophage

Tc cells get activated

Apoptosis

As a result, there is reduced clearance of infectious agents from the respiratory tract. Gaps in the protective epithelium provide other pathogens with access to other cells.

Influenza Antigenic Changes

Antigenic Drift

Source: http://www.globalsecurity.org/security/ops/hsc-scen-3_flu-antigenic.htm

Antigenic Shift

Treatment

Vaccine Antiviral Drugs Ex. Baxter vaccine (inactivated virus vaccine)

Inhibitors of the viral M2

matrix protein ion channels

Ex. Amantadine Rimantadine

Neuraminidase inhibitors

Ex. Oseltamivir :TAMIFLU Zanamivir :RELENAZA

Treatment

Influenza Vaccines

Influenza Vaccines• Inactivated vaccine (TIV)

• flu shot (injection) • Purified virus chemically inactivated by formalin or propiolactone• trivalent (three strains; usually A/H1N1, A/H3N2, and B)• non-replicating virus• contain much more antigen than live vaccines • works by putting into the bloodstream those parts of three strains

of flu virus that the body uses to create antibodies• Live attenuated vaccine (LAIV)

• nasal spray (FluMist®) • Live virus, completely devoid of pathogenicity • provide continuous antigenic stimulation• works by inoculating against those same three strains that have

been genetically modified to minimize symptoms of illness.• Each of the three strains is a reassortant of internal proteins of a

master donor virus (MDV) that contains the CA and TS phenotypes• surface proteins (HA, NA) are from wild-type influenza virus.

Flu vaccine production (Overview)

• Strain selection• Preparation of seed virus• Seed passaging and selection• Large scale production• Purification and testing• Packaging and shipping

Strain selection and Preparation of seed virus

2 gene segments representing the HA and NA antigens are selected from the target strain.

6 gene segments come from a lab virus strain that confer high growth capacity in eggs.

SEED VIRUS:a hybrid is formed which contains the inner components of the laboratory strain, and the outer components of the pandemic strain

Seed passaging and Selection

Purification and Testing

Large – scale production

• Accredited laboratories distribute seed viruses to manufacturers to begin the production process.

• Once the seed virus has been received, the working seed virus can be prepared by passaging the seed virus in eggs.

• These passages are necessary to determine the optimum growth conditions to improve virus yield in the industrial environment.

• Millions of specially - prepared chicken eggs are used to produce the vaccine. Throughout the year, fertilized eggs are delivered to the manufacturer. Each egg is injected with the working seed.

• The eggs are incubated for several days to allow the virus to multiply. After incubation, the virus – loaded fluid is harvested.

• Manufacturers test the vaccine concentrate using specially prepared reagents provided by WHO Collaboratoring Centers to measure the quantity of virus produced and guarantee the optimal dosage of ready to use vaccines.

• A limitation of current vaccines is that the antigenic regions of HA are highly susceptible to continuous mutation in circulating epidemic virus strains.

•Thus, the currently available influenza vaccines need to be updated every year to match the antigenicity of the virus strains that are predicted to circulate in the next season.

•However, current vaccines would not be effective in preventing the spread of a new pandemic strain containing a substantially different HA protein. Therefore, new approaches are being investigated to develop broadly crossprotective vaccines, focused primarily on type A influenza viruses

Limitations

1. The extracellular domain of M2 (M2e) is highly conserved among multiple influenza A viruses, indicating that M2 is an attractive antigenic target for developing a universal influenza vaccine. Also, an inactivated influenza vaccine supplemented with M2 VLP(virus like particles) prevents disease symptoms.

2. HA is a homotrimeric molecule consisting of a disulfide-linked globular head of HA1 and a stem domain composed of part of HA1 and all of HA2. It possesses conserved structural features in the HA2 segment involved in anchoring to the viral membrane. It has recently been recognized that this segment, termed the stalk, is a potential target for inducing broadly cross-reactive immunity.

Universal influenza vaccine

Challenges

• Non – egg – based production•Targeting generation of virus specific CTLs•Increasing immunogenicity and adjuvants•Rapid production•Dosage

References• Novel vaccines against influenza viruses: Virus Research,

Volume 162, Issues 1–2, December 2011, Pages 31-38. S.M. Kang, J.M. Song, R.W. Compans

• The Influenza Virus Enigma: Cell, Volume 136, Issue 3, 6 February 2009, Pages 402-410Rachelle Salomon, Robert G. Webster

• The 2009 A (H1N1) influenza virus pandemic: A review: Vaccine, Volume 28, Issue 31, 12 July 2010, Pages 4895-4902Marc P. Girard, John S. Tam, Olga M. Assossou, Marie Paule Kieny

Thank you for your attention