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Broadly reactive influenza antibodies are not limited by germinal center competition with high affinity antibodies

Enhancing the generation of broadly reactive influenza antibodies is a pertinent goal towards developing a universal influenza vaccine. While antibodies that bind conserved influenza epitopes have been identified in humans, the frequency of these antibodies is typically very low. The predominant theory is that antibodies specific for conserved influenza epitopes are limited in germinal centers by competition with high affinity antibodies specific for the variable region of the virus. Here, we show that reducing germinal center formation and removing competition with high affinity antibodies was not sufficient to increase broadly reactive influenza antibodies or enhance protection against distinct influenza subtypes. These data disprove the prevailing hypothesis that broadly reactive influenza antibodies are rare due to competition in germinal centers. Additionally, levels of IgM antibodies specific for the variable region of HA persisted in mice in the absence of germinal centers, further demonstrating that immunodominance can be established independent of germinal centers. Our data also highlight the protective capacity of germinal center-independent IgM antibodies, which are not typically considered when testing correlates of protection, and offer an alternate target for delivering a universal influenza vaccine. IMPORTANCE It is estimated that 250,000 – 650,000 individuals worldwide die each year from seasonal influenza infections. Current vaccines provide little protection against newly emerging strains. Thus, considerable effort is focused on enhancing the generation of broadly reactive influenza antibodies in order to develop a universal influenza vaccine. However, broadly reactive antibodies are rare and the factors that limit their generation are not completely understood. Our data disprove the prevailing hypothesis that broadly reactive influenza antibodies are rare due to completion in the germinal centers with antibodies to the variable HA head. Understanding the factors limiting antibodies specific for conserved regions of the influenza virus is imperative for developing a universal vaccine, which could potentially circumvent a global pandemic

is a pertinent goal towards developing a universal influenza vaccine. While 25 antibodies that bind conserved influenza epitopes have been identified in 26 humans, the frequency of these antibodies is typically very low. The predominant 27 theory is that antibodies specific for conserved influenza epitopes are limited in 28 germinal centers by competition with high affinity antibodies specific for the 29 variable region of the virus. Here, we show that reducing germinal center 30 formation and removing competition with high affinity antibodies was not 31 sufficient to increase broadly reactive influenza antibodies or enhance protection 32 against distinct influenza subtypes. These data disprove the prevailing 33 hypothesis that broadly reactive influenza antibodies are rare due to competition 34 in germinal centers. Additionally, levels of IgM antibodies specific for the variable 35 region of HA persisted in mice in the absence of germinal centers, further 36 demonstrating that immunodominance can be established independent of 37 germinal centers. Our data also highlight the protective capacity of germinal 38 center-independent IgM antibodies, which are not typically considered when 39 testing correlates of protection, and offer an alternate target for delivering a 40 universal influenza vaccine. 41 IMPORTANCE: It is estimated that 250,000 -650,000 individuals worldwide die 42 each year from seasonal influenza infections. Current vaccines provide little 43 protection against newly emerging strains. Thus, considerable effort is focused 44 on enhancing the generation of broadly reactive influenza antibodies in order to 45 develop a universal influenza vaccine. However, broadly reactive antibodies are 46 rare and the factors that limit their generation are not completely understood. Our 47 data disprove the prevailing hypothesis that broadly reactive influenza antibodies 48 are rare due to completion in the germinal centers with antibodies to the variable 49

