MAVS regulates the quality of the antibody response to ... · 12/15/2019 · 2 1 Abstract 2 A key difference that distinguishes viral infections from protein immunizations is the

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MAVS regulates the quality of the antibody response to West-Nile Virus

Marvin O’Ketcha, Cameron Larsona, Spencer Williamsa, Jennifer L. Uhrlauba, Rachel Wonga, b,

Neha R. Deshpandea, and Dominik Schentena, 1

a Department of Immunobiology, University of Arizona, Tucson, AZ 85724

bDivision of Biological and Biomedical Sciences, Washington University in St. Louis,

Saint Louis, MO 63110

Author contributions: M.O. and D. S. designed research; M. O., C. L., and S.W. performed

research; J. L. U., R. W., and N. R. D. provided critical reagents and assistance; M. O. and D. S.

analyzed and interpreted the data; D. S. directed the overall project and wrote the manuscript.

1 To whom correspondence should be addressed. Email: [email protected]

The authors declare no conflict of interest.

.CC-BY-NC-ND 4.0 International licenseavailable under a(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

The copyright holder for this preprintthis version posted December 15, 2019. ; https://doi.org/10.1101/2019.12.15.875906doi: bioRxiv preprint

https://doi.org/10.1101/2019.12.15.875906

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2

Abstract 1

A key difference that distinguishes viral infections from protein immunizations is the 2

recognition of viral nucleic acids by cytosolic pattern recognition receptors (PRRs) such as RNA-3

sensing Rig-I-like receptors (RLRs). Insights into the specific functions of cytosolic PRRs in the 4

instruction of adaptive immunity are therefore critical for the understanding of protective immunity 5

to infections. West Nile virus (WNV) infection of mice deficent of MAVS, the essential RLR 6

signaling adaptor, results in a defective adaptive immune response. While this finding suggests a 7

role for RLRs in the instruction of adaptive immunity to WNV, it is difficult to interpret due to the 8

high WNV viremia, associated exessive antigen loads, and pathology in the absence of a MAVS-9

dependent innate immune response. To overcome these limitations, we have infected MAVS-10

deficient mice with a single-round-of-infection mutant of WNV called RepliVAX (RWN). RWN-11

infected MAVS-deficient (MAVSKO) mice failed to produce an effective neutralizing antibody 12

response to WNV despite normal titers of antibodies targeting the viral WNV-E protein. This defect 13

occurred indepedently of antigen loads or overt pathology. The specificity of the antibody 14

response in RWN-infected MAVSKO mice remained unchanged and was still dominated by 15

antibodies that bound the neutralizing lateral ridge (LR) epitope in the DIII domain of WNV-E. 16

Instead, MAVSKO mice produced IgM antibodies, the dominant isotype controlling primary WNV 17

infection, with lower affinity for the DIII domain. Our findings suggest that RLR-dependent signals 18

are important for the quality of the humoral immune response to WNV. 19



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Introduction 1

The features that separate protective adaptive immune responses from similar responses 2

that fail to protect from infection have yet to be clearly delineated. Aside from important factors 3

such as antigen structure or antibody specificity, it is widely believed that many aspects that define 4

protective immunity are instructed by the innate immune system (1–4). Presumably, pathogens 5

and live vaccines trigger multiple pattern recognition receptors (PRRs) that induce the optimal set 6

of signaling molecules for the regulation of protective adaptive immune responses. In contrast, 7

adjuvant-based subunit vaccines likely represent incomplete mimics of live vaccines that fail to 8

replicate the necessary set of regulatory signals. As the nature and functions of these signals are 9

incompletely understood, rational vaccine design is still facing considerable challenges. 10

Consistent with this view, the highly successful live yellow fever vaccine YF-17D activates multiple 11

PRRs that collectively define the cytokine profile and magnitude of both CD4 and CD8 T cell 12

responses as well as antibody responses (5–7). Likewise, recognition of RNAs uniquely 13

associated with live bacteria can promote the magnitude and vaccine efficacy of T-dependent 14

antibody responses (8–10). Vaccination with live attenuated microbes is therefore still often 15

considered the best way to elicit effective long-lasting cellular and humoral immunity (4, 11–13). 16

A key feature that distinguishes viral infections from immunizations with subunit vaccines 17

is the activation of cytosolic RNA or DNA-sensing PRRs during the course of viral infections. The 18

Rig-I-like receptor (RLR) family of PRRs include the ubiquitously-expressed RNA-sensing 19

helicases RIG-I and MDA5, which recognize microbial RNA in the cytosol (14). Both receptors 20

rely on the adaptor protein MAVS for the transmission of their signal and the induction of 21

proinflammatory cytokines and interferon responses (14–16). RLRs clearly play an essential 22

function in the regulation of innate immunity to many RNA viruses as mice deficient of components 23

of the RLR signaling pathway often suffer from uncontrolled viral replication and succumb to the 24

infection. However, a clear understanding of RLR function in adaptive immunity has remained 25



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elusive as the viral uncontrolled replication complicates efforts to separate intrinsic functions of 1

RLRs from the confounding variables of antigen load and pathology. 2

Infection of MAVS-deficient mice (MAVSKO mice) with pathogenic West-Nile Virus (WNV), 3

a single-stranded RNA virus of the flavivirus family, results a dysregulated adaptive immune 4

response (17, 18). Specifically, MAVSKO mice generate poorly neutralizing antibodies against 5

WNV, even though they have higher WNV-specific antibody titers than wild-type controls, 6

suggesting that MAVS may play a role in quality control of the antibody response to WNV (17). 7

This finding was surprising because WNV-mediated TLR activation should be sufficient for the 8

generation of humoral immunity to WNV. However, interpreting this result as evidence for a direct 9

link between MAVS-induced signals and the quality of neutralizing antibody responses is 10

challenging because MAVS deficiency also leads to a significant increase in WNV viremia (>1000-11

fold) that causes severe pathology and death as well as an excess of viral antigens (17). It remains 12

therefore unclear whether MAVS directly contributes to humoral immunity to WNV. 13

To overcome the limitations of high viral titers associated with WNV infections of mice with 14

deficiencies in innate signaling pathways, we infected MAVSKO mice with a single-round-of-15

infection mutant of WNV called WNV-RepliVAX (RWN) (19). This mutant virus carries a deletion 16

in the capsid gene and fails to generate infectious viral particles but produces all other viral 17

proteins and RNA. Using this system, we show here that MAVSKO mice fail to generate effective 18

neutralizing antibody responses to RWN even under conditions of similar antigen loads. We show 19

that this defect is caused a T-dependent antibody response that is directed at the neutralizing 20

epitope of WNV but displays a lower affinity for this epitope. Our data therefore suggest that 21

MAVS-dependent signals directly influence the quality of the antiviral antibody response against 22

WNV by calibrating the affinity of the neutralizing antibodies. 23



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Material and Methods 1

2

Mice. MAVSKO mice, MHCIIKO mice, and Rag2KO mice were kept on a C57BL/6 background under 3

SPF conditions (20–22). Experimental mice and wild-type controls were cohoused immediately 4

following weaning. The mice were analyzed between 7-12 weeks of age and involved both sexes. 5

