Single-dose avian influenza A(H5N1) Clade 2.3.4.4b hemagglutinin–Matrix-M nanoparticle vaccine induces neutralizing responses in nonhuman primates

1 Single- dos e a vian influe nz a A(H5N1 ) Clade 2.3.4. 4b he ma g glutinin – Ma trix -M n anopa rticle v accine induc es n eutr alizing r e spon s es in nonh um an pri ma tes Nit a P at el 1 , Asma R ehman 1 , J essic a F . T r o s t 1 , Rhonda Fl or es 1 , Zach Long acr e 1 , Mim i Guebre-Xabier 1 , Haixia Zhou 1 , Bin Zhou 1 , K elsey Jac obson 1 , Desheng Jiang 1 , Xiaoyun Bai 1 , Rafia Khatoon 1 , Thomas K ort 1 , Jim Nor t on 1 , M . Madh angi 1 , Melinda H ersey 1 , Ann M. Green e 1 , Filip Dubovsky 1 , Gale Smith 1* 1 N o vavax , Inc ., Gai the r sbur g , MD 20878, USA * Corr esponding Author : Gale Smith, g s m i t h @ n o vavax . co m Author Emails: npat el@nov a v ax.com , arehman@nov a v ax.c om , rflor es@nov a v ax. c om , j t ro s t @ n o vavax . co m , zlong acr e@nov a v ax.com , mguebre-x abie r@no vavax . co m , hzhou@no vava x . co m , bzhou@no vavax . co m , k j a co b s o n @ n o vavax . c o m , d j i a n g @ n o vavax . co m , x b a i @ n o vavax . co m , tk o rt@nov av a x. c om, jnort on@nov a v ax. c om, maddy em [email protected] om , m h e rs e y @ n o vavax . co m , a g re e n e @ n o vavax . co m , f dubovsky@no vavax . co m , g s m i t h @ n o vavax . co m W ord c ou n t: 5021 (<5000, e x cludi ng methods, abs tr ac t, re f erences, figur e l eg ends ) Abs tr act wor d count: 185/200 w or ds R e f e r enc es: 47/70 Title: 16/15 w or ds .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 November 21, 2024. ; https://doi.org/10.1101/2024.11.21.624712doi: bioRxiv preprint 2 ABS TR A CT With th e r ec ent rise in c as es of highly pa t hog enic a vian influenz a (HP AI) A(H5N1) clade 2.3.4 .4b inf ecti on in humans and animals, th ere is an associat ed increase in t he risk of human-t o-hu man tr ansmission. In this s tudy , w e char ac t e riz e rec ombin ant A(H5N1) A/ Americ an Wi g eon/Sou th Car o lina/22/000345- 001/2021 (A/ A W /SC/2021 ) clade 2.3.4.4 b v accine. Purified r e c ombina n t A/ A W/S C/2021 HA trimer s upon f ormula tion with M atrix-M™ adjuv an t , sa ponin-cholest e r ol-phospholipi d ic osah ed r al particl es, non- c ov alently anchored to the vertic es of th e Matrix-M f orming A(H5N1) HA – M atrix-M nanopar ticles (H5- MNP s). In naïv e mice, t w o intr anasa l (IN) or in tramuscular (IM) doses of A/ A W/SC/ 2021 H5-MNP v accine induced r obust a n tibody- and cell-medi ated immune r espo nses, including neu tr ali zing an tibo dies ag ainst A(H5N1). In non-human prim a tes (NHP s) primed with season al influenz a v accine, a single IM or I N dose of the A/ A W/SC/2021 H5-MNP v accine in duced g eome tric mean se rum A(H5N1) clade 2.3.4 .4b pseudovirus neutr ali zing ti t er s of 1:1160 and 1:54, r esp ectively; abov e t he g e ner al ly accept ed ser oc on ver ting neutralizing ti t e r of 1:40. Immuniz ation with H5-MNP v accine indu ced an ti body r espons es ag ains t conser v ed epi t opes in the A(H5N1) HA st em, v e s tigial e s ter ase s ubdomain, and r ecep t or bin ding sit e . This nov el A(H5N1) H5-MNP IN and IM v accine w as immuno g enic in r ode n ts an d NHP s as a pot e n ti al A(H5N1) pandemic si ngle-dose v accine. .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 November 21, 2024. ; https://doi.org/10.1101/2024.11.21.624712doi: bioRxiv preprint 3 INT R ODU CT ION The emer gence of highly pa t hog en ic a vian influenz a (HP AI) A(H5N1) viruses mak es the dev elo pment of saf e and e f f ective v accines a public heal t h priority , which is being pur sued ac tiv el y by the Unit ed S t ates Biomedic al Adv anced R es ear ch a nd Dev e lopment Auth ority 1 . The fir st c ase of HP A I A(H5N1) tr ansmission t o a human w as r epo rted in 1997 in Hon g K ong 2 . Since then, th e W o rld Heal th O rg aniz a ti on has document ed ov e r 800 c ases of A(H5N1) inf ection in humans with a f at ali ty r at e of ov er 50% 3 . R ecent outbreak s of A(H5N1) clade 2.3 .4.4b influenz a ha ve r e ached panz o otic lev els wit h inf ections in wild bir ds, poul try , and 48 mammalian specie s acr oss 26 c oun t ries increasing th e pand emic public health risk 4 . A r ece n t spik e in A(H5N1) inf ecti on s in the Uni t ed S t ates has occurred since early 2024, including appr o xim at ely 508 dairy c ow herds, 158 flock s of bir ds (c ommer cial and backy ar d) , and 52 human c ases (all link ed t o exposu r e t o inf ec t ed dai ry cow s or poult ry) as of Nov ember 2024 5,6 . Although t he current risk of a pandemic fr om A(H5N1) clade 2.3.4.4b remains low f or humans, ongoing tr ansmission ev e n ts among animals in close c ont ac t with hu mans will incr ease th e possibility of human-t o-human tr ansmissibility with pand emic potential 7,8 . Her e , w e describ e a nov el HP AI A(H5N1) clade 2.3.4 .4b HA – Matri x-M saponin adj uv an t nan opar ticle (H5- MNP) v accine. R ec ombina n t full-leng t h HA pr o t ein w as produced in insec t cells fr o m the A(H5N1) clade 2.3.4.4b A/ Ame ric an Wig e on/South Car o lina/22/000345-001/20 21 (A/ A W/S C/202 1) s tr ain which is among the eme r ging a vian HP AI influenza viruses and designated as a c andi dat e vaccine virus with a possible risk f or human-t o-human tr ans mission 9 . Purified r ec ombin ant A/ A W/SC/2021 HA pr e fusion trimer s link ed t o pol y sorb at e 80 (HA-PS80) nanoparticles were mix e d with Matri x-M™ adjuv an t, c omposed of saponins fr om th e Quillaja saponoria Moli na t r ee c o-f ormulat ed wit h cholest er ol and phospholipids f orming a c ag e-lik e i c osah edr al s t ructu r e with an ap pr o xim at e dia met er of 40–50 nm 10 . Upon mixing HA-PS80 nanopar ticles with Ma t rix-M adjuv ant, a non-c ov al ent asso cia tion of H5 HA trimer s via th eir C-t e rminal tr a nsmembr a ne domain with M a tri x-M w as obser v ed , f orming the H5-MNP 50–55 nm particles . W e show mice immuniz ed with the A/ A W/SC/2021 H5-MNP v accine by either intr amuscular (IM) or in tr an asal (IN) r ou t es exhibi t ed high hom ologous hemag glutina ti on inhibiti on (HAI ) and pseudovirus neutr ali zing antibody ti t er s an d amplified an tigen-specific CD4 + T cell r esponses when c ompa r ed t o r espons es in placebo-t r e at ed anima ls. Fu rthe rmore, nonhuman p rimat es (NHP s) primed with quadriv ale n t na nopar ticle influenz a v acci ne (qNIV) e xhibi t ed limi t ed n eutralizing a n tibody responses ag ains t H5N1 A/ A W/SC/2021, but a single subsequent IM o r IN dos e of A/ A W/SC/ 2021 H5-MNP v accine .