IL-15 enhances cross-reactive antibody recall responses to seasonal H3 influenza viruses in vitro

Background: Recently, several human monoclonal antibodies that target conserved epitopes on the stalk region of influenza hemagglutinin (HA) have shown broad reactivity to influenza A subtypes. Also, vaccination with recombinant chimeric HA or stem fragments from H3 influenza viruses induce broad immune protection in mice and humans. However, it is unclear whether stalk-binding antibodies can be induced in human memory B cells by seasonal H3N2 viruses. Methods: In this study, we recruited 13 donors previously exposed to H3 viruses, the majority (12 of 13) of which had been immunized with seasonal influenza vaccines. We evaluated plasma baseline strain-specific and stalk-reactive anti-HA antibodies and B cell recall responses to inactivated H3N2 A/Victoria/361/2011 virus in vitro using a high throughput multiplex (mPlex-Flu) assay. Results: Stalk-reactive IgG was detected in the plasma of 7 of the subjects. Inactivated H3 viral particles rapidly induced clade cross-reactive antibodies in B cell cultures derived from all 13 donors. In addition, H3 stalk-reactive antibodies were detected in culture supernatants from 7 of the 13 donors (53.8%). H3 stalk-reactive antibodies were also induced by H1 and H7 subtypes. Interestingly, broadly cross-reactive antibody recall responses to H3 strains were also enhanced by stimulating B cells in vitro with CpG 2006 ODN in the presence of IL-15. H3 stalk-reactive antibodies were detected in CpG 2006 ODN + IL-15 stimulated B cell cultures derived from 12 of the 13 donors (92.3%), with high levels detected in cultures from 7 of the 13 donors. Conclusions: Our results demonstrate that stalk-reactive antibody recall responses induced by seasonal H3 viruses and CpG 2006 ODN can be enhanced by IL-15.