All experiments were performed in accordance with the Institutional Animal Care and Use 6

Committee (IACUC) of the University of Arizona. 7

8

Virus production and mouse infections or immunizations. The single-round-of-infection 9

mutant of WNV (WNN-RepliVAX, RWN) has been described before (23). This WNV-NY-derived 10

virus carries an inactivated capsid gene and requires passaging over capsid-expressing BHK 11

cells for the production of infectious virions. BHK cells were infected with 0.05 MOI of RWN and 12

the supernatants were harvested 48-96 hrs later. RWN titers were determined by infecting fresh 13

Vero cells with serial dilutions of RWN and subsequent intracellular staining for infected cells with 14

a biotinylated humanized anti-WNV-E antibody (hE16-biotin) followed by Streptavidin-horseradish 15

peroxidase (HRP) (24) or, alternatively, with purified hE16, followed by anti-human IgG2a 16

antibody. HRP-positive cells were detected with the TrueBlue substrate. Unless otherwise noted, 17

mice were infected with RWN subcutaneously in the footpads with a dose of 1 x 105 Pfu per 18

mouse. When indicated, mice were infected with 5 x 105 Pfu RWN and 1x 106 Pfu RWN or 19

immunized with 50 µg Ovalbumin and 5 µg LPS in Incomplete Freund’s Adjuvant (all Sigma 20

Aldrich, St. Louis, MO). 21

22

Antibodies and other reagents. Antibodies against CD3e, CD4, CD8, CD11b, CD11c, CD25, 23

CXCR5, PD-1, CD19, B220, and FoxP3 were purchased from BD Biosciences (San Diego, CA), 24

ThermoFisher (Waltham, MA), or Biolegend (San Diego, CA). PNA was obtained from Vector 25



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Laboratories (Burlingame, CA). E641:I-Ab tetramers and recombinant DIII and DIII-K307E/T330I 1

were kindly provided by M. Kuhns (University of Arizona) and D. Bhattacharya (University of 2

Arizona), respectively (25, 26). Anti-PE microbeads were purchased from Miltenyi Biotec (Auburn, 3

CA). 4

5

Surface and intracellular staining. Cells were stained with indicated antibodies for 15 min on 6

ice for cell surface staining. For CXCR5 and E641:I-Ab tetramer staining, cells were incubated for 7

45 min at room temperature. E641:I-Ab+ cells were subsequently enriched with anti-PE-8

microbeads according to manufacturer’s instructions. Staining for intracellular antigens were 9

performed with the BD Bioscience or Thermofisher (for FoxP3) intracellular staining kits. Cells 10

were analyzed on a LSRFortessa flow cytometer (BD Bioscience, San Diego, CA) and the FlowJo 11

software (Tree Star). 12

13

Detection of WNV-E protein. For the detection of WNV-E antigen levels in RWN-infected mice, 14

1-2 x 107 cells from the dLNs were isolated 24 hrs after infection and cultured for an additional 24 15

hrs in vitro. Subsequently, supernatants and cells were subjected to one freeze-thaw cycle at -80 16

°C to release RWN virions from the infected cells. After a centrifugation step, the supernatants 17

were incubated with anti-mouse IgG2a-coupled LEGENDplex beads (Biolegend) that had been 18

coated with the anti-DII/III E60 antibody at a concentration of 1 µg/100 µl of beads. Binding of 19

WNV-E protein to the beads was detected by flow cytometry with the biotinylated anti-DIII hE16 20

antibody, followed by SA-PE. Samples from naïve mice or standards with and without 21

recombinant WNV-E protein were used as controls. 22

23

Quantitative PCR. RNA was isolated from the dLNs following infection with RWN and converted 24

into cDNA. Quantitative PCR was performed using the PerfeCTa SYBR Green Fastmix 25



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(Quantabio, Beverly, MA) on a StepOne Real-time PCR system (Applied biosystems, Foster City, 1

CA). PCR products were amplified with the following primer pairs: Cytokines: IL-1b-F 5’-2

TGAGCACCTTCTTTTCCTTCA and IL-1b-R 5’-TGTTCATCTCGGAGCCTGTA; IL-6-F 5’-3

GTTCTCTGGGAAATCGTGGA and IL-6-R 5’-TTTCTGCAAGTGCATCATCG; TNF-a-F 5’-4

CCCCAAAGGGATGAGAAGTT and TNF-a-R 5’-TGGGCTACAGGCTTGTCACT; IFN-a4-F 5’-5

AGGACAGGAAGGATTTTGGA and IFN-a4-R 5’-GCTGCTGATGGAGGTCATT; IFN-b-F 5’-6

CACAGCCCTCTCCATCCACT and IFN-b-R 5’- GCATCTTCTCCGTCATCTCG; IFN-l2-F 5’- 7

CAGAGCCCAGGTCCCCGA and IFN-l2-R 5’-CACACTTGAGGTCCCGGGT; IFN-l3-F 5’-8

CAGAGCCCAAGCCCCCGA and IFN-l3-R 5’-CTTGAGGTCCCGGAGGAG; IFIT1-F 5’-9

GCTGAGATGTCACTTCACATGG and IFIT1-R 5’-CACAGTCCATCTCAGCACACT; IFIT2-F 5’-10

AGTACAACGAGTAAGGAGTCACT and IFIT2-R 5’-AGGCCAGTATGTTGCACATGG; ISG15-F 11

5’-GGTGTCCGTGACTAACTCCAT and ISG15-R 5’-TGGAAAGGGTAAGACCGTCCT; and 12

RSAD2-F 5’-TGCTGGCTGAGAATAGCATTAGG and RSAD2-R 5’-13

GCTGAGTGCTGTTCCCATCT; WNV proteins: E-F 5’-GGCTTCCTTGAACGACCTAA and 14

WNV-E-R 5’-CGTGGCCACTGAAACAAAAG; NS1-F 5’-CAACTCAGAATCGCGCTTGG and 15

NS1-R 5’-CTCTCGAGGATTCCATCGCC; and NS4b-F 5’-AACCCGTCTGTGAAGACAGT and 16

NS4b 5’-ATAAGCACGACAACCAACCC. 17

18

IFN Bioassay. IFN activity was measured by a standard assay quantifying the protection of cells 19

from cytopathic effects (CPE) of vesicular stomatitis virus (VSV). L929 cells were plated on 96-20

well tissue culture plates and incubated for 24hrs at 37 ᴼC. IFN-α2 standard was titrated and 21

serum samples were serially diluted before incubation with VSV on confluent L929 cells for 24 22

hours. The cells were fixed with paraformaldehyde and stained with a solution containing crystal 23

violet dye. The protection of cells from CPE by each serum sample was scored and compared to 24

IFN-α2 standard samples to establish a concentration value for type I IFN in the serum in Units/ml. 25



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Immunoglobulin ELISA. Detection of WNV-specific antibodies was based on ELISAs with 1

recombinant WNV-E, DIII, or DIII-KT as antigens. Production of these reagents followed published 2

protocols (27, 28). Briefly, DIII proteins were expressed in BL21(DE3) E. coli cells, refolded from 3

inclusion bodies by oxidative refolding, and purified by size exclusion. AviTag-DIII protein was 4

biotinylated and purified again by size exclusion. Serial dilutions of serum from RWN-infected 5

mice were applied to plates coated with recombinant WNV-E, DIII; or DIII-KT proteins. Bound 6