Introduction
Seasonal influenza vaccines do not effectively protect against novel strains that 55 emerge each year. Consequently, there is considerable interest in developing a 56 universal influenza vaccine that induces immunity to epitopes conserved across 57 different virus subtypes, thereby providing long-lasting, heterosubtypic protection 58 against multiple influenza strains. The antibody response to current influenza 59 vaccines is dominated by antibodies specific for the globular head domain of the 60 surface protein, hemagglutinin (HA) (1). Antibodies targeting the HA head 61 neutralize the virus by preventing the virus from binding host epithelial cells. To 62 escape immune detection, the influenza virus mutates key residues of the HA 63 head region, which frequently gives rise to novel strains. Thus, the most effective 64 antibodies generated by current vaccines are specific for the most variable region 65 of the virus, and therefore only provide strain-specific protection. In addition to 66 the variable HA head, broadly reactive influenza antibodies specific for 67 conserved epitopes of influenza, including those in the membrane proximal stalk 68 region of HA, have been identified in humans (2-5). Broadly reactive influenza 69 antibodies have the capacity to protect against infection with multiple influenza 70 subtypes and therefore, form the basis of a universal vaccine. However, these 71 'broad spectrum' B cell clones are extremely rare and consequently, do not 72 contribute significantly to the antibody response following vaccination (1, 6-9). 73 Indeed, it is estimated that antibodies specific for the HA head are 1000-fold 74 more prevalent than antibodies targeting conserved epitopes (10). A better 75 understanding of factors limiting antibodies specific for conserved regions of the 76 influenza virus is critical for developing a universal vaccine, which could 77 potentially circumvent a global pandemic. the draining lymph nodes or the spleen. Influenza-specific B cells are activated 80 and can differentiate into short-lived plasma cells, memory B cells, or seed 81 germinal centers (11). B cells with the highest avidity for antigen are selected to 82 develop in germinal centers through a series of interactions with T follicular 83 helper cells (T FH ), which provide essential factors that promote proliferation and 84 further hypermutation of B cells. As T FH are limiting, BCRs with the highest 85 affinity have a competitive advantage over lower affinity BCRs (11). Following 86 affinity maturation in the germinal center, B cells can differentiate into antibody-87

secreting plasma cells or memory B cells. 88
Although it is well established that the B cell response following influenza 89 infection or vaccination is heavily biased toward epitopes in the variable head 90 region of HA relative to conserved HA stalk epitopes, the mechanisms that 91 mediate this immunodominance are not completely understood (12). Several 92 factors contribute to the scarcity of broadly reactive antibodies, including steric 93 hindrance of conserved epitopes, antigen quantity, naïve B cell precursor 94 frequency, B cell receptor avidity, and immunization route (11)(12)(13)(14). Furthermore, 95 some antibodies specific for conserved epitopes on the HA stem exhibit 96 polyreactivity, which may promote deletion or anergy of B cells generating these 97 antibodies to circumvent autoimmunity (15,16). The prevailing hypothesis for the 98 paucity of broadly reactive influenza antibodies is that antibodies specific for the 99 conserved epitopes are out-competed in germinal centers by high avidity 100 antibodies specific for the HA head region (11,12). This is supported by the 101 rapamycin treatment. 158 To determine the impact of reducing germinal center formation on the responding 159 antibody response, we analyzed sera for HKx31-specific IgG and IgM, 28 days 160 following infection. Mice given anti-CD40L antibody had reduced HKx31-specific 161 IgG, but HKx31-specific IgM levels were maintained compared to isotype controls 162 ( Fig. 1, E and F). Similarly, in Bcl6 CD4 mice, HKx31-specific IgG, but not IgM, was 163 reduced compared to wildtype controls (Fig. 1, G and H). These data are 164 consistent with previous findings that long-lived IgM, but not IgG, plasma cells 165 develop in the absence of germinal center formation (24). Interestingly, although 166 germinal centers were reduced in rapamycin-treated mice compared to controls, 167 the amount of HKx31-specific IgG was similar between rapamycin and PBS-168 treated mice (Fig. 1, E and G). Moreover, as we previously reported, rapamycin-169 treated mice had more HKx31-specific IgM compared to control mice. However, 170 the increase in HKx31-specific IgM was not observed in Bcl6 CD4 mice or mice 171 treated with anti-CD40L compared to controls (Fig. 1, F and H). It is intriguing 172 that germinal centers were reduced comparably in rapamycin-treated, anti-173 CD40L-treated, and Bcl6 CD4 mice, yet only the rapamycin-treated mice displayed 174 increased levels of HKx31-specific IgM, without a reduction in IgG. These data 175 suggest that the increased HKx31-specific IgM in rapamycin-treated mice is not 176 simply due to a reduction in germinal centers and reduced class switching to IgG,177 implying that rapamycin impacts the generation of influenza-specific IgM by a 178 different mechanism. Although germinal centers were typically regarded as the 179 major site of antibody isotype switching, several groups demonstrated 180 extrafollicular isotype switching (25-28). In fact, Roco et al., recently reported 181 that the majority of isotype switching occurs prior to germinal center formation 182 not in rapamycin-treated mice suggests that anti-CD40L signals by T FH are 184 required for extrafollicular isotype switching, and that rapamycin is modulating a 185 distinct signaling pathway. Together, these results indicate effective methods to 186 limit germinal center formation following influenza infection, and interestingly, 187 these methods have distinct impacts on the antibody response. As germinal 188 center-independent IgM memory cells can be long-lived, these cells may be good 189 candidates to target for vaccine development (24,(30)(31)(32)(33). 190

