DOI: 10.5281/zenodo.3464942
title: Additional ELISA data for the manuscript "Restrained expansion of the recall germinal center response as biomarker of protection for influenza vaccination in mice"

description: This repository contains the source ELISA data for supplementory figure 4 of the manuscript "Restrained expansion of the recall germinal center response as biomarker of protection for influenza vaccination in mice" currently under review by PLOS ONE. Files include Raw OD's per plate and reported values. Purpose:<br> These ELISA's were performed to assess differences in hemagglutinin (HA) specific IgG responses in sera of mice immunized with one of three univeral flu vaccine regimens conferring different levels of protection against lethal H1N1 influenza challenge. Method:<br> Naïve 8-weeks old mice were immunized three times, at days 0, 21 and 42 with different immunogens. One cohort of animals was vaccinated received a largely protective vaccine containing a high dose (30 µg) of a trimeric full-length H1 HA antigen (FL H1#2316) that provides heterologous protection against H1N1 A/Netherlands/602/2009 while a second cohort received a minimally protective vaccine containing a high dose (30 µg) of an HA stem-based monomeric antigen (UFV#4157) derived from FL H1#2316. To control for differences in immune responses related to the dose rather than intrinsically protective properties of the used immunization antigen, a lower but partiallly protective protective dose (0.3 µg) of FL H1#2316 was used for immunizations of a third cohort, while as a negative control for survival a fourth cohort was immunized with PBS only. All the immunized proteins are based on the HA of H1N1 A/Brisbane/59/07. All immunizations, including mock-immunizations with PBS, were adjuvanted with alum. 8 animals per cohort were sacrificed at 8 different timepoints (days 4, 7, 12, 19, 25, 28, 46 and 49) to obtain serum and lymphnodes for analaysis To assess the protective efficacy of the administered vaccine regimens, 10 animals per immunized cohort were challenged intranasally with a lethal dose of 25xLD50 H1N1 A/Netherlands/602/2009. 2 days prior to the challenge, on day 68, we also took a blood sample to check the pre-challenge titers of each animal. On the serum isolated on day 4, 7, 12, 19, 25, 28, 46 49 andd 68, we performed two ELISA experiments. In one ELISA we coated the plates with the recombinant HA of A/Brisbane/59/07, homologous to the used vaccine candidates, and in the other ELISA we used recombinant HA of A/California/07/09, 99.4% homologous to the challenge strain H1N1 A/Netherlands/602/2009. Results:<br> At the end of the three immunizations we see that the IgG titers against A/Brisbane/59/07 reach similar levels in both the 30µg and 0.3µg UFV#2316 cohorts, whearas the responses of the headless UFV#4157 are slightly lower. Because the titers against the rHA A/California/07/09 in the UFV#4157 cohort is similar to both UFV#2316 regimen we can allocate the differences observed in the A/Brisbane/59/07 ELISA to the absense or availablility of the immunodominant head domain. In conclusion we do not see any differences in antibody titers that can correlate with the challenge outcome at the end of the study:<br> 30µg UFV#2316 80% protection<br> 0.3µg UFV#2316 60% protection<br> 30µg UFV#4157: 0% protection<br> PBS: 0% protection