RWN-specific antibodies were detected with biotinylated goat anti-mouse IgM, IgG, or IgG2c 7

(Southern Biotech, Birmingham, AL), followed by streptavidin-conjugated horseradish peroxidase 8

(SA-HRP) and TMB substrate (both BD Bioscience, San Diego, CA). Anti-mouse Ig(H+L) 9

(Southern Biotech, Birmingham, AL) and serial dilutions of mouse IgM and IgG2c (Southern 10

Biotech, Birmingham, AL) were used for standards. High-affinity antibodies were measured 11

similarly using plates coated with recombinant DIII alone or diluted 1:3 with BSA. After the initial 12

binding, low affinity antibodies were washed off by incubating the samples for 15 min in presence 13

of increasing amounts of NaSCN before detection with biotinylated goat anti-mouse IgM or IgG, 14

followed by SA-HRP. 15

16

Virus neutralization. The assay to measure the ability of serum to neutralize WNV has been 17

described before (29). Briefly, 2000 pfu/ml RWN were incubated with serial dilutions of serum 18

from RWN-infected mice for 2.5 hrs at room temperature and subsequently used to infect Vero 19

cells. The number of infected cells was assessed 30-48 hrs later by staining for the expression of 20

WNV-E protein using an anti-WNV-E antibody (E16-biotin). Scored was the lowest dilution factor 21

of serum necessary to achieve a reduction of 90% of infected cells compared to cells infected 22

with WNV without prior serum incubation (PRNT90). 23

24



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Statistical Analysis. All experiments were performed independently three or more times. 1

Statistical significance was determined with a Mann-Whitney test using the Prism6 software 2

(GraphPad). Number of asterisks represents the extent of significance with respect to the p value. 3



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Results 1

2

Impaired neutralizing antibody response to RWN in RWN-infected MAVSKO mice. To dissect 3

the function of MAVS in the regulation of humoral immunity, we infected MAVSKO mice and 4

MAVSWT controls with a replication-incompetent mutant of WNV called WNV-RepliVAX (RWN) in 5

(23). This mutant lacks a functional capsid gene and thus fails to produce infectious progeny but 6

generates otherwise all viral proteins and RNA. RWN-infection of MAVSKO mice in the footpads 7

led to a WNV-E-specific IgM and IgG response similar to wild-type levels on day 8 post-infection 8

(Fig. 1A-B). However, sera from RWN-infected MAVSKO failed neutralize the virus effectively when 9

compared to MAVSWT mice (Fig. 1C). Importantly, the sera from MAVSKO mice also showed a 10

neutralization defect of WNV when the amounts of RWN-specific IgM and IgG Abs were taken 11

into account, as MAVSKO mice exhibited a significantly lower neutralization index than MAVSWT 12

mice (neutralization divided by amount of virus-specific IgM + IgG) (Fig 3D). This finding was also 13

true when the neutralization index was calculated based on the IgM or IgG titers alone 14

(Supplementary Fig. S1A-B). 15

Due to the repetitive nature of the envelope proteins on viral surfaces, many viruses can 16

elicit a combination of T-dependent or T-independent antibody responses. Indeed, the primary 17

antibody response to replicating WNV is initially independent of CD4+ T cells and becomes mainly 18

T-dependent by day 10. As the kinetics of the antibody response to RWN may differ from that to 19

WNV and MAVS is known to regulate components of the complement cascade, we wanted to 20

ascertain that the observed impairment of virus neutralization by sera from RWN-infected 21

MAVSKO mice on day 8 is due to a defect of the T-dependent antibody response itself. The 22

neutralization defect of sera from RWN-infected MAVSKO mice was complement-independent 23

(Supplementary Fig. 2A-B). Thus, we infected CD4 MHCIIKO and CD40KO mice as well as wild-24

type controls with RWN and measured the ability of the sera from these mice to neutralize the 25

virus. In contrast to sera from wild-type mice, sera from either MHCIIKO or CD40KO mice were 26



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completely devoid of any neutralizing activity to RWN (Supplementary Fig. 2C-D). Together, our 1

data show that viral neutralization by sera from RWN-infected mice depends on the generation of 2

T-dependent antibody responses and is unlikely to involve significant contributions of innate 3

effector mechanisms such as the production of anti-microbial peptides or altered complement 4

activation. Instead, MAVS regulates the quality of the anti-WNV antibody response. 5

6

RWN infection of MAVSKO mice leads causes an increase in viral RNAs but not antigens. 7

To test the nature of RWN infection in MAVSKO mice compared to MAVSWT mice, we infected 8

mice with RWN and measured first the presence of viral RNA encoding WNV-E in the whole 9

draining lymph nodes (dLNs, here: inguinal and popliteal LNs). We found that the dLNs of RWN-10

infected MAVSKO mice contained significantly more viral RNA than those from MAVSWT mice (Fig. 11

2A). As RWN is restricted to a single round of infection, we conclude that MAVSKO mice produce 12

more viral RNA per infected cell without expanding the number of infected cells. RLR-mediated 13

inhibition of protein translation during infection with RNA viruses is thought to occur independently 14

of MAVS and instead may be regulated by the innate signaling adaptor STING (30). We therefore 15

determined whether the elevated levels of viral RNA in MAVSKO mice also translate into more viral 16

proteins in the dLNs. We isolated cells from the dLNs from MAVSKO mice and MAVSWT controls 17

on day 1 post RWN-infection, cultured these cells for 24 hrs in vitro, and measured the production 18

of WNV-E during that time period in a flow cytometry-based bead assay. We used samples from 19

naïve mice or assay buffer as negative controls. Cells from both MAVSKO and MAVSWT mice 20

produced significant amounts of WNV-E compared to controls. However, we did not observe 21

significant differences in WNV-E production between MAVSKO and MAVSWT mice, even though 22

we noticed a modest trend towards higher WNV-E production in MAVSKO mice (Fig. 2B-C). 23

Together, our data show that RWN infection of MAVSKO mice leads to the expression of more 24

viral RNA compared to MAVSWT mice but does not significantly alter the levels of viral antigens. 25

We conclude that RWN infection of MAVSKO mice overcomes the caveats associated with 26



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replication-competent WNV, namely severe immune-pathology and abundance of viral antigens 1

due to the uncontrolled viral replication in these mice. 2

3

Antibody-mediated virus neutralization of RWN-infected mice is independent of antigen 4

loads. It is well-known that high antigen loads can affect the quality of the antibody responses 5

(31, 32). In contrast to infections with replicating WNV, MAVSKO mice infected with RWN do not 6

produce significantly higher amounts WNV-E protein in the dLNs than MAVSWT mice, suggesting 7

that uneven antigen loads are not major drivers of the observed neutralization defect in RWN-8

infected MAVSKO mice. Nonetheless, as we noticed a trend towards higher levels of WNV-E 9

expression in MAVSKO mice (Fig. 2B, C), we tested whether an increase of the infectious dose of 10