Introduction
Worldwide, annual influenza epidemics are estimated to result in about 3 to 5 million cases of severe illness, and about 250,000 to 500,000 deaths 1,2 . Preventive vaccination is the major intervention currently used to prevent influenza infections 3,4 , and is designed to elicit IgG antibodies directed against the hemagglutinin viral surface protein (HA). Current vaccine formulations elicit a potent immune response against viruses that are closely matched to the vaccine strain, largely through targeting epitopes on the globular head of HAs of influenza A H1N1 and H3N2 subtypes and influenza B strains. However, antigenic drift of influenza virus, which is caused by an accumulation of point mutations within the HA sequences, frequently occurs in influenza A strains and this is particularly true for H3 influenza A strains 5-7 . Individuals who were infected by or vaccinated against H3 influenza viruses circulating in prior years may thus be susceptible to new viral strains.
Since jumping species to humans in 1968, H3N2 swine flu viruses have been responsible for several seasonal pandemics, resulting in both prolonged duration of the influenza season and greater disease severity 8,9 . Under the selective pressure of host immunity, H3N2 influenza virus HAs have undergone progressive antigenic drift. This became particularly problematic during the 2014-2015 influenza season, when H3N2 strains became predominant and were antigenically and genetically distinct from the A/Texas/50/2012 (A/Tex12) vaccine strain 10,11 . The resulting antigenic mismatch between the vaccine strain and circulating H3 viruses, lead to extremely low vaccine effectiveness in the northern hemisphere 12 .
In contrast to the variable HA head domain, epitopes within the HA stalk domain are highly conserved and have become a main target for development of novel treatments using either antibody-based vaccine design or passive immunotherapy. Several human monoclonal antibodies that target highly conserved epitopes on the stalk region of influenza HA display broad reactivity with group 1 and/or group 2 viruses and protect against lethal challenge with influenza viruses in vivo [13][14][15][16] . Animals immunized against H3 stalk elicited broadly cross-reactive antibodies, resulting in protection from challenges with viruses that are of the same HA subtype and/or group [17][18][19] . Others have found that vaccination with a divergent hemagglutinin can increase the frequency of B cells encoding broad influenza A-neutralizing antibodies 20 . However, stalk-reactive antibodies are rarely found in individuals vaccinated with traditional in-activated influenza virus seasonal vaccines [21][22][23] . Recent studies have detected broadly cross-reactive, anti-stalk IgG antibodies in people vaccinated with the pandemic H1N1 A/California/07/09 (pdm A/Cali09) strain 24,25 . Recombinant chimeric HA from H3 viruses have also been shown to elicit broad immune protection in mice and humans 26,27 . However, it is unclear whether stalk-binding antibodies can be induced in human memory B cells using seasonal H3N2 viruses.
Several strategies have been employed in an attempt to improve broadly cross-reactive IgG production by application of a non-specific stimulus, the most common of which is the addition of various adjuvants to promote increased antibody secretion. In addition, the application of various cytokines has also been studied to increase antibody production, including IL-15. IL-15, a member of the 4-α-helix bundle family of cytokines, signals via hetero-trimeric receptors involving the IL-2 receptor β chain (IL2Rβ ), a common γ chain (IL2Rγ c), which is also required for signaling by IL-2, IL-4, IL-7, IL-9 and IL-21, and a unique α subunit (IL-15Rα) that confers receptor specificity to IL-15 28 . Some cytokines that signal through the common IL-2Rγ chain have been shown to increase activated naive and memory B cell IgG secretion rates 29 . IL-15 signals through the activation of JAK2, p38 and ERK1/2 MAPK, SYK kinase and the NF-kB transcriptional factor 30 . Due to the common γ c and β chain, IL-15 shares certain functions with IL-2, including T cell proliferation, the generation of cytotoxic T cells, immunoglobulin synthesis by B cells and the generation and persistence of NK cells 31,32 . IL-15 has been shown to play an essential role in the proliferation of memory B cells and Ig production in vivo 33 .
In addition to promoting the proliferation, differentiation, and IgG secretion of germinal center B cells, IL-15 is also involved in the generation and maintenance of long-term serologic memory 29,34,35 . IL-15 adjuvant has been reported to increase IgG production in animals immunized with influenza vaccines 36 , and DIII antigens of Japanese encephalitis virus and West Nile virus 37 . IL-15 adjuvanted immunization with a DNA vaccine comprised of the N1 and NP genes from the H5N1 influenza virus induced early and high antibody response in chickens 38 . In addition, IL-15 participates in the homing of immature B cells and maintenance of the B cell repertoire 39 . Finally, IL-15 signaling appears to be essential to CD4 T cell and B cell activation by CpG ODN signaling through TLR9 40 , suggesting further synergy between existing vaccine adjuvants and IL-15.
As a vaccine adjuvant, IL-15 has been used for the HIV vaccine and cancer trails (www.clinicaltrials.gov: NCT00775424, NCT00115960, NCT00528489 and NCT01021059). Previous studies have shown that IL-15 promotes the survival, proliferation and Ig production of memory B cells 41,42 . In the current study, we examine human memory B cell IgG recall responses to H3N2 influenza virus in the presence of CpG 2006 ODN activation with IL-15 co-stimulation in vitro. Our results demonstrate that stalkreactive IgG antibodies induced by B cell exposure to H3 viruses in vitro, in the presence of CpG 2006 ODN, are enhanced by IL-15 co-administration. In addition, IgG antibodies elicited by H3 viruses and/or IL-15 broadly bound to influenza HAs from both group 1 and group 2 influenza strains, which suggests potential use of CpG adjuvants and/or IL-15 agonists in influenza vaccination strategies.

Study subjects
This study was approved by the Institutional Review Board at the University of Rochester Medical Center (RSRB protocol RSRB00066522). Subjects were recruited at the University of Rochester through local advertisement, and signed a written statement of informed consent prior to phlebotomy for the study. A total of 13 adults with an age range of 26 to 63 years (mean 43.7 years) were included in the study. Twelve study subjects (S1-S3, S5-S13) gave a history of being previously vaccinated with seasonal influenza vaccines, while one subject (S4) indicated that they had never received any influenza vaccine. Peripheral blood was obtained from all subjects as part of the study for B cell stimulation and analysis of baseline influenza-specific antibodies.

mPlex-Flu assay
The levels of HA-reactive IgG were measured in plasma and in vitro stimulated B cell culture supernatants using the mPlex-Flu assay, as previously described 43 . The assay panels included whole HA or the head segments of influenza group 1, group 2, B strain and chimeric HA, as listed in Table 1. Briefly, 25µL of plasma dilution (1:5000) or undiluted culture supernatants were incubated with 25µL of a panel of beads coupled with HAs at room temperature for two hours on a rotary shaker (500 rpm) in the dark. Then 150µL of phycoerythrin (PE) conjugated goat anti-human IgG (Southern Biotech, Birmingham, Al) was added and incubated at room temperature for 2 hours on a rotary shaker