Germinal center inhibition decreases high avidity H3-specific IgG 191 antibodies 192
To determine whether the synthesis of high avidity antibodies specific for HA was 193 impaired in mice with fewer germinal centers, we analyzed sera from mice 194 harvested 28 days after HKx31 infection by an H3 HA ELISA with increasing 195 concentrations of urea. Similar to our observations with whole virus, H3-specific 196 IgG levels were reduced in anti-CD40L-treated and Bcl6 CD4 mice compared to 197 controls (Fig. 2, A and B), while H3-specific IgM levels were maintained (Fig. 2,C 198 and D). This suggests that the immunodominance of the HA epitope in shaping 199 the IgM repertoire can be established independent of germinal center 200 competition. Furthermore, in both models, rapamycin-treated mice had more H3-201 specific IgM relative to PBS-treated control mice (Fig. 2, C and D). As expected, 202 the avidity of H3-specific IgG antibodies produced in the anti-CD40L-treated mice 203 was significantly reduced compared to isotype control-treated mice, with a similar 204 trend observed in Bcl6 CD4 mice relative to wildtype controls (Fig. 2, E and F). 205 These findings are consistent with the established theory that germinal centers 206 are the main site of antigen-driven affinity maturation (11). Interestingly, despite a 207 reduction of germinal centers in mice treated with rapamycin, the level of IgG 208 trend in lower amounts of high avidity H3-specific IgG antibodies relative to PBS-210 treated controls (Fig. 2, E and F). These data demonstrate that class switching to 211 IgG occurs outside germinal centers, and further highlight the role of germinal 212 centers in fine-tuning the avidity of IgG to specific epitopes. In contrast to the IgG 213 antibodies, the absence of germinal centers did not reduce the avidity of H3-214 specific IgM compared to controls, except for rapamycin-treated Bcl6 CD4 mice 215 these data indicate that inhibiting germinal center formation, via anti-CD40L or in 219 Bcl6 CD4 mice, effectively reduced the prevalence of high avidity influenza-specific 220 IgG antibodies. 221

Reducing high avidity H3-specific IgG antibodies does not promote broadly 222 reactive influenza antibodies 223
To determine whether reducing germinal center formation and the frequency of 224 high avidity H3-specific IgG antibodies gives rise to an increase in broadly 225 reactive influenza antibodies, we tested sera from mice infected with HKx31 226 (H3N2) for antibodies specific for an H5 HA protein from A/Vietnam/1203/04 227 (Vn1203) or a recombinant H5N1 virus that contains H5 and N1 from Vn1203 in 228 combination with the same internal genes as HKx31 (ΔVn1203). The frequency 229 of broadly reactive, H5-specific IgG antibodies was negligible for all groups of 230 mice (Fig. 3, A and B). IgG antibodies specific for the whole ΔVn1203 virus were 231 detectable in HKx31-infected mice; however, the reduction in high avidity anti-H3 232 IgG antibodies in mice treated with anti-CD40L did not lead to an increase in 233 broadly reactive IgG antibodies compared to control mice (Fig. 3C). Likewise, wildtype mice (Fig. 3D). Not surprisingly, given the diminished opportunity for 236 germinal center affinity maturation, the avidity of the cross-reactive, ΔVn1203-237 specific IgG antibodies was reduced in anti-CD40L-treated and Bcl6 CD4 mice 238 compared to control mice (Fig. 3, D and F). Although all groups of mice lacked 239 IgG antibodies specific for H5, all HKx31-infected mice had an increase in H5-240 specific IgM antibodies compared to naïve mice (NMS) (Fig. 3, G and I). rapamycin-treated Bcl6 CD4 mice that had a reduction in high avidity H3-specific 249 IgM antibodies (Fig. 2, G and H) also showed an increase in high avidity 250 ΔVn1203-specific IgM antibodies (Fig. 3N). Together, these results indicate that 251 limiting the formation of the high avidity H3-specific IgG antibodies by reducing 252 germinal center formation is not sufficient to increase the prevalence of broadly 253 reactive influenza antibodies. Furthermore, rapamycin alters the antibody 254 repertoire via a mechanism independent of germinal center reduction, and this 255 impact is enhanced in Bcl6 CD4 mice that lack T FH. 256