RWN can impact the anti-WNV neutralization index in MAVSWT mice. We first compared MAVSKO 11

mice infected with the standard dose of 105 Pfu per footpad to MAVSWT mice infected with a high 12

dose of 106 Pfu per footpad to ensure that the chosen increased dose for MAVSWT mice leads to 13

similar or higher levels of WNV-E protein in the dLNs. Cells from the dLNs of MAVSWT mice 14

infected with the high dose did indeed express equivalent amounts of WNV-E protein as MAVSKO 15

mice infected with the standard dose (Fig. 3A). Importantly, increasing the infectious dose in 16

MAVSWT mice did not impact neutralization efficiency of the antibody response as this did not 17

negatively impact the neutralization index (Fig. 3B). We thus conclude that the impact of MAVS 18

on the quality of the antibody response in RWN-infected animals is independent of the antigen 19

load. 20

21

Enhanced Tfh cell and GC B cell response to RWN in MAVSKO mice. In order of gain insights 22

into the potential drivers for the impaired neutralizing antibody response, we next characterized 23

the T and B cell response of RWN-infected MAVSKO mice on the cellular level. MAVSKO mice had 24

normal B cell numbers in the dLNs on day 8 after RWN infection (Fig. 4A). However, germinal 25

center (GC) B cells were more frequent in these mice (Fig. 4B). CD4+ T cell numbers were 26



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increased in the dLNs of MAVSKO mice (Fig. 4C). CD4+ T cells specific for the immuno-dominant 1

WNV epitope E641 were present in similar frequencies in MAVSKO and MAVSWT mice as shown 2

by staining with E641:I-Ab MHC class II tetramers (Fig. 4D). However, as the CD4+ T cell 3

compartment was enlarged in MAVSKO mice, these mice contained significantly more E641:I-Ab+ 4

CD4+ T cells in the dLNs than MAVSWT controls (Fig. 4D). Similarly, the frequency of CXCR5+ 5

PD-1+ Tfh cells did not change in MAVSKO mice (Fig. 4E) but Tfh numbers were increased (Fig. 6

4E). Together, our findings may imply that the qualitative defect of the antibody response to RWN 7

in MAVSKO mice is caused by an impaired recruitment of WNV-E-specific B cells with high affinity 8

into the response or their impaired selection into the plasma cell compartment, either because of 9

a defect that acts on B cells directly or a reduced selection pressure due to an increase in Tfh cell 10

number or function (33). 11

12

Altered cytokine production in the dLNs of MAVSKO mice. RLRs are major inducers of NF-13

kB-driven proinflammatory cytokines and type I and type III IFN responses. In fact, the induction 14

of IFNs in particular often depends on the activation of RLRs in infections with RNA viruses. As 15

such changes in the cytokine milieu may alter the generation of T or B cell responses in RWN-16

infected MAVSKO mice, we determined the expression of a selection of cytokines and IFNs is 17

altered in these mice. We measured the expression of cytokines, IFNs, and interferon-sensitive 18

genes (ISGs) in the whole dLNs of MAVSKO and MAVSWT mice 24 h after RWN-infection by qRT-19

PCR. We chose this time point because IFN responses peak early in viral infections and innate 20

instruction of CD4+ T cells and B cells is thought to occur at that time as well. Unexpectedly, RWN-21

infected MAVSKO mice expressed more IL-1β, IL-6, and IFN-l in the dLNs than MAVSWT controls, 22

whereas MAVSKO and MAVSWT mice expressed equal amounts of type I IFNs and TNF-a (Fig. 23

5A-B). Systemic type I IFNs in the serum of RWN-infected mice were only detectable by a type I 24

IFN-sensitive bioassay and were not significantly different between MAVSKO mice and MAVSWT 25



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controls, suggesting that IFNs mainly act locally in RWN-infected mice (Supplementary Fig. S3). 1

Finally, ISG expression was unchanged in the dLNs of MAVSKO mice (Fig. 2C). Proinflammatory 2

cytokines remained expressed at the same levels in MAVSKO and MAVSWT mice 48 h post 3

infection, while IFNs and ISGs were, as expected, downregulated (Supplementary Fig. S4). 4

Together, the data suggest that inflammatory mediators are not uniformly dysregulated in 5

MAVSKO mice and that the expression of specific cytokines known for their ability to promote Tfh 6

or B cell immunity such as IL-1β and IL-6 are is increased in these mice (34, 35). Of note, we did 7

not observe that specific cytokines, IFNs, or ISGs were downregulated in MAVSKO mice, 8

suggesting that the expression of these genes is driven by other PRRs than RLRs. 9

10

Antibodies specific for the neutralizing epitope in the WNV-E protein are efficiently 11

generated in MAVSKO mice. Alterations in the Tfh and B cell response of MAVSKO mice (Fig. 4) 12

suggested that the selection of WNV-E-specific B cells is affected in these mice. Although RWN-13

infected MAVSKO mice produce similar amounts of WNV-E-specific antibodies, they may 14

preferentially generate antibodies that are directed at non-neutralizing epitopes. The major 15

neutralizing epitope of WNV is located in the lateral ridge (LR) epitope of the domain III (DIII) of 16

WNV-E (24, 36, 37). Thus, we used recombinant DIII protein as antigen in ELISAs to test whether 17

the serum of MAVSKO and MAVSWT mice contain similar amounts of DIII-LR-specific antibodies. 18

We also measured the antibody titers specific for a mutant form of DIII with an altered LR epitope 19

(DIII-K307E/T330I) that abrogates the binding of DIII-LR-specific antibodies. We found that the 20

levels of both IgM and IgG specific for anti-DIII remained unchanged MAVSKO mice (Fig. 6A). The 21

same was true for anti-DIII-K307E/T330I antibodies in these mice (Fig. 6B). Consistent with 22

previous findings, the anti-DIII-K307E/T330I antibody titers were much lower than those specific 23

for wild-type DIII, suggesting that the majority of anti-DIII antibodies in both MAVSKO and MAVSWT 24

mice are directed against the neutralizing DIII-LR epitope (25). Importantly, the ratio of antibodies 25

bound to DIII versus DIII-K307E/T330I was also similar in MAVSKO and MAVSWT mice (Fig. 6A, 26



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B). A recalculation of the neutralization index (Fig. 1D) with the anti-DIII antibody titers reinforced 1

the notion that the neutralizing Ab response is compromised in MAVSKO mice (Supplementary 2

Fig. S5). Together, these findings demonstrate that MAVSKO mice produce antibodies against 3

WNV with similar specificities as MAVSWT mice and a lack of antibodies against the neutralizing 4

DIII-LR epitope is not responsible for the neutralizing defect of MAVSKO mice. 5

6

Decreased affinity of anti-DIII antibodies in MAVSKO mice. Given the unchanged specificity of 7

the antibody response in MAVSKO mice, we hypothesized that the defect in virus neutralization of 8

MAVSKO is caused by a lower avidity of the neutralizing antibodies in these mice. To test this, we 9

measured the avidity of the antibodies against the DIII domain by ELISA in the presence of 10

increasing amounts of NaSCN to enhance the stringency of antibody binding. For IgM, we also 11

reduced the binding avidity by diluting the recombinant DIII antigen with BSA. Consistent with 12

previous results (Fig. 1), we did not find any differences in the total anti-DIII IgM titers between 13