ELISpot assays
ELISpot assay of memory B cell IgG secretion was performed as previously described 25 . Immobilon P membrane-based 96-well plates (Millipore, Billerica, MA) were coated overnight at 4°C with 10µg/mL H3N2 HA in phosphate-buffered saline (PBS) (40 µl/well). PBS only was added to the negative-control wells. Plates were blocked with complete PRMI 1640 medium. Cells were plated at a density of 10 6 per well in U-bottom plates and stimulated with CpG 2006 ODN, six days after stimulation with A/Vic11 (H3N2, IRR catalog No: FR-1041). B cells were resuspended in complete medium containing either alkaline phosphataseconjugated goat anti-human IgG (H-L) (KPL, Gaithersburg, MD) at 0.2µg/mL and incubated for 5h at 37°C in 5% CO 2 . The plates were washed, and then HA antibody secreting cell spots were developed with an alkaline phosphatase substrate kit (Vector Laboratories, Burlingame, CA). Spots were counted using a CTL ImmunoSpot plate reader and counting software (Cellular Technology Limited, Cleveland, OH).

Statistical analysis
For multiple comparison of H3N2-specific IgG between-group (CpG 2006 ODN alone or together with H3N2 or H7N9 viruses), one-way ANOVA was performed. Correlations between H3-specific antibodies and stalk-reactive antibodies, or A/Vic11specific antibodies between plasma and CpG 2006 ODN ± memory B cells, were evaluated using the Pearson's χ-squared test. Statistical analysis was performed using SPSS11.5 statistical software, as well as Prism 5 software. For all the statistical tests performed, p<0.05 was accepted as significant.

Prevalence of H3-specific and stalk-reactive IgG
To evaluate the anti-influenza antibodies in plasma from 13 subjects and further infer influenza-specific memory B cells in their peripheral blood, we examined H3 HA-specific IgG levels using our mPlex-Flu assay. Six H3N2 strains, accommodating 45  strains were detected in all subjects. Of these, eight displayed high levels of H3-specific antibodies, while the other five had lower plasma baseline H3-reactive antibodies. Antibodies directed against A/Vic11 were high among all H3-specific IgG in 12 of the subjects. Only one subject displayed antibodies targeting the historical outbreak H3N2 strains, A/HK68 and A/PC73, which were higher than those against the more recent seasonal strains, A/Perth09, A/Vic11, A/Tex12 and A/Swi13 ( Figure 1). The emergence of H3N2-specific antibodies indicates that all subjects likely had prior exposure to H3 influenza viral antigen.
In order to determine the degree of H3 stalk-reactive antibodies induced by inactivated A/Vic11 viruses, cH5/3, a soluble HA construction that contains the stalk of H3 virus and the head of H5 virus, was used in our mPlex-flu assay. This recombinant HA protein allows for the direct detection of stalk-reactive IgG antibodies in polyclonal sera or plasma. As shown in Figure 1B, H3 stalk-reactive IgG was detected in plasma (dilution of 1:5,000) from 11 donors, with 4 to 11-fold lower than H3 strain-specific antibodies. For 4 of them, MFI values were greater than 3,000.
Since most donors had a history of receiving seasonal influenza vaccines, we also examined the H1N1 stalk-reactive antibodies. Consistent with H3 stalk results, H1 stalk-binding IgG was detected in all subjects ( Figure 1C).  Figure 2A, the number of ASCs was greater in CpG-H3 group than in CpG alone group. H3N2 strain-specific antibodies in supernatants were assessed by mPlex-Flu assay. B cells from all donors displayed rapid antibody responses to inactivated A/Vic11 viruses. Stimulation with CpG 2006 ODN and H3 viruses resulted in a significant increase in antigen-specific IgG production, compared with H3, CpG and CpG with A/Shanghai/1/2013 (A/SH13) (CpG-H7) groups ( Figure 2B). We also analyzed the relationship between the levels of A/Vic11specific antibodies in plasma and IgG production by activated B cells. No correlation between antibody recall responses and plasma baseline IgG against A/Vic11 was detected (r=0.124, P=0.687) ( Figure 2C).   Figure 2D). Interestingly, antibodies against the most recent seasonal strain A/Swi13 were lower than those against A/Vic11 ( Figure 2E).