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 November 21, 2024. ; https://doi.org/10.1101/2024.11.21.624712doi: bioRxiv preprint 4 incr ease d pseudovirus neutraliza tion r es ponses abov e a se r oc o n v er sion n eutralizi ng tit e r of 1:40 11 , with GMT s of 1160 and 56, r esp ectiv ely , which further inc r ease d t o 11,698 a nd 134 after a sec o nd IM or IN dose, respectively . A/ A W/SC/2021 H5-M NP v accine boos ter dos es in primed NHPs also induced r obust Th1 CD4 + T cell–mediated immune r esp o nses ag ains t HP A I clade 2.3 .4.4b A(H5N1) . Neu tr alizing monoclonal antibodi es ag ains t A/ A W/SC/ 2021 w er e also isol at ed fr om H5-MNP–i mmuniz ed mice that bind c onser v ed A/ A W/SC/2021 H5 HA neutr alizing epi t op es in th e r ecep t o r bindin g sit e (RBS), v es tigial es ter ase (VE) subdomain, and HA st em. T his nov el HP AI A(H5N1) r e c ombina n t HA v accine has the potential t o induce protective immunity with a single IM or I N dose . RE SUL TS 1. Gener a tion and cha r act eriz a tion of a r ec ombinan t full-len gth A(H5N1) A/ A W/SC /2021 HA pr ot ein W e designed and generat ed a full-leng th sequence (r esi dues 1–552), including c o mplet e h ead and st em domains with the C-terminus tr a nsmemb r ane domain anch or of A(H5N1) A/ A W/SC/2021 HA. A polybasic clea v ag e si t e dele tion (ΔKRRK at residues 341–344) w as inc orpor ated to impr ov e a n tigen st ability ( Figur e 1A). The an tigen w as cloned , pr oduc ed in insect cells, purified , and f ormed HA-PS 80 nanopar ticles as pr eviously described 12 . The puri ty of the A(H5N1) A/ A W/SC/2021 HA as assessed by SDS-P AGE densitomet ry ( Figur e 1B ) w as 97.02% and the par ticle siz e w as 34.78 nm in diame t e r as determined using dynamic ligh t sc a ttering (DLS) ( Ta b l e 1 ). The thermal st ability of purified p r o t ein w as ass essed by dif f erential sc anni ng c alorime try (DSC), s howing a major tr ansition with a mel ting t emp er ature ( T m ) of 52.10 °C ( Ta b l e 1 ). Purified A/ A W/SC/2021 HA trimer nanop articles wer e cha r acteriz ed by neg ativ e staining–tr a nsmission electron micr osc opy (NS-TEM) and 2D cla ss a v er ag e w as ob t ain ed ( Figur e 1C). The NS-TEM da ta show ed that, similar t o o ther known influenz a HA an tigens, A(H5N1) A/ A W/SC/2021 HA is a homotrime r gly c opr o t ein with dime nsions of ~138 Å (leng th) × 15–40 Å (r adius) 13 in a pr e fusio n s ta te associated with a PS80 det erg e n t micelle n ear i ts membr ane anchor d omain, f orming a pr o t ein –d et e r g e n t n anopa rticle . 2D classific a tion also r eveal ed the assem bly of higher-or der nan opar ticles, whe r e mor e th an one HA trimer w as associ at ed with a de ter gent micelle ( Figur e 1C). A sequence-based in silico molecul ar model w as g enerat ed f or A/ A W/SC/2021 HA and w as superimposed i n to an el ectr on d e nsity map r ec o ns tr ucted fr om the 2D classific a tion ( Figur e 1D ) r ep r ese n ting th e int ac t assembly of pr e fusio n an tigen; the HA0 s tructu r e w as mai nt ained f or e ach pr o t o mer within th e trimer . .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 November 21, 2024. ; https://doi.org/10.1101/2024.11.21.624712doi: bioRxiv preprint 5 W e obser v ed th at full-leng th HA homo tri mer s w ere link ed t o an ex t end ed membrane-embedd ed ancho r along with α-helices surr ound ed by the d et e r g e n t micell e. This is a critical f e a tu r e of the HA nanopar ticle, whe r e the n a tive-lik e s truc t ur e of the antig en is mimick ed and st abili z ed by PS80 det e r gent. This is signific an t f or immuno g enicity , as accur at e antig en r ep r ese nt ati on enables the g enera ti on of an t ibodies that tar g e t v ario us HA epitopes upon v accin a tio n. Over all , the d a ta sug g es t th at our r e c ombina n t full-leng th A/ A W/SC/20 21 HA r ec o ns ti tu t ed in PS80 deter gent e nables a r ange of orienta ti ons f or higher-or d er nan opar ticl e assemblies (r osettes), pr ese n ti ng a w ell -de fined, flexible s tructu r e via a membr a ne-lik e anchor . 2. Char act eriz a tion of A(H5N1) A/ A W /SC/2 021–Matrix-M nanoparticles (H5-MNP s) W e next char ac t e riz ed th e time-d epend e n t inter actio ns of the influenz a v accine A (H5N1) A/ A W/SC /2021 HA nanopa rticle ( Figur e 2A) when mix ed with Ma t rix-M adjuv a n t ( Figur e 2B). Purif ied A/ A W/SC/2021 HA nanopar ticles w e r e mi x ed with M atrix-M a t an app r o xim at ely 40:1 molar ra tio (c o rr espo nding t o 120 µg /mL: 75 µg / mL c oncen tra tion ra ti o by mass) and the bioph y sic al p r ope rties were char acteriz ed using high pr essure–siz e e x clusi on chr om a togr aph y (HP-SE C) and 2D classific a tion by NS-TEM t o in v estig at e th e kinetics of an tigen–M atrix-M associ a ti on. HP-SE C show ed tha t at time 0, A(H5N1) HA and Matri x-M eluted fr om the c olumn as separat e p eaks c orr esp onding to r esp ective r e t e n tion ti mes with apex es a t 8.719 min and 7.662 min. After 12 h of incuba tio n, th e elu a te show ed a H5-M NP peak with a shor t er r etention tim e of the ap e x (7.584 min) and a lar ger peak a r ea , indica ting th at the particle siz e of H5-MNP bec ame la r ger and app r o xim at ely 95.5% of HA w as bound t o M atrix-M a t th is tim epoint based on the ar ea un der the cur v es ( Figur e 2C). NS-TEM 2D classific a tion ena bled th e visualiz at ion of this Matrix-M – HA association ( Figur e 2D ,E ), wher e HA t rimer s arr an g ed on the M a t rix-M icosahe dr al c age surf ace at the v er tices in a he ad-to-s tem orie nt atio n ( Figur e 2F ) demons tr ating a direct int eraction bet w een the A/ A W/SC/2021 HA tr ansmembr ane dom ain and the lipi d:chole st er ol bil a y er surf a ce v ertices of M a t rix- M. The mechanism unde rlying this phen omenon will be in v estig at ed in futu r e stu dies. B y char ac t e rizing MNP s’ siz e, s truc ture, and f orm a tion , w e c an better unde r st and th eir bioph y si c al p r oper ties as a v accine drug pr oduct . 3. The H5-MNP v accine is immunog e nic in naïv e mice T o ev alua t e the in vivo immunog e nicity o f the H5-MNP v accine, w e immuniz ed naï v e B ALB/ c mice with a tw o-dose prima ry seri es of H5-MNP adminis ter e d via the I M or IN r ou t e ( Figur e 3A) and ev alua ted an tib ody- and cell-mediat e d immune r es ponses to v accina ti on. Hemag glutin a ti on inhibiting (HAI) .