MAVSKO and MAVSWT mice (Fig. 7A). However, upon dilution of DIII with BSA and increasing 14

concentration of NaSCN, we observed a successive reduction of the anti-DIII IgM titers from sera 15

of RWN-infected MAVSKO mice while sera from MAVSWT mice retained their ability to bind to DIII 16

significantly better (Fig. 7A). The quantification of these results, in which we expressed the 17

amount of high avidity anti-DIII IgM as percentage of total anti-DIII in each mouse, confirmed this 18

result (Fig. 7B). In contrast to the anti-IgM response of MAVSKO mice, we did not observe any 19

differences in the anti-DIII IgG response of these mice compared to MAVSWT controls (Fig. 7C-20

D). The latter observation was consistent with the notion that the primary antibody response to 21

WNV is dominated by IgM whereas somatically mutated high-affinity IgG responses emerge late 22

in the primary response and are mainly required for protection from secondary challenges (38–23

40). Together, these results show that MAVS-deficiency results in a qualitatively inferior primary 24

antibody response due to a reduced affinity to the neutralizing LR epitope of WNV-E protein. In 25



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light of the defective IgM response, which consists mostly of unmutated antibodies, these findings 1

also imply that MAVS directly affects the recruitment of B cells into the immune response. 2



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Discussion 1

In contrast to the better-known functions of transmembrane PRRs, the mechanisms by 2

which cytosolic PRRs regulate adaptive immunity are still poorly understood. Previous work 3

suggested that the detection of viral RNA by RLRs and their signaling adaptor MAVS is an 4

important step in the control of adaptive immunity to pathogenic WNV (17, 18, 41). While these 5

results seemingly established a link between MAVS signaling and the generation of protective 6

adaptive immune response, it was difficult to attribute a specific role to MAVS in the regulation of 7

such responses as the unrestricted viral replication in the absence of MAVS-dependent innate 8

immune defenses results in strongly elevated levels of viral antigens and RNAs as well as severe 9

pathology and death (17). To overcome these complications, we have used a replication-10

incompetent mutant of a pathogenic WNV called RepliVAX-WN (RWN) to study the functions of 11

MAVS in anti-viral humoral immunity. 12

Although the RLR/MAVS signaling pathway is known to regulate the release of serum 13

components such as specific members of the complement cascade, we show here that the 14

MAVS-dependent effects on WNV neutralization depend on the presence of T-dependent 15

antibodies, consistent with the previously observed important contribution of T-dependent 16

antibodies to the overall humoral immune response to WNV (42, 43). MAVS-deficiency did not 17

alter the overall nature of the antibody response as both the choice of immunoglobulin isotypes 18

and, importantly, the choice of WNV-E epitopes remained the same. Instead, the lack of MAVS 19

reduced the overall affinity of the IgM response, the main isotype responsible for protection during 20

primary infection, to the neutralizing LR epitope of the DIII domain despite otherwise normal anti-21

DIII antibody titers (36, 37, 44–48). This feature is the likely cause for the impaired WNV 22

neutralization in MAVSKO mice, as antibody affinity defines the antibody occupancy rate on the 23

WNV virion and thus is a major factor that determines WNV neutralization (49). 24

High antigen loads and the associated abundant formation of immune complexes have 25



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been recognized since the early days of B cell immunology for their potential to impair the overall 1

affinity of the antibody response (31, 32). While such a scenario may contribute to the impaired 2

antibody response in the context of uncontrolled replication of pathogenic WNV, it is an unlikely 3

factor in infections of MAVSKO mice with RWN. Here, the replication-incompetent nature of this 4

WNV mutant results in a similar cellular tropism in mice as WT controls, which differs from WNV-5

infected mice with deficiency in type I IFN responses (50). Despite elevated levels of viral 6

transcripts in the dLNs, we observed only a modest increase of WNV-E protein production in 7

MAVSKO mice early in the infection. These findings are consistent with a recent study indicating 8

that the RIG-I/MDA5-mediated suppression of protein translation following infection with RNA 9

viruses depends on the signaling adaptor STING instead of MAVS (30). The mere increase in the 10

infectious dose did not alter the quality of the neutralizing antibody response. Thus, the defect of 11

the neutralizing antibody response in RWN-infected MAVSKO mice is not a consequence of a 12

fundamental change in the antigen load and instead is likely caused by an altered immune 13

regulation by cytokines or other signaling molecules in the absence of MAVS. Consistent with this 14

view, a recent study of MAVSKO mice infected with a replicating non-pathogenic strain of WNV did 15

not impair the quality of the antibody response, even though these mice exhibited increased viral 16

titers (51). 17

At the present time, it remains unclear how MAVS regulates the quality of the antibody 18

response. Although we did not resolve the cytokine profile of the individual cell populations in the 19

dLNs, our data nonetheless indicate that several cytokines are upregulated in RWN-infected 20

MAVSKO mice, presumably through the activity of other PRRs such as TLRs . One possibility may 21

be that the altered cytokine environment in the dLNs of RWN-infected MAVSKO mice directly 22

influences the ability of B cells to become activated. Indeed, both type I and type III IFNs can 23

promote B cell responses directly by facilitating B cell receptor activation or indirectly by regulating 24

the B cell-intrinsic activity of TLR7 and other PRRs (52–54). Such signals may facilitate the 25

activation of low affinity B cell that usually would be prevented from participating in the response. 26



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In this context, it is interesting that MAVS affects the quality of the neutralizing antibody response 1

to pathogenic strains of WNV, whereas it does not seem to play the same function in infections 2

with non-pathogenic strains (51). A key difference between these viral isolates is their divergent 3

interference with the IFN signaling pathway, further pointing towards a function of IFNs in the 4

regulation of the anti-WNV antibody response (55). 5

An additional possibility is that altered cytokine profiles may lead to the promotion of an 6

enhanced Tfh cell response. Indeed, we observed increased numbers of antigen-specific CD4+ T 7

cells as well as Tfh cells in RWN-infected MAVSKO mice. Here, the observed upregulation of IL-1 8

and IL-6 may help CD4+ T cells to overcome Treg-mediated suppression and promote their 9

differentiation into more effective Tfh cells (35, 56, 57). The consequence of this scenario may 10

therefore be a reduced competition of cognate B cells for Tfh cell help that leads to the entry of 11

low affinity B cells into the immune response. Such a checkpoint is usually recognized in the 12

context of a GC response. Here, the selection of high affinity B cells depends on their more 13

successful access to limited Tfh cell help and becomes less stringent when T cell help is abundant 14

(33, 58). However, a similar checkpoint is thought to occur already at the T-B border before 15

cognate B cells re-enter the B cell follicle (59). The reduced affinity of neutralizing IgM in MAVSKO 16

mice could therefore support the idea that MAVS already prevents the recruitment of low affinity 17