A/Vic11 stimulation induced cross-reactive IgG
We then tested the IgG induced by inactivated H3N2 viruses binding to recombinant HAs from influenza A strains, H1, H2, H5, H6, H7, H9, and B strains. For H1, six strains accommodating As shown in Figure 3A, antibodies binding to group 1 and B strains, induced by the group 2 subtype, H3 viruses, were detected in supernatants derived from 8 and 4 donors, respectively, with increases of median of 3.6 to 166.4-fold. IgG in supernatants derived from 8 donors displayed broad cross-reactivity to HAs from 6 different H1N1 strains. Anti-A/Jap57 IgG was detected in supernatants of activated B cells from 3 donors, while anti-A/Viet04, anti-A/TW13 and anti-A/gfHK99 IgG were detected in B cells from 2 donors. For group 2, increases in levels of IgG against H7N9 strains, A/rheaNC93, were detected in 4 donors.  anti-B/Mass12 were not detected. As shown in Figure 3B, the largest response was enhancement of anti-H3 B cell recall responses, and a broad, but lower, increase in responses to other more molecularly distant strains also occurred. Notably, significant increases in IgG binding to chimeric HAs containing the H3, H1, and H7 HA stalk segments, but not the H7 or H5 head segments, were observed, strongly suggesting the presence of broadlycross-reactive stalk antibodies targeting the conserved stalk regions ( Figure 3B).

IL-15 enhanced cross-reactive IgG secretion to H2N2 A/Victoria/361/2011 + CpG 2006 stimulation
To assess if IL-15 has an influence on B cell recall responses to H3N2 viruses, we added IL-15 to cell cultures with CpG 2006 ODN and BPL inactivated A/Vic11 influenza virus, or with CpG 2006 ODN alone. The supernatants at day 6 were measured for IgG production against influenza A strains, including group 1 and group 2, B strains and chimeric HA proteins by mPlex-Flu assay. Fold change values were calculated as described in Figure 3. As shown in Figure 5A, cross-reactive antibody responses to the specific A/Vic11 viruses were enhanced in cell culture derived from all donors upon IL-15 stimulation.
First we analyzed the influence of exogenous IL-15 on the in vitro B cell recall production of A/Vic11-specific IgG from all subjects. All subjects showed increased B cell secretion of A/Vic11 HA-specific antibodies after IL-15 treatment (median 13.1).   (Figure 5B). We next analyzed the relationship between strain-reactive IgG and HA stalk types, which revealed increases in stalk-reactive IgG against H1, H3, and H7 stalk regions that increased greatly with IL-15 + CpG 2006 ODN stimulation in vitro. (Figure 5C).

IL-15 increased secretion of stalk-reactive IgG
Although stalk-reactive antibodies induced by seasonal H3N2 viruses were detected in this study, these antibodies were lower than those against entire H3 HA. To assess whether H3 stalk-reactive antibodies can be enhanced by IL-15, we measured IgG binding to cH5/3. As shown in Figure 6A, anti-cH5/3 IgG increased after costimulation with CpG 2006 ODN, H3 viruses and IL-15, compared with CpG-H3 and CpG groups. Since most influenza subtypes, either influenza A, including group 1 and group 2 subtypes, or influenza B strains, share conserved stalk epitopes, we also evaluated IgG against H7 and H1 stalk using cH5/1 (containing H5 head and H1 stalk) and cH4/7 (containing H4 head and H7 stalk). Anti-cH5/1( Figure 6B) and anti-cH4/7 ( Figure 6C) IgG were higher in CpG-H3-IL-15 groups than in CpG-H3 and CpG alone groups. H7 and H1 stalk-reactive antibodies were upregulated along with H3 stalk-reactive antibodies.

