ELISA quantification of anti-HA IgG

antibodies in the plasma of UFLU vaccinated

cynomolgus macaques
Sarah Jones
Mentor Sandra Dross
Spring Quarter 2021
Influenza A virus https://viralzone.expasy.org/223

Virus characteristics
- Orthomyxoviridae family
- 13.5 kb -ssRNA enveloped genome
- 8 segments
- Codes for 11 proteins
- NP encapsidated

Proteins & variability

- HA (hemagglutinin) and NA (neuraminidase) on surface of virion
- Matrix (M), nucleoprotein (NP), polymerase complex (PA, PB1, PB2) proteins
- Matrix 2 protein extracellular domain (M2e) = 23 aa res and highly conserved
- M2 ion channel with nuclear export protein (NEP)
Infection and Variability
Infection
- Virus HA + sialic acid receptor on epithelial respiratory cells
- Immune response to viral surface antigens HA and NA
- Antibody responses against HA variable head region primarily

Shift and Drift

- Shift = major change = new HA proteins
- New influenza A virus can cause pandemics
- Drift = small mutations = alterations in antigenic proteins
- But maintains function
- Drift is reason behind need for seasonal vaccines
Modified from Jason Smith, UW Dept
Microbiology, 2021
Influenza vaccines
Current seasonal vaccines
- Live attenuated and inactivated
- Goal to induce humoral responses against variable surface antigens
- Based on annual predictions for circulating viruses
- Lack cross-reactivity

Universal vaccines
- Tries to solve problem of annual drift
- Goal to induce immune response to conserved regions
- Need to overcome immunodominance against variable region HA
R44 UFlu study

goal to determine immune response

and protective potential of the
universal influenza A DNA vaccines
UFlu-1 and -2 against a variety of
influenza A viruses by measuring
humoral and cellular responses

S Dross, May 2021 R44 Orlance Study Design Update

R44 Universal Flu Vaccine (UFlu-1 & -2)

Multi antigen (MA) universal DNA vaccine

- Nucleoprotein (NP)
- Matrix 1 (M1)
- Matrix 2 (M2e)
- Stem-region antigens (HA)

NP plasmid @ vax 3 and 4

Administered with Gene Gun

(particle-mediated epidermal delivery)
Modified from Jason Smith, UW Dept Microbiology, 2021
R44 Uflu ELISAs
ELISAs (enzyme-linked immunosorbent assay)
- Detect and quantify specific soluble substances in a sample
- Especially used to measure antibody titers

UFlu ELISAs
- Measure antibodies in plasma against whole HA recombinant protein
- Expect presence anti-HA IgG in infected and vaccinated animals
- Want to compare titers between vaccinations (3rd vs 4th)
- Look for cross-reactivity with infected animals against different HAs
My project - questions

How do anti-HA IgG titers in plasma compare between groups?

How do anti-HA IgG titers in plasma compare between the third and
fourth vaccination in mock and vaccinated animals?
My project - hypotheses / expectations

Predict that there will be anti-HA IgG antibody response in all

animals with a greater response in the vaccinated group compared to
the mock and control groups

Predict that titers after the 4th vaccination will be similar or lower
than after the 3rd vaccination
 Indirect ELISA protocol

1. Dilute and plate antigen and standard cyno-IgG → incubate overnight 4C

2. Wash (PBST) and block with 5%milk/PBS

3. Wash (PBST) and plate plasma samples + PBS

4. Wash (PBST) and plate secondary antibody

5. Wash (PBST) and add Sureblue substrate

6. Quench reaction with HCl

7. Read plates at 450 nm

Indirect ELISA wells
plate 100 uL diluted (1X PBS) HA stem antigen in each experimental well

recombinant
protein/antigen
Indirect ELISA wells
add 50 uL diluted plasma samples to each experimental well

anti-HA IgG

recombinant
protein/antigen
Indirect ELISA wells
add 100 uL diluted goat anti-monkey antibody + HRP to all wells

enzyme

secondary
antibody

anti-HA IgG

recombinant
protein/antigen
Indirect ELISA wells
add 100 uL Sure Blue Peroxidase to all wells

enzyme substr
ate
color ch
secondary ange
antibody

anti-HA IgG

recombinant
protein/antigen
Results

*OD values plotted rather than concentrations (ng/mL)

Results

graph from S Dross

Future ELISAs / Next Steps
More ELISAs:
- Anti-HA IgG in plasma against other HA proteins
- Anti-HA IgG in other sample types
- Antibodies against other proteins
- Different types of antibodies
- More timepoints and animals

Future goals:
- Try to see if vaccine can neutralize multiple strains
- Look for more possibilities of cross-protection with infection and
vaccination
Sources
Koday, M. T., Leonard, J. A., Munson, P., Forero, A., Koday, M., Bratt, D. L.,... Fuller, D. H. (2017). Multigenic
DNA vaccine induces protective cross-reactive T cell responses against heterologous influenza virus in
nonhuman primates. PloS one, 12(12), e0189780. https://doi.org/10.1371/journal.pone.0189780

Mezhenskaya, D., Isakova-Sivak, I. & Rudenko, L. M2e-based universal influenza vaccines: a historical
overview and new approaches to development. J Biomed Sci 26, 76 (2019).
https://doi.org/10.1186/s12929-019-0572-3

Ogawa, L.M., Vallender, E.J. Genetic substructure in cynomolgus macaques (Macaca fascicularis) on the island of
Mauritius. BMC Genomics 15, 748 (2014). https://doi.org/10.1186/1471-2164-15-748

SIB Informatics. “Orthomyxoviridae.” ViralZone, viralzone.expasy.org/223.

“Types of Influenza Viruses.” Centers for Disease Control and Prevention, Centers for Disease Control and
Prevention, 18 Nov. 2019, www.cdc.gov/flu/about/viruses/types.htm.

2018 DNA prime / rVSV boost Multi-Antigen Universal Influenza Vaccine Grant
Questions?
Multi antigen (MA) universal DNA vaccine
- Nucleoprotein (NP)
- Matrix 1 (M1)
- Matrix 2 (M2e)
- Stem-region antigens (HA)

UFLUA-1
- A/G1-Stem-M2e-M1 + A/G1-LAH-M2e-NP

UFLUA-2
- A/G2-Stem-M2e-M1 + A/G2-LAH-M2e-NP