B cells into the anti-WNV immune response at this early stage, thus facilitating the production of 18

an antibody response with overall higher affinity to the neutralizing DIII-LR epitope. IgM is the 19

major isotype responsible for humoral immunity during primary WNV infection, whereas class-20

switched and somatically mutated IgG antibodies are thought to be more relevant for secondary 21

WNV infection (39). RLR ligands can promote increased immunogenicity and affinity maturation 22

of IgG responses in protein immunizations (60). Although we noted enlarged Tfh cell and GC B 23

cell compartments in MAVSKO mice, we did not observe a reduction of the affinity of DIII-LR-24

specific IgG antibodies in the mice titers in contrast to IgM. Such a lack of phenotype in the IgG 25

response may not be surprising because RWN-infected cells and MAVS-dependent signals 26



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20

disappear quickly from the draining lymph after infection in our experimental system. Nonetheless, 1

our findings provide a mandate to explore the regulation of GC responses by MAVS in more detail 2

using replicating pathogenic WNV strains. 3

Regardless of the specific circumstances that promote the production of low affinity 4

antibodies in RWN-infected MAVSKO mice, it is likely that this effect is caused by an absence of 5

MAVS in myeloid cells. This argument rests on the finding that myeloid cells are much more 6

frequently infected by WNV (50). This view is also attractive because myeloid cells and DCs in 7

particular orchestrate the early events of CD4+ T cell responses. Recent results with extracellular 8

bacterial infections demonstrate that phagocytosed microbial RNA can be sensed by a 9

combination of transmembrane PRRs such as TLR3 and cytosolic PRRs such as NLRP3 that 10

cooperate to induce IL-1 and type I IFN for the regulation of Tfh responses (8–10). Such regulatory 11

circuits have been shown to affect primarily the magnitude of the antibody response, whereas our 12

study demonstrates a role for the sensing of microbial RNA in the quality of the antibody response. 13

Nonetheless, these findings may therefore set a precedent for the general recognition of microbial 14

RNAs by phagocytes and provide a conceptual basis for the understanding of RLR signaling 15

pathway in the regulation of antibody responses. Future experiments will address the questions 16

about the origin and nature of the MAVS-dependent signals required for the regulation of the 17

antibody response to WNV. 18



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Acknowledgments 1 2

We thank Drs. Deepta Bhattacharya, Michael Kuhns, and Janko Nikolich-Žugich for critical 3

reagents, technical help, and overall comments and suggestions. This work was supported by the 4

Arizona Biomedical Research Foundation and the National Institutes of Health through 5

R56AI130044. 6



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Figure Legends 1

2

Figure 1. Impaired neutralizing antibody response in RWN-infected MAVSKO mice. (A, B) 3

WNV-E-specific IgM (A) and IgG (B) on day 8 after infection with RWN (105 pfu/footpad) as 4

measured by ELISA. (C) Virus neutralization by sera of infected MAVSKO and MAVSWT mice. 5

RWN was incubated with serial dilutions of sera prior to infection of Vero cells in vitro. The number 6

of infected cells was determined 30 h later by staining with an anti-WNV-E antibody. The reduction 7

of infected cells by 90% was scored (PRNT90). (D) Neutralization index on day 8 post infection. 8

The index normalizes virus neutralization to the total amount of WNV-E-specific antibodies in 9

MAVSKO and MAVSWT mice. The index was calculated by dividing the dilution factor (PRNT90) of 10

each mouse by the total amount of WNV-E-specific IgM and IgG of the same mouse. The data 11

were normalized across multiple experiments to MAVSWT mice. Each dot represents one mouse, 12

the lines represent the median. **, p <0.005; ***; p < 0.0005; n.s., not significant; Mann-Whitney 13

test. 14

15

Figure 2. Similar levels of WNV-E protein in RWN-infected MAVSKO and MAVSWT mice. (A) 16

Viral RNA levels in the dLNs on day 1 post infection with RWN (105 pfu/footpad) as measured by 17

qPCR using primer pairs located in the WNV-E or NS4b genes of the viral genome. Data were 18

normalized to the RNA level of RWN-infected MAVSWT mice. (B, C) Production of WNV-E protein 19

in cells from the dLNs of RWN-infected MAVSKO and MAVSWT mice. Cells from the dLNs were 20

isolated 24 hrs after infection and cultured for an additional 24 hrs in vitro. The amount of WNV-21

E protein in the combined cell lysates and supernatants was quantified by flow cytometry using 22

an anti-WNV-E bead assay. Samples from naïve mice or assay buffer were used as controls. (B) 23

A representative experiment is shown. Shaded area represents the background staining of 24

samples from uninfected animals, red lines represent RWN-infected animals. (C) Statistical 25

summary of geometric means of multiple independent experiments. The data were normalized to 26



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29

the background staining of uninfected mice in each experiment. Each dot represents one mouse; 1

the line is the median; n.s., not significant, ***, p < 0.0005, Mann-Whitney test. 2

3

Figure 3. Increased antigen production does not impair virus neutralization. (A) Comparison 4

of the viral load in the dLNs of MAVSKO mice infected with 105 Pfu RWN and MAVSWT mice 5

infected with an increased dose of 106 Pfu RWN. Cells from the dLNs were isolated 24 hrs after 6

infection and cultured for an additional 24 hrs in vitro. The amount of WNV-E protein in the 7

combined cell lysates and supernatants was quantified by flow cytometry using an anti-WNV-E 8

bead assay. (B) Increased doses of RWN infection do not impair virus neutralization in MAVSWT 9

mice. Mice were infected with indicated doses of RWN. The amount of WNV-E-specific IgM and 10

IgG as well as PRNT90 were determined in order to calculate the neutralization index. Shown are 11

the combined data of 3 experiments. Each dot represents one mouse, the line represents the 12

median. ns, not significant; Mann-Whitney test. 13

14

Figure 4. Enhanced GC B and CD4+ T cell response to RWN in MAVSKO mice. The cellularity 15

of the dLNs from MAVSKO and MAVSWT mice was analyzed 8 days post RWN infection (105 16

pfu/footpad) by flow cytometry. (A) Total numbers of CD19+ B cells. (B) Left panels: Frequency 17

of Germinal Center (GC) B cells. Right panel: Total GC B cell numbers of 5 experiments 18

normalized to the average of MAVSWT mice in each experiment. (C) Absolute number of CD4+ T 19

cells. (D) Left panels: Frequency of E641:I-Ab class II tetramer+ CD4+ T cells specific for the 20

immunodominant E641 epitope derived from WNV-E. Right panel: Total E641:I-Ab+ CD4+ T cells 21

numbers of 3 experiments normalized to the average of MAVSWT mice in each experiment. (E) 22

Left panels: Frequency of CXCR5+ PD-1+ T follicular helper (Tfh) cells. Right panel: Total Tfh cell 23

numbers of 5 experiments normalized to the average of MAVSWT mice in each experiment. 24

Frequencies are shown as mean ± SD. Cell numbers: Each dot is one mouse, lines are the 25

medians. *, p < 0.05; ***; p < 0.0005; n.s., not significant; Mann-Whitney test. 26