Discussion
The majority of adults possess pre-existing IgG antibodies against influenza viruses from prior infection and/or vaccination 51 , primarily directed against the immunodominant globular head domain of the HAs. Antibodies against the conserved, immuno-subdominant HA stalk domain are generally detectable at very low levels, if at all, despite being broadly protective against multiple influenza strains and subtypes 52 . In this study, we analyzed strain-specific and H3 stalk-reactive antibodies in plasma from donors, and found high levels of anti-H3 specific IgG in all donors. Of these, 12 subjects had high IgG levels against the recent seasonal H3N2 strain A/Vic11, likely by vaccination or infection. One donor, who had never received a seasonal influenza vaccine, showed much lower IgG binding to A/Vic11, but higher anti-A/HK68 IgG levels. Interestingly, 11 of 13 subjects had detectable H3 stalk-reactive IgG at levels only 4-11 fold lower than H3 strain-specific IgG antibodies. The presence of pre-existing stalk-reactive antibodies suggests that stalk-specific memory B cells exist in memory B cell pool, a hypothesis supported by the results from the in vitro B cell stimulation ELISpot experiments.
Immunological memory against influenza following immunization is the corner-stone for prophylactic vaccination programs 53 . Most adults possess a low (0.1-1.0% of total IgG memory B cells) but consistent base line of influenza virus-specific memory B cells 25 . Stalk-reactive memory B cells have generally been reported at very low frequencies, suggesting minimal effective protection 16,20,54 . Interestingly, broad cross-reactive antibodies have been detected in participants who received the pdm A/Cali09 vaccine 24 , which is distinct from the prior seasonal H1N1 strain. Competition of memory B cells responding to either common or rare antigens is hypothesized to regulate the appearance of cross-reactive IgG against influenza HA 20,24 . Repeat exposure to common influenza strains primarily boosts head-reactive responses and limits the expansion of B cells secreting broadly neutralizing antibodies. In contrast, repeated exposure to diverse influenza strains boosts antibodies against highly conserved influenza HA regions, such as the the stalk 24 or a conserved Ca 2+ -binding region 55 on the globular head.
In this study, we focused on enhancing the memory B cell recall response to H3N2 subtype influenza viruses following H3 stimulation in vitro in the presence of CpG 2006 ODN ± IL-15. Class switched IgG antibodies against a panel of H3 strains spanning 45 years from 1968 to 2013 were analyzed. Increases in antibodies against recent seasonal strains were present in all subjects, while induction of IgG antibodies binding to historical H3 strains (prior to the birth year of the subjects) only occurred in 11 subjects. This is consistent with the prevailing hypothesis that antigenic drift leads to low vaccine efficacy 5-7,10 . Decreases in responses to Anti-A/ Swi13 also explain the low vaccine efficacy in 2014-2015 influenza season. Moreover, we found that antibodies induced by H3 viruses, especially in the presence of IL-15, broadly bound to group 1 HAs, with moderate reactivity against group 2 and B influenza subtypes.
These results are consistent with findings by other groups of crossreactive IgG that react against both H1 and H3 influenza strains, primarily against epitopes on the HA stalk regions 19,21 .
CpG 2006 ODN stimulation has been shown to drive in vitro differentiation of both CD27 − and CD27 + human B cells to the plasma cell phenotype 29,44 . Antibody production rates after CpG 2006 ODN stimulation appear to be modulated by IL2Rγ signaling cytokines 29 . In vivo, mouse vaccine studies have noted that CpG adjuvanted influenza vaccination increases anti-HA IgG titers in young, but not older mice 56 . Our results suggest that the stimulation of B cells in vitro with inactivated H3 influenza in combination with CpG 2006 ODN and IL-15 not only stimulate increased anti-HA IgG, but appears to increase levels of secreted crossreactive IgG compared with CpG alone. IL-15 has been reported to overcome immundominance of antigens in CD8 T cell activation 57 . Further work will need to be done to determine if the addition of IL-15 to CpG adjuvanted influenza vaccines would boost protective anti-HA IgG production in vivo in older individuals.
Prior characterization of broadly neutralizing IgG antibodies by other groups has demonstrated that cross-activity results from stalk-reactive antibodies 26,58 . Using the mPlex-Flu assay, we found that moderate to high levels of H3 stalk-reactive antibodies could be induced in vitro after CpG 2006 ODN stimulation of memory B cells from 8 subjects (53.3%). These H3 stalk-reactive antibodies emerged along with IgG that bound the stalk regions of H1 and H7 subtypes, which share conserved epitopes with H3 viruses. Using a liner correlation model, we found that levels of antibodies binding to A/HK68 were positively related to H3 stalk-reactive antibodies, suggesting that clade cross-reactivity was likely due to the conserved epitopes in H3 stalk. This correlation also strongly existed between IgG antibodies binding to the H3 stalk and anti-A/HK68 antibodies responding to inactivated H7N9 viruses, suggesting that H3 stalk-specific memory B cells responded to H7N9 subtypes. Interestingly, stalk-specific IgG recall responses were not seen in memory B cells from the infected/un-vaccinated participant (S4), although they had detectable IgG antibodies against historical outbreak H3 strains after in vitro CpG 2006 ODN + IL-15 stimulation.
Although the production of stalk-reactive antibodies indicated activation of stalk-specific memory B cells, we found both strain cross-reactive and stalk-reactive IgG antibodies present at much lower levels than H3 strain-specific anti-HA IgG antibodies. Given the desirability of inducing broadly cross-reactive anti-HA stalkreactive IgG antibodies, vaccination strategies to achieve this goal need to be developed. In this study, we evaluated the effect of supplemental IL-15 on B cell recall responses to inactivated A/Vic11 viruses. We demonstrated that the B cell recall responses to H3 viruses were enhanced by CpG 2006 ODN + IL-15, with increases of 6.9 to 15-fold in IgG production. Broadly cross-reactive IgG antibodies were observed to bind to group 1, group 2 and B strain influenza HA subtypes following CpG 2006 ODN + IL-15 stimulation, with median of increase of 22.3-fold for group 1, 21.4fold for both group 2 and 17.6-fold for B subtypes compared to CpG 2006 ODN stimulation alone. This demonstrated IL-15 greatly augmented recall secretion of broadly cross-reactive anti-influenza HA IgG antibodies. Not surprisingly, there was a negative correlation between antigenic sequence dissimilarity of the stimulating influenza strain and recall IgG antibody levels.