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 November 21, 2024. ; https://doi.org/10.1101/2024.11.21.624712doi: bioRxiv preprint 6 g eomet ric mean ti t e r s (GMT) ag ains t A/ A W/SC/2021 w er e analy z ed in sera c ollec ted tw o w e ek s after th e primary seri es; all doses an d r egimens of the H5-MNP v accine elici t ed r ob us t HA I an tib ody titer s, with ser oc on ver sion obs er v ed in all immuniz e d animals, while ti t er s in t he place bo gr o up w er e un detectable (Figur e 3B). Serum HAI titer s w e r e signific antly higher f ollowing immuniz a tion wit h 100i:jμg H5-MNP (IN) c ompared to 1 μg H5-MNP (IM) (p = 0.048). How ev er , no st atis t ic ally signific ant diff er e nces w e r e obser v ed between t iter s in th e tw o I N do se lev els, indica ting that M a tri x-M has an an tigen-sparing e f f ec t (p > 0.05). Similarly , there w ere no st atist ic ally signific an t dif f erences in A/ A W/SC/2021 pseudovirus neutr ali zing ti t er s in ser um c ollec t ed tw o w eek s aft e r th e primary se ries ( Figur e 3C) acr oss the H5-MNP tr e atment gr oups (p > 0.05), and all anim als e xhibi t e d ser ocon v er si on, indicating t ha t the H5-MNP r egimens g en era ted equiv al ent lev els of functional a n tib odies in mice. As e xp ecte d, neutralizing ti t er s w er e un detectable in t he placeb o gr oup. T o ev alua t e mu c osal a n tibody responses t o immuniza tion , anti-A/ A W/SC/2021 HA immunoglobulin A (IgA) and Immunoglobulin G (IgG) r espon ses w er e de termined in b r onchoalveola r la v ag e (B AL) fluid c ollec t ed t w o w e ek s aft e r the p rimary se ries ( Figur e 3D). IgA titer s w e r e unde t ec table in th e placebo- tr e at ed c ontr ols and in the H5-MNP 1 μg (IM) tr e a tme n t gr ou p, indica ting that IM adminis tra tio n of H5- MNP v accine w as ine f f ectiv e a t genera t in g measur able muc osal IgA antibody resp onses in the lower r espirat ory tract of mice a t this dose leve l. How ev er , el ev at ed a n ti-A/ A W/SC/2021 HA IgA r esp onses w er e obs er v ed after IN adminis tration, w ith both IN H5-MNP t r e a tme n t gr o ups e x hibiting equiv ale n t an ti-A/ A W/SC/2021 HA IgA ti t er s (p > 0.0 5). An ti-A/ A W/SC/2021 HA IgG r espons es in B AL w ere measur able in all H5-M NP tr e atment gr o ups but w ere unde t ec t able in plac ebo an imals. Anti- A/ A W/SC/2021 HA IgG tit er s in B AL w e r e signific an tly higher in t he 10 μg (IN) gr oup c ompared to ti t er s in the 1 μg (IM) H5-MNP tr eatment gr oup (p = 0.028). There f or e , IN admini s tra ti on of the H5-MNP v accine success fully elicit ed a n ti g en-specific IgA and IgG a n tibo dy r espons es in the low e r r espirat ory tract in mice, while IM administr ation generat ed IgG r esp onses but n ot IgA r espons es in t his c ompartm ent, as e xpec t e d. R eg ar ding T cell r espo nses, a n tigen-speci fic Th1 and Th2 cyt okine r esponses were analy z ed in CD4 + T cells fr om the splee n ( Figur e 3E) and the lung ( Figur e 3F ) c ollect ed 2 w e ek s aft e r t he primary ser ies. I n spleen e f f ec t or CD4 + T cells, s ta ti s ti c ally signific an t increases in numb er s of antig e n-specific polyfunctional T cells (IFN-0i:j + , IL-2 + , o r TN F-α + ) upon A/ A W/SC /2021 HA s timula tio n w er e obser ved in the 1 μg (IM) and 10 μg (IN) H5-MNP–tr e at e d gr oups c ompar e d t o pl acebo –t r e at ed a nimals (p = 0.015 and 0.014, r esp ectiv ely; Figur e 3E ). In the gro up adminis tered 1 μg (IN) H5-MNP , the a n tigen-specific .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 November 21, 2024. ; https://doi.org/10.1101/2024.11.21.624712doi: bioRxiv preprint 7 polyfunctional CD4 + T cell number GM was 30.4-f old higher than th e GM in the pl acebo gr oup, bu t this dif f erence w as no t s ta ti s tically signific an t (p > 0.05). A similar tr end was obser v ed when e x amining individual Th1 cyt okine expression (IFN-0i:j , IL-2, or TNF-α) in spleen e f f ec t o r cells upon s timul a tion with A/ A W/SC/2021 HA ( Figur e S1A ); c ompared t o v alu es in the pl acebo gr oup , signific ant incr e ases in IFN-0i:j + CD4 + T cell number s w er e obser v ed af t er immuniz a tion with 1 μg (IM) or 10 μg (IN) H5-MNP (p = 0.018 and 0.013, respectively), and signific an t i ncr eases in IL-2 + and TN F-α + cell number s w er e obser ved aft e r immuniz a tio n with 10 μg (IN) H5-MNP (p = 0.010 and 0.004, respectively). An increase of IL-4 + CD4+ T cells (Th2 cyt okine) w as obser v ed aft er i mmuniz a tion wit h 1 μg (IM) H5-MNP (p = 0.002), but positive cell number s w e r e signific antly low er tha n all the Th1 cyt okin e + CD4 + T cell number s, indic ating a Th1- biased response ( Figur e S1). Immuniz a tio n with 1 μg (IN) H5-MNP did not r esult in s t atistic ally signific ant incr eases in individual cy t okine p ositiv e CD4 + T cell number s c ompar ed t o numbe r s in the placeb o gr oup. In lung r eside n t CD4 + T cells, IN admini s tra tio n of 10 μg H5-MNP r esult ed in a signi fic an tly higher numb er of an tigen-specific polyfunctional T cells c ompared to aft e r IN admini s tra tio n of 1 μg H5-MNP dose (p = 0.0043) (Figur e 3F ). Individual Th1 cyt okine (IFN-0i:j, IL-2, or TNF-α) or Th2 cyt okine (IL-4) r esponses in lung cells upon s timulation with A/ A W/SC/2021 HA c an be f ound in Fig S1B . Signif ic ant incr e ases in IFN- 0i:j + cell number s w e r e se en in animals im muniz ed with a 10 μg H5-MNP dose (IN) ov er thos e immuniz ed with a 1 μg H5-MNP dose (IN) (p = 0.004 3). No othe r signific ant dif f e r ences in indi vidual Th1 or Th2 cyt okine exp r ession w e r e obser ved. 4. The H5-MNP v accine is immunog e nic in NH P s primed with seasonal influenz a v accine ( qNIV) T o c onfirm the immunog enicity of the H5 -MNP v accine in a non-human primat e ( NHP) model with an immune backgr ound mimicking tha t of t he human popul a tio n, w e n e x t in v estig ated the a n tib ody- and cell-mediat ed immune r espons es to tw o IM or I N doses of the H5-M NP v accine in Rhesus mac aqu es p r i m e d w i t h t h e M a t r i x -M – a d j u va nt e d N o vavax q u a d r i va l e n t n a n o p a r t i c l e i n f l u e n za v accine (qNIV) c ont aining HAs fr om 2023-2024 seasonal influenz a s tr ains ( Figur e 4A). Immuniz a ti on with tw o doses of qNIV r esul ted in unde t ec t able H AI ti t e r s ag ains t A(H5N1) A/ A W/SC/2021 in all NHP s ( Figur e 4B). Adminis tra ti on of a single IM dose of th e H5-MNP v accine (60 µ g HA with 75 µg Ma tri x-M adjuv ant) r esul t ed in de t ec t abl e HAI (A/ A W/SC/2021) tit er s in se rum in three of fiv e animals (tw o animals e xhibi ting ser o c on v er si on), and a second IM dose of th e H5-MNP v accine (60 µg H A with 75 µg Ma trix-M adjuv an t) pr oduce d detectable HA I (A/ A W/SC/2021) tit er s in se rum of all fiv e animals (all animals e xhibi ting ser o c on v er si on). In c o n tras t , t w o IN doses of th e H5-MNP v accine (240 µg HA or 60 µg HA, r espec tiv ely , with 75 µg Ma tri x-M adjuv an t) did not induc e detectable se rum HAI r espons es. .