Limiting high avidity antibodies in intact germinal centers is not sufficient 257 to boost broadly reactive influenza antibodies 258
Our study highlights that influenza-specific IgM and IgG antibodies can be 259 maintained independent of germinal centers. It is possible that germinal center-260 outside of the germinal center. Additionally, within germinal centers, broader IgM 262 repertoires may develop in the absence of high avidity antibodies. Therefore, we 263 sought to limit high avidity antibodies while keeping the germinal centers intact. 264 To this end, we infected Aicda -/mice, which cannot undergo somatic 265 hypermutation or isotype class switching, and therefore cannot produce IgG or 266 high avidity antibodies in the germinal centers (34). The Aicda -/and wildtype 267 C57BL/6 mice were infected i.p. with HKx31, and antibodies in the sera were 268 analyzed 28 days later. As expected, Aicda -/mice did not have any HKx31-269 specific IgG following infection, in both rapamycin and PBS-treated mice (Fig. 270 4A). In addition, the level of HKx31-specific IgM increased in Aicda -/mice treated 271 with rapamycin or PBS compared to wildtype mice, which likely reflects 272 accumulation of IgM due to a block in class switching to IgG (Fig. 4B). Similar to 273 anti-CD40L-treated or Bcl6 CD4 mice lacking germinal centers, Aicda -/mice that 274 lack high avidity IgG did not have an increase in broadly reactive IgM antibodies 275 relative to wildtype mice ( Fig. 4C), suggesting that removal of the high avidity IgG 276 antibodies was not sufficient to increase broadly reactive antibodies. 277 Remarkably, Aicda -/mice treated with rapamycin had the highest levels of 278 ΔVn1203-specific IgM antibodies and the highest avidity antibodies compared to 279 all other groups of mice ( Fig. 4D), which was similar to rapamycin-treated Bcl6 CD4 280 mice. Together, these data confirm that removing high avidity H3-specific 281 antibodies is not sufficient to promote the generation of broadly reactive influenza 282 antibodies. Additionally, rapamycin enhances the generation of broadly reactive 283 antibodies via a mechanism other than removing competition from high avidity 284 antibodies.

subsequent heterosubtypic influenza infections 287
Our previous work demonstrated that rapamycin enhanced protection to 288 heterosubtypic influenza infections when given during vaccination with an H3N2 289 virus (18). This protection was B cell-dependent and transferred to naïve mice via 290 serum, indicating that rapamycin enhanced protection by altering the antibody 291 response. When analyzing the antibody response to the whole virus, there is only 292 a modest increase in the levels of ΔVn1203-specific IgM antibodies in mice 293 treated with rapamycin compared to control mice (Fig. 3). However, we 294 previously demonstrated that rapamycin altered the specificities of both the IgG 295 and IgM antibodies generated during infection, and there were significant 296 differences in antibodies specific for particular epitopes in the rapamycin and 297 PBS-treated mice (18). Thus, although mice that lack high avidity IgG antibodies 298 via germinal center or Aicda deletion did not have an increase in the levels of 299 broadly reactive influenza antibodies, altered antibody specificities may enhance 300 heterosubtypic protection. Therefore, we tested whether restricting the production 301 of high avidity IgG antibodies in mice infected with an H3N2 virus impacted 302 survival following a secondary challenge with an H5N1 virus (ΔVn1203). Mice 303 were infected i.p. with HKx31 and treated with either rapamycin or PBS as a 304 control for 28 days. The following day, mice were given an intranasal challenge 305 with ΔVn1203 and monitored for survival and weight loss. As we reported 306 previously, rapamycin treatment significantly increased survival compared to 307 PBS-treated controls (Fig. 5). However, germinal center reduction via anti-CD40L 308 treatment or in Bcl6 CD4 mice was not sufficient to enhance protection against a 309 heterosubtypic virus (Fig. 5