Conclusions
In conclusion, broadly cross-reactive anti-influenza stalk-binding IgG antibodies exist in individuals exposed to influenza strains. Seasonal H3N2 virus exposure, through vaccination or infection, can induce memory B cells that bind to the conserved stalk region of HAs. In vitro recall responses to these stalk-reactive antibodies can be enhanced by IL-15. These results suggest the potential for IL-15 augmentation of adjuvant to overcome immunodominance of influenza HA head region epitopes as a potential vaccine boosting strategy to increase levels of broadly cross-reactive anti-influenza HA IgG antibodies.

Competing interests
No competing interests were disclosed. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

2.
3. , in the presence of in vitro CpG2006 ODN, are enhanced by IL-15 co-administration. In addition, IgG antibodies elicited by H3 viruses and/or IL-15 broadly bound to influenza HAs from both group 1 and group 2 influenza strains, which suggests potential use of CpG adjuvants and/or IL-15 agonists in influenza vaccination strategies. The use of IL-15 to enhance responses to immunization has been studied previously (see for example, PMID:24706798 which the authors could cite). However in that study, it was T-cell rather than antibody responses that were found to be protective. In this context, it would be important in a future study to examine whether the induced, cross-reactive antibodies show any protective activity in an appropriate assay. Overall, the manuscript is clearly written and interesting to read. However, the authors could consider the following additional points to improve clarity.

Open Peer Review
Please include list of abbreviations used in the manuscript. In the result section, authors mention "As shown in Figure 1B, H3 stalk-reactive IgG was detected in plasma (dilution of 1:5,000) from 11 donors, with 4 to 11-fold lower than H3 strain-specific antibodies. For 4 of them, MFI values were greater than 3,000". However, the maximum MFI values given in data set is 1250. Also there is no caption for Figure 1C Is the work clearly and accurately presented and does it cite the current literature? Yes

Is the study design appropriate and is the work technically sound? Yes
Are sufficient details of methods and analysis provided to allow replication by others? Yes

If applicable, is the statistical analysis and its interpretation appropriate? Yes
Are all the source data underlying the results available to ensure full reproducibility? Yes

Are the conclusions drawn adequately supported by the results? Yes
No competing interests were disclosed.

Competing Interests:
I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
The benefits of publishing with F1000Research: Your article is published within days, with no editorial bias You can publish traditional articles, null/negative results, case reports, data notes and more The peer review process is transparent and collaborative Your article is indexed in PubMed after passing peer review Dedicated customer support at every stage For pre-submission enquiries, contact research@f1000.com