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 November 21, 2024. ; https://doi.org/10.1101/2024.11.21.624712doi: bioRxiv preprint 8 As anoth er measu r e of functional a n t ibo dy r esponses t o immuniz ation, n eutralizi ng r esponses in ser a w er e ev alu at ed in NHP s thr oughou t th e study . Primary immuniz ation with q NIV in duced cr oss- neutr ali zing ti t er s in NHP s ag ainst A/ A W/SC/2021 pseudovirus with a GMT of 146, abov e the generally accept ed s er ocon v er ting neu tr alizing ti te r of 1:40 in eigh t of ten animals ( Figur e 4C), though thes e ti t e r s w aned t o below 1:40 in nine of t en anim als aft er two months. A single I M or I N dose of H5-MNP v accine signific an tly incr e ased pseudovirus ne utralizing titer s ag ainst A/ A W/SC/2021 by 9 2.2- and 4.3-f old t o GMT s of 1160 and 54, r esp ectiv ely , when c ompar e d t o ti t er s b e f or e H5-M NP adminis tra tion (Study Da y 83) (p = 0.0003 and 0.008). This fir s t H5-MNP boos t r esul t ed in 100% ser o c on v e r s ion aft er an IM dose and 80% ser oc on ver sion after an I N dose . A sec ond IM or I N dose of H5-MNP v accine furthe r incr e ased induced pseudovirus neu tr alizing ti t e r s a g ains t A/ A W/SC/2021 t o GMT s of 11698 and 134, r esp ectiv ely (p = 0.0003 and 0.0007), c or r espo nding t o 1 00% ser oc on v e r sion aft e r administr ation via either r ou t e. Signific an tly higher pseu dovirus neutraliz ing tit e r s w ere obser v ed af t er I M administr ation c omp ar e d t o titer s aft e r IN a dminis tra ti on (Da y 97, 13 9, and 153, p = 0.008). These results indi c at ed that a single IM dose or two IN dos es of H5-MNP g enerated potentially pr o tectiv e a n tib ody r espo nses ag ains t A/ A W/SC/2021 in animals primed with s easonal influenz a v accine , and th a t imm uniz ation via the IM r oute resulted in higher functi onal a n tibo dy tit e r s c ompared t o immuniz ation via t he IN route. T o measur e HA-binding a n tibody respons es, ser a w e r e also t e s ted f or anti-A/ A W/SC/2021 HA IgG an tib odies. Th e qNIV prima ry series r esul t ed in me asur able a n ti-A/ A W/SC/2021 H A IgG ti t e r s on Da y 35, which had signific an tly decreased by Da y 83 (p = 0.02) ( Figur e S2A ). Animals imm uniz ed with one or tw o doses of IM H5-MNP , bu t not I N H5-MNP , show ed signific an tly increased se rum Ig G ti t er s compared to pr e-boost ti t e r s on Da y 83 (p < 0.0001, p 0.05 , r esp ectiv ely). W e also in v estig at ed cel lular immune r es ponses ag ains t A/ A W/SC/2021 HA using peripheral blood mononuclear cells (PBMCs) c ollected fr o m the NHP s primed with M a t rix-M adjuvant ed qN IV , f ollow ed by tw o IM o r IN dos es of H5-MNP ( Figur e 4 D). Measurable polyfuncti onal (tripl e Th1 cyt okine posi tiv e) an ti g en-specific CD4 + T cell r esponses ag ains t A/ A W/SC/2021 HA w er e e licited in NHP s primed with a tw o-dose prima ry seri es of 2023-2024 seasonal flu v accine (p = 0.0007; Da y 0 v s. Da y 35). F ollowing one H5-MNP boos ter dos e, w e obser ved a signific an tly great e r CD4 + T cell r esponse in the gr oup v accin a ted IM c ompa r ed t o th e gr oup v accinated IN (Da y 97; p = 0.016) and r esponses w ere a lso signific an tly higher in the IM gr o up than in the I N gr oup afte r tw o doses (Da y 153; p = 0.016). Analy si s of individual cyt okine r espons es show ed th a t IM administr atio n of one or tw o dos es of H5-MNP occ asionally pr oduced .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 November 21, 2024. ; https://doi.org/10.1101/2024.11.21.624712doi: bioRxiv preprint 9 s ta tistic ally signific a n t increases in indivi dual Th1 cyt okine exp r ession c omp ar e d to IN administr ation (Da y 97: p = 0.0079 f or IFN-0i:j, 0.016 f or IL-2; Da y 153: p = 0.032 f or IFN-0i:j, 0.032 f o r TNF-α; Fig S1C ). Next, we ev aluated muc osal responses to H5-MNP boos ter dos es in the upp er an d low er r espi r at o ry tr act (Fig S2B-E). T w o w eek s after a singl e IM dose of H5-MNP , anti-A/ A W/SC/202 1 HA IgA ti t er s in t he upper respir atory tr ac t (nasal w ash; G MT = 0.371) w er e signific a n tly higher t han ti t er s af t er the qN IV primary seri es (GMT = 0.072; p = 0.045). Aft e r the se c ond I M H5-MNP boost e r do se, anti-A/ A W/SC/2021 HA IgA ti t e r s in nasal w ash incr e ased furt her to a GMT of 1.16, corresponding t o a 3.1-f old rise in GMT c ompared to ti t er s after t he fir s t H5-M N P boos t, tho ugh this dif f e r ence was not st atistic ally signific ant (p > 0.05). After one IN H5-MNP boost, a n ti -A/ A W/SC/2021 HA IgA titer s in nasal w a sh w er e no t signific an tly dif f e r e n t t han ti t e r s aft er the qNIV primary seri es (p > 0.05), though t he sec ond IN H5-MNP boos t did e lev at e t iter s signific antly c om par ed t o titer s after th e prima ry series (G MT = 0.540; p = 0.011) (Figur e S2B ). In the low er r espi r at o ry tr a ct (B AL), one or two IM doses of H5-MNP r esul t ed in a n ti- A/ A W/SC/2021 HA IgA tit er s of 0.163 an d 0.208, r esp ectiv ely , which w ere signific an tly higher than t iter s aft er th e qN IV primary seri es (p = 0.001 and 0.0001, r esp ectiv ely). The fir st and se c ond IN b oost er d oses of H5-MNP incr eased th e anti-A/ A W/SC/2021 HA IgA ti t er s in BAL 1.8- and 2.0-f old c ompared to ti t er s aft er th e qN IV primary seri es, tho ugh the se incr eas es w ere not sta tistically signific an t (p > 0.05) ( Figur e S2C). Similarly , IM adminis tration of one or tw o dos es of H5-MNP signific an tly increased a n ti- A/ A W/SC/2021 HA IgG tit er s in the upp er and low er respir atory tr ac ts c ompa r ed to Da y 35 tit er s (p = 0.040 and 0.017; p = 0.0028 an d 0.0016, r espec tiv ely), but I N adminis tration of on e or tw o dos es did not r esult in an y signific ant increase in muc o sal IgG r esp onses, and IgG responses were undetectable in B AL aft er I N immuniz ation (p-v alues >0.05) ( Figur e S2D ,E ). 5. H5-MNP v accina tion g ener a t es neutr alizing an tibody r esponses t ar g eting epit op es in the HA r ecept or binding sit e, v es tigial es t er ase subdomain, and s t em T o char act eriz e epi t ope-specific neu tr aliz ing poly clonal an t ibody r esp onses t o H5-MNP v accine in animal models, w e isol at ed A/ A W/SC/2021 HA-specific monoclonal antibodies (mAbs) by h ybridoma t ech nology fr om H5-MNP v accina t ed mice . As seen i n Ta b l e 2 , mAb NVX.361.4 e x hibi t ed HA I activity (endpoint ti t er of 250 ng /mL ) ag ains t A/ A W/SC/2021 HA and othe r A(H5N1) HA pr o t eins, and s t r o ng neutr alizing ac tivity ag ains t A/ A W/SC/2021 (pVN 50 of 3.57 ng /mL) and other A(H5N1) HA proteins. mA b NVX.73.2 e xhibi ted undetect a ble HA I activity (endpoi n t ti ter gr eater th an 500 ng /mL) ag ains t A/ A W/S C/2021 HA and other A(H5N1) HA pr oteins, and mod er ate neu tr alizing activity ag ainst A/ A W/SC/2021 ( pVN 50 of 42.80 ng /m L) and othe r A(H5N1) HA pr o t eins . .