Proficiency tests to evaluate the impact on assay outcomes of harmonized influenza-specific Intracellular Cytokine Staining (ICS) and IFN-É£ Enzyme-Linked ImmunoSpot (ELISpot) protocols.

The magnitude and quality of cell-mediated immune responses elicited by natural infection or vaccination are commonly measured by Interferon-É£ (IFN-É£) Enzyme-Linked ImmunoSpot (ELISpot) and Intracellular Cytokine Staining (ICS). To date, laboratories apply a variety of in-house procedures which leads to diverging results, complicates interlaboratory comparisons and hampers vaccine evaluations. During the FLUCOP project, efforts have been made to develop harmonized Standard Operating Procedures (SOPs) for influenza-specific IFN-É£ ELISpot and ICS assays. Exploratory pilot studies provided information about the interlaboratory variation before harmonization efforts were initiated. Here we report the results of two proficiency tests organized to evaluate the impact of the harmonization effort on assay results and the performance of participating FLUCOP partners. The introduction of the IFN-É£ ELISpot SOP reduced variation of both background and stimulated responses. Post-harmonization background responses were all lower than an arbitrary threshold of 50 SFU/million cells. When stimulated with A/California and B/Phuket, a statistically significant reduction in variation (p < 0.0001) was observed and CV values were strongly reduced, from 148% to 77% for A/California and from 126% to 73% for B/Phuket. The harmonizing effect of applying an ICS SOP was also confirmed by an increased homogeneity of data obtained by the individual labs. The application of acceptance criteria on cell viability and background responses further enhanced the data homogeneity. Finally, as the same set of samples was analyzed by both the IFN-É£ ELISpot and the ICS assays, a method comparison was performed. A clear correlation between the two methods was observed, but they cannot be considered interchangeable. In conclusion, proficiency tests show that a limited harmonization effort consisting of the introduction of SOPs and the use of the same in vitro stimulating antigens leads to a reduction of the interlaboratory variation of IFN-É£ ELISpot data and demonstrate that substantial improvements for the ICS assay are achieved as comparable laboratory datasets could be generated. Additional steps to further reduce the interlaboratory variation of ICS data can consist of standardized gating templates and detailed data reporting instructions as well as further efforts to harmonize reagent and instrument use.

Can Geographically Targeted Vaccinations Be Ethically Justified? The Case of Norway During the COVID-19 Pandemic.

This article discusses the fairness of geographically targeted vaccinations (GTVs). During the initial period of local and global vaccine scarcity, health authorities had to enact priority-setting strategies for mass vaccination campaigns against COVID-19. These strategies have in common that priority setting was based on personal characteristics, such as age, health status or profession. However, in 2021, an alternative to this strategy was employed in some countries, particularly Norway. In these countries, vaccine allocation was also based on the epidemiological situations in different regions, and vaccines were assigned based on local incidence rates. The aim of this article is to describe and examine how a geographical allocation mechanism may work by considering Norway as a case study and discuss what ethical issues may arise in this type of priority setting. We explain three core concepts: priority setting, geographical priority setting and GTVs. With a particular focus on Norway, we discuss the potential effects of GTV, the public perception of such a strategy, and if GTV can be considered a fair strategy. We conclude that the most reasonable defence of GTV seems to be through a consequentialist account that values both total health outcomes and more equal outcomes.

Functional and Binding H1N1pdm09-Specific Antibody Responses in Occasionally and Repeatedly Vaccinated Healthcare Workers: A Five-Year Study (2009-2014).

Background: In 2009, a novel influenza A/H1N1pdm09 emerged and caused a pandemic. This strain continued to circulate and was therefore included in the seasonal vaccines up to the 2016/2017-season. This provided a unique opportunity to study the long-term antibody responses to H1N1pdm09 in healthcare workers (HCW) with a different vaccination history. Methods: HCW at Haukeland University Hospital, Bergen, Norway were immunized with the AS03-adjuvanted H1N1pdm09 vaccine in 2009 (N=55) and divided into groups according to their vaccination history; one vaccination (N=10), two vaccinations (N=15), three vaccinations (N=5), four vaccinations (N=15) and five vaccinations (N=10). HCW are recommended for influenza vaccination to protect both themselves and their patients, but it is voluntary in Norway. Blood samples were collected pre- and at 21 days, 3, 6, and 12 months after each vaccination, or annually from 2010 HCW without vaccination. ELISA, haemagglutination inhibition (HI) and microneutralization (MN) assays were used to determine the antibody response. Results: Pandemic vaccination induced a significant increase in the H1N1-specific antibodies measured by ELISA, HI and MN. Seasonal vaccination boosted the antibody response, both in HCW with only the current vaccination and those with prior and current vaccination during 2010/11-2013/14. We observed a trend of increased antibody responses in HCW with only the current vaccination in 2013/14. A two- and three-year gap before vaccination in 2013/14 provided a more potent antibody response compared to annually vaccinated HCW. Conclusions: Our long term follow up study elucidates the antibody response in HCW with different vaccination histories. Our findings contribute to our understanding of the impact of repeated vaccination upon antibody responses.

Repeated Influenza Vaccination Boosts and Maintains H1N1pdm09 Neuraminidase Antibody Titers.

Antibodies to influenza surface protein neuraminidase (NA) have been found to reduce disease severity and may be an independent correlate of protection. Despite this, current influenza vaccines have no regulatory requirements for the quality or quantity of the NA antigen and are not optimized for induction of NA-specific antibodies. Here we investigate the induction and durability of NA-specific antibody titers after pandemic AS03-adjuvanted monovalent H1N1 vaccination and subsequent annual vaccination in health care workers in a five-year longitudinal study. NA-specific antibodies were measured by endpoint ELISA and functional antibodies measured by enzyme-linked lectin assay (ELLA) and plaque reduction naturalisation assay. We found robust induction of NA inhibition (NAI) titers with a 53% seroconversion rate (>4-fold) after pandemic vaccination in 2009. Furthermore, the endpoint and NAI geometric mean titers persisted above pre-vaccination levels up to five years after vaccination in HCWs that only received the pandemic vaccine, which demonstrates considerable durability. Vaccination with non-adjuvanted trivalent influenza vaccines (TIV) in subsequent influenza seasons 2010/2011 - 2013/2014 further boosted NA-specific antibody responses. We found that each subsequent vaccination increased durable endpoint titers and contributed to maintaining the durability of functional antibody titers. Although the trivalent influenza vaccines boosted NA-specific antibodies, the magnitude of fold-increase at day 21 declined with repeated vaccination, particularly for functional antibody titers. High levels of pre-existing antibodies were associated with lower fold-induction in repeatedly vaccinated HCWs. In summary, our results show that durable NA-specific antibody responses can be induced by an adjuvanted influenza vaccine, which can be maintained and further boosted by TIVs. Although NA-specific antibody responses are boosted by annual influenza vaccines, high pre-existing titers may negatively affect the magnitude of fold-increase in repeatedly vaccinated individuals. Our results support continued development and standardization of the NA antigen to supplement current influenza vaccines and reduce the burden of morbidity and mortality.