, A-D). Likewise, deletion of high avidity antibodies in 310
Aicda -/mice did not enhance protection following an H5N1 infection (Fig. 5, E  311 and F). These data indicate that simply reducing germinal center formation or immunity. Notably, the mice that received anti-CD40L and Bcl6 CD4 mice treated 314 with rapamycin were protected from ΔVn1203 infection at levels similar to 315 rapamycin-treated wildtype mice even though these mice had minimal HKx31-316 specific or cross-reactive IgG antibodies. This suggests that rapamycin enhances 317 heterosubtypic immunity in a manner independent of germinal center formation 318 and high avidity IgG antibodies. Furthermore, the fact that wildtype, anti-CD40L-319 treated, and Bcl6 CD4 mice given rapamycin had higher levels of HKx31-specific 320 IgM than the PBS-treated cohorts, which was also observed for cross-reactive 321 IgM antibodies, supports the notion that immunity to conserved portions of 322 influenza virus may be enhanced by increasing broadly reactive IgM antibodies. 323 The protective role of IgM antibodies in rapamycin-treated mice is most evident 324 by the survival of Aicda -/mice treated with rapamycin, which completely lack IgG 325 antibodies. Further studies to assess the protective capacity of rapamycin 326 through germinal center-independent IgM antibodies may provide valuable 327 insight into enhancing immunity to conserved influenza epitopes. Together, our 328 data show that reducing germinal center formation and limiting the prevalence of 329 high avidity antibodies is not sufficient to enhance heterosubtypic immunity to 330 influenza viruses. 331 In contrast to the general dogma that broadly reactive antibodies are rare 332 due to favored selection of high avidity antibodies in germinal centers, our data 333 demonstrate that restricting development of high avidity IgG antibodies is not 334 sufficient to increase the prevalence of broadly reactive influenza antibodies. The 335 fact that antibodies specific for the conserved regions of influenza did not 336 develop in the absence of high avidity antibodies implies that other factors limit 337 the development of broadly reactive antibodies. The precursor frequency of the variable region, indicating that precursor frequency does not limit broadly 340 reactive antibodies (17). Moreover, in the absence of the HA variable region, 341 antibodies specific for the conserved stalk are generated, demonstrating the 342 immunogenic potential of these epitopes (1, 17

Virus and Infections 369
The HKx31 and ΔVn1203 viruses were constructed using the eight-plasmid 370 reverse genetics system (44)

H3-, H5-, and Vn1203-Specific ELISA with Urea 401
Microtiter plates (Nunc) were coated with H3 or H5 protein at 35ng/well for IgG 402 assays or 70ng/well for IgM assays or lysed whole Vn1203 diluted in PBS at 403 10ng/well for IgG assays, or 20ng/well for IgM assays, overnight at 4 o C. Plates The reaction was stopped via addition of 1N sulfuric acid. Absorbances were 415 measured at 450 and 550nm in a microplate reader (Molecular Devices). 416 Background signal (550nm) was subtracted from Absorbance (OD450) values to 417 give the relative OD. An avidity index (AI) value was calculated for sera using 418 either 2M or 3M of urea, in accordance with the rate of antigen-antibody 419 destabilization, and compared to no urea, or diluent alone 420 (AbsUREA/AbsDiluent) (45). 421

Fig. 2. Germinal center inhibition decreases high avidity H3-specific IgG 644
antibodies. Sera from anti-CD40L or isotype-treated mice were taken 28 days 645 after immunization with HKx31 and analyzed by ELISA for (A) H3-specific IgG or 646 (C) H3-specific IgM. Sera from wildtype or Bcl6 CD4 mice were taken 28 days after Mean ± SEM of n = 9 to 11. NMS and ΔVn1203-infected control n = 3 to 6. 675