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 November 21, 2024. ; https://doi.org/10.1101/2024.11.21.624712doi: bioRxiv preprint 10 T o iden tify the epi t opes t arg e t ed by thes e neutralizing antibodi es (NVX.73.2 and NVX.361.4), cry o-EM w as utiliz ed. B oth a n tib odies show ed bin ding t o th e globular h ead region of HA-A / A W/SC ( Figur e S3 ). Epit op e mapping s tudi es utilizing molecu lar modelling c ombin ed with cry oEM ma ps. mAb NVX.73.2 w as f ound t o bind in th e A/ A W/SC/2021 HA v es tigial e s ter ase (VE) subdomain, with cri tic al residues be tw e en 60-70 (NGVK PLILK D), 90 -95 ( PEW S YI), and 284-290 (GVE Y G H CN)(ST QKAIDGVTNKVN-) making dir ect c ont act with HC-CDRs (hea vy chain-c omplement ari ty determining region) and L C-CDR3 (li gh t chain- c ompleme n tarity de t e rmining r egion 3) ( Figur e 5A), c orr esponding t o th e VE subdomain based on H3 numbering , which is c onser v ed among H 5 subtypes. The VE domain is loc at ed be t w een th e RBS in HA1 and the membrane-pr oximal st em region of HA2 ( Figur e 1A ). mAb NVX.361.4 w as r ev eal ed to bind t o A/ A W/SC/2021 HA in the r ecep t or bindin g sit e (RBS) of the globular he ad region a bov e the VE subdomain 14 with critic al r esidues be tw e en amino acids (ETSL GV ); 105–107 (GAP) ; 118–121 ( KKN D); bound to HC-CDR3 and r esidues 151–155 (E EQT N); 187–118 ( -GQ ) bound t o L C-CD R2 ( Figur e 5B). These epitope studies confirmed th a t H5-MNP v accina tion e licits a br o adly neutralizing response by g enera t ing an tib odies th at bind both the RB S and VE subdomains of HA head region. T o c onfirm the in vivo g en era tion of a n tib ody r esponses ag ain s t th e A/ A W/SC/2021 HA subdomains, ser a c ollec t ed fr om th e NHP s tudy describ ed i n Figur e 4 w er e analy z ed in r eal-time c o mpetition binni ng s tudies ( Figur e 5C). Competitiv e a n tib odi es w ere induced in ser a ag ai ns t all three mAbs (CR6261 t arg eting t he HA st em r egion, NVX.73.2 tar g e ting the VE subdomain, and NVX.361.4 t a r g eti ng the RBS) in NHP s aft er th e qNIV prima ry seri es, and a single IM dose of H5-MNP v accine increased c ompe titive an tib ody equiv ale n t (CAE) by 6.5- t o 7.0-f old ag ains t all mAbs c ompa r ed t o v alu es on Da y 35, which w er e signific an t f or th e HA VE subdomain- and RBS-t arg eting a n tib odies (p = 0.023 and p = 0.013, r espec tiv ely). A similar t r end w as obser ved aft er the se c ond IM d ose, whe r e CAEs f or all mAbs r emained 31.1- t o 32.3-f old higher c omp ar e d t o v al ues on Da y 35, with signific an tly higher CAEs ag ains t HA he ad an tib odies (p = 0.0001 and p = 0.0001 , r e spectiv ely). IN a dminis tra ti on of H5-MNP r esul t ed in less r ob us t CAEs ag ains t the HA stem-t a r g eti ng mAb: aft er th e fir s t I N boost, th e lev el of antib odies c ompeti tiv e ag ains t HA st em mAb CR6261 decreased 1.5-f old c ompar ed t o levels aft er the pri mary series, a nd aft er the second IN b oost, CAE ag ains t t his mAb decr eas ed ano the r 1.3-f old, though th e se decr e ases w e r e no t s ta tistic ally signific a n t (p > 0.05). F or the tw o antibodi es c ompeti tiv e ag ain s t t he H A head (VE subdomain and RBS), aft er the fir st IN bo ost er d ose, CAE ag ains t mAb NVX.73.2 increased 2 .8-f old and CAE ag ains t mAb NVX.361.4 incr e ased 10.0-f old, tho ugh neithe r of these dif f erences w e r e statis ti c ally signific an t c ompared to CAE on Da y 35 (p -v alues > 0.05). These CAE v alues did not chan g e signific an tly after th e sec ond I N boost e r dose (p-v alues > 0.05). Ov er all, the lev els of c ompe ting antibodi es ag ains t HA .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 November 21, 2024. ; https://doi.org/10.1101/2024.11.21.624712doi: bioRxiv preprint 11 c onser v ed epi t opes in RBS , VE, and s tem r egions f or all th r ee mAbs w e r e 7 .0- t o 1 1.4-f old higher in animals administ ered two IM doses of H 5-MNP v accine c ompar ed t o levels aft e r t w o IN doses , though the only st atis ti c ally signific ant dif f e r ence w as f or an ti-HA st em mAb CR6261 (p = 0.026). DISCUSSIO N Her e , w e describ e a new link ed pr o tein-a djuv an t nanop articl e v accine f or HP AI A( H5N1) clade 2.3.4.4b made fr om r ecombinant full-leng th A/ A W/SC/2021 HA trimer s link ed t o Matrix- M adjuv an t, f orming nov el H5-MNP s. The A(H5N1) HA pre fusi on trimer s anch ored to Matri x-M adjuv an t of f er th eor e tical adv ant ages. The a n tigen densi ty and loa d of the v accine are high, which allow s r o bus t activa tio n of an ti g en-prese n ting cells (APCs) 15,16 . Furthermore, th e H5-MNP s ensure antig en an d adjuv an t a r e c o- deliv ered int o endosomes , then f ollowin g the disruption of en dosomal membr an es by the r ele ase of saponins in Matri x-M, some of the a n ti g e n c ould be releas ed int o the cy t oplasm o f APCs. This ma y help the a n tigen a v oid H A pr o t eol y sis, enabl e in ter acti on with MHC I, and induc e CD8 + T cell r esponses . Future s tudi es will in v es tiga te th e bioph ysic al mechanisms underlying this spo n ta neous Matri x-M anchoring phenom enon obser ved with A (H5N1) HA an tig e n, which w as not pr evio usly obser v ed f or other antig ens mi x ed with M atrix-M incl uding SAR S-CoV-2 spik e 17 . Futur e in vivo s tudies will also seek t o dissect whethe r thes e MNP s e x hibit adv a n tag es f or cellula r r esp onses bey ond th e r obus t B c ell immunity demons trat ed f or M a t rix-M– adjuv ant ed v accines 12,18-21 . In this s tudy , w e p r obed the a n tib ody- and cell-mediat ed immunity genera ting cap abilities of th e new A/ A W/SC/2021 H5 -MNP v accine in vivo in a naïv e mouse model a nd a season al influenz a-primed NHP model. In mice, immuniza ti on with a two -dose series of an A/ A W/SC/2021 H5-MNP v accine, either IM or IN, induce d r obust antibody HA I ti t er s an d pseudovirus neutr alization ti t e r s tw o week s aft e r v accination . HAI assa y s measu r e th e binding of head-specific an tibodi es t a r g e ting the R BS th a t inhibits r ed bl ood cell (RBC) a g glutina tion activity 22 . How ev er , n eutr alization activity t arg e t ed t ow ard oth er c onser ved epi t op es, such as the VE subdomain or th e HA st e m, is critic al f or br oad a nd dur abl e functi onal immunity since the head domain accumul at es muta ti ons at a f as t er rat e th an th e HA st em 23 . Thus, while it is desir abl e f or nov el v accine c andid at es t o be e f fic aciou s in g enera ting r obu s t HA I antibody ti t e r s , eliciting both h ead- specific and br oade r neut r alizing antibod y r esponses ma y be par amou n t f or br o ad v accine e f fic acy acr oss multiple influenz a sub types. Addi t ionally , mice that r ec eiv ed th e intr anas al H5-MNP v accine e xhibi t e d incr eas ed anti-A/ A W/SC/2021 HA IgA ti t e r s c or r el a ting with t heir admin is tr ation i nt o th e muc ous membr anes of th e r espirat o ry tract. B y c o n t r as t , anti-A/ A W/SC/2021 HA IgG titer s w e r e robus t in all H5-MNP tr e a tm ent gr oups sug g es ti ng muc osal immune activ ation f ollowing both IM a nd IN .