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Figure 5. Enhanced production of cytokines and interferon-stimulated genes (ISGs) in the 1

dLNs of MAVSKO mice. (A) Expression of IL-1β, IL-6, and TNF-α mRNA RWN-infected MAVSKO 2

and MAVSWT mice. (B) Expression of type I and type III IFNs mRNA in RWN-infected MAVSKO 3

and MAVSWT mice. (C) Expression of representative ISGs in RWN-infected MAVSKO and MAVSWT 4

mice. (A-C) mRNA was isolated from whole dLNs cells of mice 24h after infection with 105 Pfu 5

RWN per footpad and measured by qPCR. Shown is the expression over that of dLNs from naïve 6

WT mice. Expression of GAPDH was used to normalize the samples. Shown are the combined 7

data of 4 experiments using 8-12 mice/genotype **, p < 0.005; ***, p < 0.0005; Mann-Whitney 8

test. 9

10

Figure 6. Normal specificity of the antibody response of MAVSKO mice to the neutralizing 11

lateral ridge (LR) epitope in the WNV-E DIII domain. (A, B) The antibody response of RWN-12

infected MAVSKO and MAVSWT mice was measured 8 days post infection. Shown are the titers of 13

IgM (A) and IgG (B) specific for the WT DIII domain or the mutant form DIII-KT containing loss-14

of-function mutations in the neutralizing LR epitope (DIII-K307E/T330I). Ratios represent the 15

excess of antibodies directed at the neutralizing epitope over the amount of non-neutralizing 16

antibodies directed at the DIII domain. Combined data of 4 experiments. Each data point is one 17

mouse, the line is the median; n.s., not significant; Mann-Whitney test. 18

19

Figure 7. Impaired affinity of the IgM response to the DIII domain of WNV-E protein in RWN-20

infected MAVSKO mice. (A, B) DIII-specific IgM titers from RWN-infected mice were measured 21

by ELISA in the presence of BSA to reduce binding avidity and increasing amounts of NaSCN to 22

enhance stringency of binding. Recombinant DIII protein was used as antigen. Shown are the 23

absolute titers (A) and the titers as fraction of the total amount of DIII-specific antibodies in the 24

absence of BSA and NaSCN for each mouse (B). (C, D) Same as before for DIII-specific IgG. 25



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Shown are the combined data of 4 experiments. Each dot represents one mouse, the line is the 1

median. *, p < 0.05; ***, p < 0.0005; n.s., not significant; Mann-Whitney test. 2

3

4



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Supplementary Figures 1

2

Supplementary Figure S1. Defective neutralization by serum from RWN-infected MAVSKO 3

mice. (A-B) Neutralization index calculated based on serum-specific levels of WNV-ENV-specific 4

IgM (A) or IgG (B) titers on day 8 after infection. The data were normalized across multiple 5

experiments to WT mice. Each dot represents one mouse, the lines represent the median. **, p 6

<0.005; ****; p < 0.00005; n.s., not significant; Mann-Whitney test. 7

8

Supplementary Figure S2. Serum neutralization of RWN is mediated by a T-dependent 9

antibody response. (A) Virus neutralization before and after heat-inactivation (HI) to exclude 10

complement-mediated effects. Shown are the dilution factors that resulted in a 90% reduction of 11

infection of target cells with RWN in vitro (PRNT90) and the neutralization index that accounts for 12

the anti-WNV-E IgM and IgG titers in each mouse. (B) MHCIIKO and (C) CD40KO mice as well as 13

WT controls were infected with 105 Pfu of RWN in the footpads. Serum was collected 8 days later 14

to measure the dilution factor Shown are the combined data of two independent experiments. 15

Each dot is one mouse. *, p < 0.05; **, p < 0.005; ***, p < 0.0005; ****, p < 0.00005; Mann-Whitney 16

test. 17

18

Supplementary Figure S3. Normal production of systemic type I IFN in the serum of RWN-19

infected MAVSKO mice. Serially diluted serum samples from day 1 of RWN-infected mice were 20

used to protect L929 cells from the cytopathic effects (CPE) of vesicular stomatitis virus (VSV) in 21

vitro. Samples from mice immunized with OVA + LPS were used as positive controls. All samples 22

were compared to samples treated with increasing doses of recombinant IFN-α2 as standards. 23

Shown are the combined data from two experiments. Each dot represents one mouse. n. s., not 24

significant; Mann-Whitney test. 25

26



https://doi.org/10.1101/2019.12.15.875906


33

Supplementary Figure S4. Production of cytokines and interferon-stimulated genes (ISGs) 1

in the dLNs of MAVSKO mice on day 2 post RWN infection. (A) Expression of IL-1β, IL-6, and 2

TNF-α mRNA RWN-infected MAVSKO and WT mice. (B) Expression of type I and type III IFNs 3

mRNA in RWN-infected MAVSKO and WT mice. (C) Expression of representative ISGs in RWN-4

infected MAVSKO and WT mice. (A-C) mRNA was isolated from whole dLN cells of mice 24h after 5

infection with 105 Pfu RWN/footpad and measured by qPCR. Shown is the expression over that 6

of dLNs from naïve WT mice. Expression of GAPDH was used to normalize the samples. Shown 7

are the combined data of 4 experiments using 8-12 mice/genotype **, p <0.005; ***, p < 0.0005; 8

M-W test. 9

10

Supplementary Figure 5. Neutralization index for sera of RWN-infected MAVSKO mice based 11

on the titers of DIII-specific antibodies. (A) Virus neutralization with sera from MAVSKO and 12

MAVSKO mice. (B) Neutralization index for the same mice based on the DIII-specific IgM and IgG 13

titers (Dilution factor divided by the total amount of DIII-specific IgM and IgG). Each dot represents 14

one mouse, the line is the median. Shown are the combined data of two experiments. **, p <0.005; 15

****, p <0.00005; Mann-Whitney test. 16



https://doi.org/10.1101/2019.12.15.875906


ng/m

lanti-WNV-E IgM

**

WT MAVSKO0.1

110

1001000

10000

ng/m

l

anti-WNV-E IgGn.s.

WT MAVSKO

A B

Neutralization index

WT MAVSKONor

mal

ized

to W

T

DC

Seru

m d

ilutio

n

Neutralization

***

WT MAVSKO

Figure 1

110

1001000

10000100000

0

5000

10000

15000

0.0

0.5

1.0

1.5***



https://doi.org/10.1101/2019.12.15.875906


WNV-E

WT MAVSKO

Control InfectedWT MAVSKOControl

ns.****

rela

tive

WN

V-E

expr

essi

on

[Geo

Mea

n]

B C

WNV-E NS4b

20

40

60

020406080

100 *** ***A

WT MAVSKO WT MAVSKO

Fold

ove

r WT

Fold

ove

r WT

0

Figure 2

**

10 1 10 2 10 3 10 4 10 5

0

20

40

60

80

100

10 1 10 2 10 3 10 4 10 5

0

20

40

60

80

100

0

1

2

3



https://doi.org/10.1101/2019.12.15.875906


ns.ns.

