A recombinant vaccine against the H5N1 influenza virus

Presented by: Steven Mitchell

BackgroundInfluenza is an infectious disease that arises within

mammals and birds.

The influenza virus is categorized into three groups: Influenza A: infects birds, swine, and humans Influenza B: infects seals and humans Influenza C: infects swine and humans

Influenza is named using the system H_N_ H for hemagglutinin; N for neuraminidase

Traditional vaccinations cover only H3N2, Influenza B, and H1N1 strain variants

Step 1Health officials globally surveys year-round which viral strains will be most prominent for a year

Step 2Officials analyze and target the most dominant strains and are submitted to the FDA, who will choose the three most worthy candidates

Step 3Each virus is separately created then combined to form the final vaccine. Millions of chicken eggs are specially designed to incubate one strand per egg

Step 4Viral fluid from the eggs is purified and the virus is inactivated. The virus is cleaved, and fragments are combined to form the vaccine

Step 5The vaccine is packaged into vials and syringes after each batch, or lot, is approved individually by the FDA

Problems with traditional vaccines

Not all infectious agents can be grown in culture

Production of animal/human viruses require animal cell culture, which is expensive

Yield and production rate of viruses is relatively low

Batches of vaccine may not be killed, leading to inadvertent disease transmission. Attenuated strains may revert, and so constant testing is needed

Most vaccines have a limited shelf life and require refrigeration

Recombinant vaccine to prevent H5N1

HPAI-H5N1 (bird flu) is an influenza A subtype virus that is highly pathogenic and has a high mortality rate

Current efforts toward a vaccine have been unsuccessful due to traditional methods unable to handle the rapid mutation of hemagglutinin (HA), a diverse surface protein present on all influenza viruses

Recombinant DNA technology may provide a preventative treatment for this pathogen

Diagram of an influenza virus

Principles of H5N1 recombinant vaccine

The Fc portion of IgG is an important fusion tag for expressing several viral proteins, such as SARS and HIV

The Fc portion helps folding of the viral protein to enhance binding of antigen-presenting cells. The Fd (foldon sequence of T4 phage fibritin) sequence was also expressed to further promote proper folding

Two vaccines were created by hybridizing the HA protein to either the Fc sequence of IgG, or Fc plus Fd

Structure of HA protein and recombinant Fc

and Fdc protein

The HA protein of H5N1 consists of a signal peptide, HA1, HA2, and a protease cleavage sites

The SP sequence was replaced by IL2ss sequence in the Fc vector to create HA1-Fc protein

HA1-Fdc was created by attaching HA1 to the Fd C-terminus, followed by the Fc vector

ResultsTransfected HEK 293T cells were used as

expression vectors for HA1-Fc and HA1-Fdc proteins

293T cells were used because of their ability to amplify transfected plasmids. Other cell lines could be used for substitution (such as CHO cells)

Proteins were extracted from the supernatant medium, purified, then analyzed by SDS-PAGE and a western blot

Schematic of a subunit vaccine

Western blot for HA1 fusion protein

The purified HA1-FC and HA1-Fdc proteins were analyzed using a western blot with an anti-HA mAb. Purified proteins were separated by 10-20% Tricine gels and transferred to nitrocellulose. Proteins were blocked overnight, then incubated with anti-HA mAb for 1 hour. Blots were incubated with HRP-conjugated goat anti-mouse IgG for 1 hour.

ResultsMice injected with the HA1-Fc and HA1-Fdc

recombinant proteins were analyzed via ELISA for antibody responses

Both proteins induced IgG antibody responses specific to the proteins

Subsequent injections with different clade’s HA proteins showed an antibody response (pseudoviruses)

IgG antibody response to HA1-Fc and HA1-Fdc vaccine treatments

Left: Reactivity of IgG antibody with HA1-Fc and HA1-Fdc proteins. Time scale was 10 days between first vaccination and boostersRight: The ability of IgG to bind to the proteins and the control

Results The antibodies produced were found to reduce the viruses

pathogenic qualities such as reproductive capabilities

The antibodies were effective for a wide array of live H5N1 strains, as well as H5N1 pseudoviruses, including: HK/156 VN/1194 SZ/406H HK-HA 1194-HA QH-HA XJ-HA AH-HA

These three avian flu strands are highly pathogenic in humans

Antibodies present from heterologous strains of

H5N1

Graphs show various strains of live influenza being introduced into mice after being vaccinated. The neutralizing antibodies, NAb, block biological effects the virus has on it’s host cells. Hemagglutination antibodies, HI Ab, disable the binding ability of the influenza virus.

Viral challenge in vaccinated mice

Left: Lethal H5N1 virus challenge in vaccinated mice. Three different clades were tested for 21 days. Note the difference in survival rate between proteins HA1-Fc and HA1-FdcRight: Detection of viral RNA copies by quantitative reverse-transcription PCR (Q-RT-PCR) in lung tissues of H5N1 challenged mice

Histopathological changes in lung tissue

Lung tissue samples were collected five days after all mice were sacrificed. All sections were stained with hematoxylin and eosin and observed under light microscope. Tissues are fixed in paraffin wax.

OverviewTargeting HA, the main surface protein of the

virus, would provide a feasible means of producing an effective virus

The HA gene was fused with the Fc of IgG antibodies, or with Fc and Fd (foldon) to promote trimeric folding

Both subunit vaccines were shown to be effective for a wide variety of avian influenza strains. However, the HA1-Fdc vaccine proved to be the most effective

Methods and Materials Construction, expression, and purification of recombinant HA1-Fc and

HA1-Fdc proteins Genes encoding HA1 of H5N1 were amplified by PCR using full-length HA as the

template and inserted into the Fc expression vector. The Fd sequence derived from bacteriophage T4 fibritin was fused to the C-terminus of HA1 sequences by PCR with overlapping primers. The recombinant proteins were expressed in 293T cells using calcium phosphate method. The recombinant proteins were purified by protein A affinity chromatography.

Western Blot The purified proteins were analyzed by SDS-PAGE and western blot using an anti-HA

mAb. The proteins were transferred to nitrocellulose membranes. After blocking, blots were incubated with HA specific mAb for 1 hour. Blots were incubated with HRP-conjugated goat anti-mouse IgG for 1 hour. Signals were visualized with ECL reagents.

ELISA The antibody response was evaluated by ELISA in collected mouse sera. 96-well ELISA

plates were coated with recombinant HA1-Fc and HA1-Fdc fusion proteins, HA1 protein without Fd and Fc, and inactivated H5N1 virus and blocked overnight with non-fat milk. Bound antibodies were incubated with HRP-conjugated goat anti-mouse IgG for 1 hour. The reaction was visualized by TMP and stopped by 1N H2SO4.

Further informationhttp://www.ted.com/talks/seth_berkley_hiv_and_flu_th

e_vaccine_strategy.html

http://www.who.int/csr/disease/avian_influenza/country/cases_table_2010_12_09/en/index.html

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