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 November 21, 2024. ; https://doi.org/10.1101/2024.11.21.624712doi: bioRxiv preprint 12 adminis tra tio n of H5-MNP . Similar e f f ec ts w er e obser ved in Th1 + CD4 + T cell activ a t ion, wher e in antig en- specific lung Th1 + CD4 + T cell activ a tion was r obus t f ollowing IN administra tion whi le a br oad er immune activ a ti on (spleen) w as obser v ed f ollowing both IM and IN administra tion of th e H5-MNP v accine. These r esults a r e consist e n t with t he function al , accessibility , and r espirat o ry immune activ a ti on ben e fits obser v ed by other I N v accines (r eview ed in 24,25 ). Ov er all, th e H5-MNP v accine pr od uced neutralizing r espons es ag ains t A(H5N1) in naïv e mice f ollowing both a s t anda r d IM v accin a tio n and a less in v asiv e IN v accina tion , th a t has po tential t o be self-adminis tered in the event of a pandemic. Next, we e xp lored whethe r a season al influenz a v accine w ould elici t immunity ag ains t A(H5N1) in NHP s. Aft e r tw o dos es of seasonal flu v accine, A/ A W/SC/2021 HAI an tibodies were und et ec t abl e in NHP s, and though A/ A W/SC/2021 neutr alizing antib odies w ere de t ec t able sho rtly after seaso nal flu v accina tion, an tib ody titer s quickly w aned below sero c on v er sio n lev els, indicating th a t previou s seasonal influenz a immuniz a tio n ma y not be suf ficient to prot ec t individuals ag ainst an A(H5N1) influenz a pand emic. Similarly , pr evious s tudi es ha v e shown so me pr otectio n of seasonal influenz a v acci nes alone ag ain s t A(H5N1), but this often requires multiple doses, which is not optimal in a pand em ic. F or ex ample, unadjuv ant ed t riv ale n t seas onal influenza v accine w as shown t o of f e r some pr o t e ction ag ains t A(H5N1) influenz a challenge in mice, but only afte r tw o or three IM immuniza ti ons 26 . Neu tralizing an ti bodies and cr oss-r eactive cellula r immune r esp onses ag ains t A(H5N1) w ere also de t ec t able in mice f ollowing par e nt er al immuniza ti on with tw o d oses of an inactiv at ed se asonal influenz a v acci ne 27 . Furth ermore, low lev els of A(H5N1) neutr alizing antibo dies ha v e be en de t ec t ed in se rum fr om people immuniz ed with seasonal influenz a v accine 28 . Impo r t antly , these studies as well as our own r esul ts emphasiz e th a t , while the seaso nal influenz a v accine do es pr ovide short-liv e d, limited neu tr alizing respo nses ag ains t A(H5N1), this c ov er a g e is unlik ely to be suf ficient in the ev e n t of an A(H5N1) pand emic. In the same NHP s described above prime d with qNIV (c ont aining 2023-2024 influenz a s tr ains), a single IM or I N dose of an A/ A W/SC/2021 H5-M NP v accine incr eased n eutralizing antibo dy tit e r s ag ains t t he homologous A(H5N1) s tr ain, with geome tric mean ti ter s abov e the 1:40 th r esh old . A single IM or IN dose w as suf ficien t t o seroc on vert 100% or 80 % of qNIV-primed NHP s, and tw o I N dose s w er e requi r ed t o induce neut r alizing responses in 100% of qNIV-primed NHP s. R obust a n tigen-spec ific CD 4 + T cell r espons es w ere also obser v ed in PBMCs aft er th e H5-MNP boost. T ak e n t o g eth er , the results demons trat e the po t e n ti al application of H5-MNP as a pandemic v accine in a pr e-i mmune population . Our results a r e c onsi s tent with thos e of a pr evious s tudy , wher e an inactiva ted wh ole virus A(H5N1) v accine w as adminis tered in mice aft er t w o par e nt er a l priming doses of inactiv ated seasonal influenza .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 November 21, 2024. ; https://doi.org/10.1101/2024.11.21.624712doi: bioRxiv preprint 13 v accine incr ease d the a n ti body ti t er s ag ai ns t A(H5N1) s tr ains compared t o a single dose of the A(H5N1) v accine alone 27 . Ou r findings highligh t th e potential use of th e A/ A W/SC/2021 H5-MNP v accine as a pandemic v accine, alt hough it is s till uncl ear if additio nal doses w oul d be required in adults with a more es tablished immune r ep er t oire f ollowing inf ections and v accinations t o seas onal influenz a, c onsid ering s tudies in p r e-immune human popul atio ns adminis ter e d inactiv ated influenz a vir us v accines obser v ed that tw o o r th r ee IN doses were r eq uir e d t o induce se r oc o n v er sion r espo nses 29,30 . R eg ar ding muc osal r espons es, anti-A/ A W /SC/2021 HA IgA an tibody ti t er s in th e up per respir at o ry tr ac t (nasal w ash) of NHP s incr eased signific antly aft er on e IM boo s ter dos e or tw o IN b oos ter dos es of the H5-MNP v accine; une xp ectedly , ti t er s we r e higher af t er the I M dose th an aft e r th e IN dose . IM adminis tra tio n of H5-MNP also signific antly incr eased anti-A/ A W/SC/2021 HA IgA and IgG a n tibo dy titer s in the low e r r espirat o ry tr act (BAL), but I N adminis tra ti on did not signific a n tly ind uce IgA antibodi es in this c ompar tme n t. Th ese NHP r esul ts c o n tr a s t our findings in mice and also contras ts with c on ventional par adigms that IN immuniza ti on pr oduc e s superior muc os al r espo nses tha n IM im muniz a ti on 24 . As with all influenz a HAs, th e major surf a ce gly c opr o t ein HA is r esponsibl e f or mos t elicited humor al immune r espons es, but t he immunodom inan t h ead domain mu ta tes r apidly . Thus , v accines ideally elicit immunity ag ains t more c onser ved epi t op es on the HA p r o t ein t o increase the lik eli hood th a t t he v accine will pr ot ec t ag ains t h omologous and drifted A(H5N1) s tr ains. For ex ample , clinic al s tudies ha ve shown cr oss-pr otectiv e immun e r espo nses ag ai ns t he t erologous A(H5N1) s tr ains in pr evi ously une xpose d adults immuniz ed with single IM injection of adj uv an ted A(H5N1) v accine (deriv ed fr om A/turk ey/T urk ey/1/05 HA) 11 . W e e xpec t our nov el H5-M NP v accine will elicit cr oss-neutralizing responses ag ains t oth er A(H5N1) s tr ains because NHP s primed wi th a high dose, adjuv a n ted qN IV v accine f ollow ed by one or two IM doses of the H5-M NP v accine boost e d the lev els of neu tr alizing a n tib odies th at t a r g e t highly c onser v ed cri tic al r esidu es within HA RBS , VE subdomain, and s tem, and th e mAbs w e isolat ed fr om H5- MNP v accina ted mice show ed cr oss-neu t r alizing activity ag ains t tw o he t erologous influenz a A(H5N1) s tr ains. Fu rth ermo r e, he t e r osubtype cross-r eaction h as been shown in some s t udi es t o b e mediated by neutr ali zing antibodies that tar g e t th e H5 HA 28,31,32 . Competition binning s tudies d emons trat ed induc tion of A(H5N1) neutr alizing antibodi es aft e r priming with a qNIV v accine in NHP s. In p articular , NVX.361.4 mAb binding the HA R BS is c onsist e n t wit h its s tr ong n eutr ali z ation activi ty since t he RBS binds t o sialic acid f or cellular binding 33 . NVX.73.2 mAb, which binds the VE subdomain, e xhibi te d less pot e n t neutr ali zing activity than NVX.361.4 mAb . The VE subdomain has c onser v ed epi t o pes within influenz a subtypes and ma y be important f or br ea dth of r eac tivity ag ains t H5- or subtype-s pecific r esponses . The .