1 x 105 1 x 1065 x 105Pfu

A B

Figure 3

0

1

2

3

WTMAVSKO

1 x 105 1 x 106Pfu

ns.

neut

raliz

atio

n in

dex

rela

tive

expr

essi

on

0

1

2

3

4



https://doi.org/10.1101/2019.12.15.875906


PNA

Fas

WT MAVSKO

WT MAVSKO

***

gated on CD19+

B

WT MAVSKO05

10152025

CD19+ Cells

[x 1

06]

n.s.

A

0.19 ± 0.04 % 0.24 ± 0.07 %

E64

1:I-A

b -P

E

E641:I-Ab-APC

WT MAVSKO

WT MAVSKOWT MAVSKO0

10

20

30 *

gated on CD4+ DUMP-

CD4+ CellsC D

WT MAVSKO

PD

-1

CXCR5gated on CD4+ DUMP-

3.8 ± 2.24% 3.3 ± 1.2 %

0

1

2

3

4

[fold

incr

ease

]Normalized

Tfh Cell numbers

WT MAVSKO

E***

[x 1

06]

1.7 ± 0.8% 2.9 ± 1.1 %

0

1

2

3

[fold

incr

ease

][fo

ld in

crea

se]

NormalizedGC B cell numbers

0

1

2

3*

Normalized E641:I-Ab+ CD4+ T cell numbers

Figure 4



https://doi.org/10.1101/2019.12.15.875906


Figure 5

WT MAVSKO

IL-1βFo

ld o

ver n

aive ***

WT MAVSKO

IL-6

Fold

ove

r nai

ve ***

WT MAVSKO

TNF-α

Fold

ove

r nai

ve

ns.

WT MAVSKO

IFN-βFo

ld o

ver n

aive

ns.

WT MAVSKO

IFN-α4

Fold

ove

r nai

ve ns.

WT MAVSKO

IFN-λ2

Fold

ove

r nai

ve **

WT MAVSKO

IFN-λ3

Fold

ove

r nai

ve

***

A

B

C

Fold

ove

r nai

ve

WT MAVSKO

IFIT2 ns.

050

100150200

Fold

ove

r nai

ve

WT MAVSKO

IFIT1ns.

0

100

200

300

Fold

ove

r nai

ve

WT MAVSKO

ISG15ns.

050

100150200250

Fold

ove

r nai

ve

WT MAVSKO

Viperinns.

0200400600800

1000

010203040

050

100150200

01234

05

101520

05

10152025

0

50

100

150

020406080



https://doi.org/10.1101/2019.12.15.875906


A

B

1

10

100

1000

10000

1

10

100

1000

10000

MAVSKOWT

MAVSKOWT

αDIII-WT IgM

αDIII-WT IgG

ng/m

lng

/ml

1

10

100

1000

10000

1

10

100

1000

10000

MAVSKOWT

MAVSKOWT

αDIII-KT IgM

αDIII-KT IgG

ng/m

lng

/ml

20

40

60

0

02

864

10

MAVSKOWT

MAVSKOWT

αDIII-WT / αDIII-KT IgM

αDIII-WT / αDIII-KT IgG

ratio

ratio

n.s. n.s. n.s.

n.s. n.s. n.s.

Figure 6



https://doi.org/10.1101/2019.12.15.875906


200 m

M

200 m

M

400 m

M

400 m

M

600 m

M

600 m

M

1000

mM

1000

mM

1

10

100

1000Ig

M [n

g/m

l]ns. *** *** *** *** ***

DIII 1:3 BSADIII

NaSCN

0.0

0.2

0.4

0.6

0.8

% Ig

M D

III b

indi

ng

ns. * * * *

0 mM

0 mM

200 m

M

200 m

M

400 m

M

400 m

M

600 m

M

600 m

M

1000

mM

1000

mM

DIII 1:3 BSA

0 mM

0 mM

0 mM

0 mM

NaSCN

A B

WT MAVSKO

1

10

100

1000

10000

IgG

[ng/

ml]

200 m

M

200 m

M

400 m

M

400 m

M

600 m

M

600 m

M

1000

mM

1000

mM

NaSCN 0 mM

0 mM

ns. ns. ns. ns. ns.

0.0

0.2

0.4

0.6

0.8

200 m

M

200 m

M

400 m

M

400 m

M

600 m

M

600 m

M

1000

mM

1000

mM

NaSCN

% Ig

G D

III b

indi

ng

ns. ns. ns.*C D

Figure 7



https://doi.org/10.1101/2019.12.15.875906


0

1

2

3

4

0

1

2

3 ******

Nor

mal

ized

to W

T

Neutralization index based on WNV-E-specific IgM

Neutralization index based on WNV-E-specific IgG

WT MAVSKO WT MAVSKO

Nor

mal

ized

to W

T

A B

Supplementary Figure S1



https://doi.org/10.1101/2019.12.15.875906


0

2000

4000

6000

8000Se

rum

dilu

tion

Neutralization before HI

01000200030004000500060007000

Seru

m d

ilutio

n

Neutralization after HI

0

1

2

3

4

Nor

mal

ized

to W

T

Neutralization Index before HI

0.0

0.5

1.0

1.5

2.0

Nor

mal

ized

to W

T

Neutralization Index after HI

WT MAVSKO WT MAVSKO

WT MAVSKO WT MAVSKO

** **

***

A

Seru

m d

ilutio

n

Neutralization

0

1000

2000

3000

4000

WT MHCIIKO

****

B

Seru

m d

ilutio

n

***

C

WT CD40KO0

1000

2000

3000

4000

Neutralization




https://doi.org/10.1101/2019.12.15.875906


0

500

1000

1500

U/m

l IFN

a2

WT MAVSKOWTWT

RWNOVA+LPS

Naive

ns.




https://doi.org/10.1101/2019.12.15.875906


0

5

10

15

20

0100200300400500

0.0

0.5

1.0

1.5

02468

10

0

1

2

3

4

0

10

20

30

0

20

60

40

0.0

0.5

1.0

1.5

0

5

10

15

20

10

0

20

30

40

10

0

20

30

40

WT MAVSKO WT MAVSKO WT MAVSKO

WT MAVSKO WT MAVSKO WT MAVSKO

WT MAVSKO WT MAVSKO WT MAVSKO WT MAVSKO

WT MAVSKO

IFN-βIFN-α4 IFN-λ2 IFN-λ3

IL-1β IL-6 TNF-α

IFIT2 IFIT1 ISG15 Viperin

ns.ns. ns.

ns.ns.ns.ns.

ns. ns.**

A

B

C

Fold

ove

r nai

veFo

ld o

ver n

aive

Fold

ove

r nai

ve

Fold

ove

r nai

veFo

ld o

ver n

aive

Fold

ove

r nai

ve

Fold

ove

r nai

veFo

ld o

ver n

aive

Fold

ove

r nai

ve

Fold

ove

r nai

veFo

ld o

ver n

aive




https://doi.org/10.1101/2019.12.15.875906


**

02000400060008000

1000012000

Seru

m d

ilutio

n

0.000.250.500.751.001.251.501.75

Nor

mal

ized

to W

T

WT MAVSKOWT MAVSKO

****A B




https://doi.org/10.1101/2019.12.15.875906


MAVS regulates the quality of the antibody response to ... · 12/15/2019 · 2 1 Abstract 2 A key difference that distinguishes viral infections from protein immunizations is the

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