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 November 21, 2024. ; https://doi.org/10.1101/2024.11.21.624712doi: bioRxiv preprint 14 s t em domai n is mor e highly c onser ved a mong influenz a A subtypes than the he a d domain 34 and c ould induce br oad er cr oss-reactive immunity both within and be tw e en subtypes, whic h ma y support pr otectio n fr om nov el, eme r ging s tr ains . This nov el A/ A W/SC/2021 5 -MNP pr esents adv ant ages when c ompa r ed with o the r A(H5N1) v accines that are licensed o r in dev elopm ent. A re cen t an aly sis of pr eviously s t ockpil ed A(H5N1) v accines ag ains t s tr ains of A(H5N1) fr om the ea rly 2000s ( A/Vietnam/1194/2004; clade 1, and A/In donesia/5/2005; clade 2.1) sug g es ts that th ese v accines do elicit some an tib ody-mediat ed immuni ty ag ains t currently cir cula ti ng A(H5N1) s tr ains, th ough this is lik ely not as e f f ectiv e as a str ain-specific v accine 35 . R ecent v accine dev elopme n ts include a nasal spra y A(H5N1) v accine ag ains t A/Vietnam/1203/2004 appr ov ed f or use in the Eur op ean Union 36 and a no v el mRNA lipid nan opar ticle v accine f or t he A(H5N1) 2.3.4.4b clade A/ As trakhan/3212/2020 in pr eclini c al dev elopm ent 37 . Impor t a n tly , our nov e l A/ A W/SC/2021 H5 - MNP v accine ma y include f a v or able temper ature s tability f or st or age and transpo rt (2–8 °C) using a w ell- es tablished, n on-r epli ca tiv e , pr o t ein-b ased v accine technology . Our findings indic ate th a t a single I M dos e of an A/ A W/SC/2021 H5-MNP v accine migh t ser v e as an e f f ec tiv e pan demic v accine in individuals with pr e-exis ti ng seasonal influenz a H A i mmunity fr om v accina tion o r inf ection . T w o I N doses mi gh t be c o nsidered as an a t-home self-ad minis t e r ed b oost er in seasonal influenz a-prim ed individuals du ring a pandemic and a single I N H5-MNP v accine dose c ould b e v aluable f or pandemic prepa r edn ess. METHO DS Vaccine Cons tructs The wild type HA and neuraminidase (NA ) pr ot ein se quences of influenz a A/ Ame ri c an Wigeon/South Car olina/USD A-000345-001/2021 (A(H5 N1)) and wild type sequences of HA inclu ded in qNIV (A/Wisc onsin/67/2022 (H1N1), A/ Darwin/6/2021 (H3N2), B/ Aus tria/1359417/2021 (B/Vict oria), and B/Phuk et/3073/2013 (B/Y amag a t a)), w ere downloaded fr om publish ed sequenc es in the GISA ID Epiflu dat abase (accession numb er s EPI_ISL_18 133029, EPI_ISL_15928538, EPI_ISL_156 3628, EPI_ISL_983345, and EPI_ISL_161843, respec tiv ely). All H A and NA genes and the A(H5N1) M1 g en e w ere c odon optimiz ed f or high-lev el e xp r ession in Spodoptera frugiperda (Sf9) insect cells 38,39 . The HA and NA genes w er e ch emic ally s yn thesized by GenScrip t (Pisc a t aw a y , NJ, USA) and the A(H5N1) M1 g ene w as s yn thesiz ed by Gen eAr t AG (R egensbur g , German y). As pr eviously describ ed, HA n anopar ticles w e r e pr oduced using full leng th HA g ene clon e d betw e en BamHI and Hin dII I sites downs tream fr om a .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 November 21, 2024. ; https://doi.org/10.1101/2024.11.21.624712doi: bioRxiv preprint 15 polyhedrin pr om oter in pBac1 baculoviru s tr ans f e r v ec t or ( Millipo r e Sigma, Bill erica, MA , USA ) 40 . T o in troduce th e ΔKRRK dele tion in th e A/ A W/SC/2021 HA g ene (c orr esp onding to residues 341-344), the QuikChang e® Ligh tning si t e-directed mutag enesis kit w as us ed (Agilent, Sant a Clara, CA, USA). F or H A nanopar ticles, pB ac1 plasmids c ont aini ng each HA g en e w ere c o-trans f ected int o S f22A cells with the flashBac™ GOLD bacmid c ont aini ng Autographa californica mul tinuclea r polyhedrosis virus g enome (Oxf or d Expr ession T echnol ogy , Oxf or d, U K) and X-tr emeGENE™ HP r e ag e n t (R och e, Indian apolis, I N, USA). Influenz a VLP w er e pr oduc ed using full leng th HA and NA g enes sp ecific f or each s tr ai n c ombined with influenz a A/Indon esia/05/2005 M1. HA, N A and M1 genes w e r e clon ed between BamHI and HindI II sit es downs t r e am fr om a polyhedrin pro mot e r in the p F astBac1™ baculovirus tra ns f er v ec t o r (In vitr ogen, Carlsbad, CA, USA) and subclon ed int o t ri ple t an dem v ec t or s including HA/N A/M1 g enes as previously r epo rted 41 . R ecombinant baculovirus exp r essing H5, N1, and M1 g enes were g en e r at ed using th e Bac-t o- Bac™ baculovirus e xp r ession s y st em (In vi tr ogen, Carlsbad , CA, USA). F o r e xp r essio n of VLP , Sf22A cells w er e tr ansf ec t ed with B acmid using X-tr e meGENE HP r ea g ent (R oche, Indi anapolis , IN, USA). SDS-P A GE and W es t ern Blot ting R ec ombina n t A(H5N1) A/ A W/SC/2021 H A pr o t ein w as purifi ed by TMAE anion e xchang e, Cap t o Blue , and Capt o Len t il lectin af finity chr om at ogr ap h y . Purified HA pr o t ein w as ev alu at ed by r educed 4 –12% gr adien t SDS-P AGE st ain ed with SimplyBl ue™ Saf eS t ain Coomassie r eagent (Ther mo Fisher Scientific, W altham, M A , USA), and the id entity of HA w as c onfirmed by w es tern blo t using sheep a n ti-H5 HA a nt i b o d y ( N o vavax , Inc , Gai ther sb ur g , M D , USA). The purity of HA w as determine d by densit omet ry using a Bio-Rad GS-900 Calibr ated Densitometer (Bio-Rad, He r cules, CA , USA). Electr on Micr os cop y Electr on micr oscopy w as perf ormed at th e cry oEM f acility in the Univer sity of Ma r yland (IBBR, UMB R ockville, MD , USA) using the FEI Ar cti c a Electr on Microsc op e (Thermo Fisher Scie n tific, USA), operat ed a t 2000i:jkV equipped wit h G a tan K3® dir e ct elect r on de t ec t or . Samples w e r e ei the r subject ed t o NS-TEM pr ocedu r es or single pa rticle analy sis f or high r esoluti on dat a . F or a naly sis of HA p r otein alon e or H5- MNP s, 120 µg /mL of H5N1 HA pr ot ei n w as analy z ed alon e or mi x ed with 75 µg /mL of Ma trix M-in 25 mM NaP , 300 mM NaCl, 0.01% PS80 and incubat ed a t 4 °C un til t est e d. F o r visualiz a tio n of mAb binding t o H A, mAbs w e r e digest ed into fr agments (F ab) using a kit and f ollowing the manuf actur er ’ s ins tr uctions (Pier ce F ab Pr eparation kit, The rmoFishe r Scien tific). The F abs w er e mi x ed wit h purified HA and incubat ed f or 30 min a t a 1 :10 molar ra ti o (an tigen: a n tibody F ab, in 1X PBS buf f e r be f ore NS-TEM grid pr eparation. .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 November 21, 2024. ; https://doi.org/10.1101/2024.11.21.624712doi: bioRxiv preprint 16 F or NS-TEM; 4 µL of undilut ed (10-f old dilut ed whe n r equi r ed) sample w as d eposi t ed on a glow dischar g ed c arbo n c ontinuo us grid f ollow ed by a 1-min incuba tion at r oom t empe ra tu r e (25 °C). The grid w as then w ash ed in deioniz e d w at e r thre e times with g e n tl e blo t ting and 35 µL dr oplets of 1% ur an yl f orma te (UF) dy e w er e ad ded twice with a 10–30-s incuba tion each time . Ex cess s tain w as r emov ed by g entle blo t ting and the grids wer e d ried a t 25 °C ov ernigh t . Images of each grid wer e acqui r ed a t multiple magnific ations t o assess th e ov e r all dis trib ution of th e sample . High-magnific a ti on imag es w e r e acquir ed a t a 79,000 nomin al magnific ati on, 0.769 Å/pix el. The ima g es w e r e acqui r ed a t a nominal de f ocus of −2.00i:jµm t o −1 .50i:jµm with t o tal elect r on doses of ~36.90i:j e/ Å2. F o r class a v er aging , particles w er e id entified fr om ×92,000 high-magni fic a tio n imag es, f ollow ed by alignment a nd 2D classific a tion (Ta b l e S 1 ). F or cry oEM single pa rticl e analy sis, 3 µL sample w as applied t o glow dischar ged Q uan tif oil® R1.2/1.3 Cu 300 mesh grid (Electr on Micr osc opy Scie nces, P A, USA). The sampl es w e r e th en pl ung ed int o liqu id ethane using a Vitrobot ™ Mark IV (Ther mo Fisher Scientific, USA) using g ap check pr ocedures a t 100% humidity a t 12 °C and w ere subseque n tly flash fr o z en in liquid ethan e, clipped, an d s t o r ed in liquid nitr ogen at th e Univ er si ty of Maryland, B altimore-IBBR CryoEM f acility . Da ta sets w er e c ollec t ed as summariz ed in Ta b l e S 1 . 2D Clas sifica tion and 3D r econs truction The NS-TEM or Cry oEM dat a sets wer e tr ans f er r ed t o Cry oSP AR C V4.3.0 softw a re f or pr ocessing 42 . Blob pick er w as used t o ob t ain ini tial par ticle s t ack s with 100-400 Å siz e limit and pa rticles extracted with a 512 pix el bo x (f or HA nanopar ticle wit h or without F abs) or 100-1000 Å (t o acc ommodate Matrix-M particles) extracted with 1024 pi x el box subjected to f ourier cr o p down t o 1 28 pix els o r 256 pix els r espec tiv ely . 2D classes r esembling known s tructure of the hemag glutinin pr o tein w ere selec t ed fr om initial 2D classific a ti on and used t o create t empl at es, r e-pick par ticles using templat e pick er an d subjec t r esulti ng particle st ack t o sev eral cy cles of 2D classific a tion and filtering. Following r ounds of 2D classific a tion , the b es t 2D class w as used t o create a 3D classific a tion yielding map . Model building The s truct ur al model of HA-A/ A W/SC/2021 and HA with mAbs w as pr edic t ed usin g AlphaF old 43 . Pr edicted models w e r e then fitt e d t o the c orresponding EM maps using USCF Chi mer aX 44 . The surf ace, c ar t oon , and s tick represe n ta ti on of s truc tur al models wer e p r ep ared in USCF Chimer aX 44 . Dynamic Ligh t Sca t t ering Dynamic ligh t sc a ttering w as pe rf ormed using Z et asiz er Ult r a (Malv er n P analytical L t d, UK) a t a pr o t ei n c oncentra tion of 1.0 mg /mL in low v olu me disposable ZEN0040 cuv ett e (Malv e rn P analytic al L t d , UK). .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 November 21, 2024. ; https://doi.org/10.1101/2024.11.21.624712doi: bioRxiv preprint 17 The Z-a v er ag e siz e and poly disper sity inde x (PDI) w er e obt ained using the c umulan t ana ly sis with r epe at abili ty . Int e nsity w eighed siz e dis tribu tion w as obt ai ned by the g ene r al method a t 25 °C, r espec tiv ely . Diff er en tial Scanning Calorimetry (DSC) DSC w as perf ormed using Micr oCal PE A Q-DSC (Malv ern P analytic al Lt d, UK). Samp le (a t a c onc entr ation of 1.0 mg /mL ) w as deg assed f or 15 min ( 16 Hg /mm) a t 25°C prior t o an aly sis. DSC measur eme n t w as initiated immediately aft er sample d eg as sing and pr otein sample w as sc a nned fr o m 20°C t o 100°C a t 1°C /min using f ormula tion buff er as a re f e r e nce. Acquired d a ta w as buf f er sub tr ac t ed , c on v er ted to molar heat c apaci ty (ΔH), and baseline c or r ec t e d using Micr oCal PE A Q-DSC Softw ar e (Malv ern P analyti c al L t d, UK). The obser v ed peak tr a nsitions a r e re ported at melti ng t emp er ature (T m ) a t th e ma ximum he a t c apacity (obser v ed und er each p eak; ΔH cal). H i g h P re s s u re - S i ze E xc l u s i o n C h rom ato g ra p hy ( H P - S EC ) A/ A W/SC/2021 HA (30 µg /mL ) w as mix ed with Ma t rix-M (150 µg /mL ), f ollow ed b y incuba tion a t 25 °C f or 12 h. F or HP-SE C analy sis, the mix ture w as char act e riz ed after 2, 4, 6, 8, and 12 h. An Acclaim SE C 1000 c olumn (ThermoFisher Scie n tific, Frederick, MD , USA) w as equilibrat ed wit h 25 mM Sodium Phosphat e, 300 mM Sodium Chloride , 0. 01% PS80, pH 7.2 mobile phase a t 0 .3 mL/min a t 25 °C. 85 µL of the mix ture w as injected to the HP-SE C column f or HA and Matrix-M bin ding kinetics analy sis. 85 µL of HA (30 µg / mL) and 85 µL o f Ma trix-M (1 50µg /mL ) w er e inject ed i nt o th e c olumn individually as c on t r ols a t t he same run ning c ondi tions abov e . Da ta analy sis and calculation of th e per c ent HA bound t o M a t rix w as perf ormed using Agile n t Op enLab CDS2 softw ar e (Agilent T echnologies, Sa v a g e, MD , USA). The per ce n tag e of HA bou nd to Matri x w as calculat ed by c ompari ng the surf ace a r ea of bound HA t o H A alone a t tim e 0 (T0). Animal E thics St a t emen t The r epo rting in this manuscrip t f ollow s the r e c ommend a tio ns in the AR RIVE guidelines. The mous e s tudies wer e c onduc t ed a t Noble Lif e Sci ences (S yk es ville, MD , USA). Animals w e re maint ained and tr e at ed ac c or di ng t o An imal W elf are Act R egulations, th e US Public Healt h Ser vice Of fice of Labor at ory Animal W elf are P olicy on Humane Car e a nd Use of Labor at o ry Animals, Gui de f or Car e and Use of Labor at o ry Animals (Ins ti tu t e of Labora to ry Animal R esou r ces, Commission on Lif e Sciences, N a tion al R esear ch Council, 1996), and A AALACi accr edi t ation . Mouse studies wer e appr ov e d by Noble Lif e Sciences Institu tional A nimal Car e and Use Commit t e e (IACUC). The s tudy in rhesu s mac aques w as c onduc t ed a t T e x as Biom edic al Resea r ch Ins ti tu t e (San Antonio , TX, USA). Animals w er e mai nt aine d a t .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 November 21, 2024. ; https://doi.org/10.1101/2024.11.21.624712doi: bioRxiv preprint 18 T e x as Biomedical R esea r ch Institu te f or t he entire in-lif e por tion of th e s tudy and w er e treated acc o r ding t o Animal W elf ar e Act regulations and th e Guide f or th e Car e and Use of Labora to ry Animals (2011). Rhesus mac aqu e s tudi es w e r e app r ov ed by T e x as Biomedic al R ese ar ch In s ti tute IA CUC. Mouse Study Design F emale B ALB/ c mice ( Mus musculus ; 10– 11 w eek s old, weighing 16–20 g; En vigo , Fr ede rick, MD , USA) w er e p r ovided ad libit um access t o f ood and w at er . The mice w e r e r and omiz ed int o f our gr oups (n0i:j=0i:j3– 10 per gr oup) and immuniz ed by either i n tr amuscula r (IM) injection or intr an asal (IN) adminis tr ation with tw o doses of ei ther f ormul a tio n buff er (placebo; IN; n = 3) or A(H5N1) A/ A W/SC/22 /2021 HA ( N o vavax , Inc ., this c o ns t ruct used f or im muniz a ti on did not poss ess the ΔKRRK muta tion) with M a tri x-M adjuv an t (Nov a v ax, AB, Uppsala, SE) on S tudy Da y 0 and 21 (n = 10 ). The thr ee tr e a tme n t gr o ups r eceived 10i:jμg of A/ A W/SC /2021 HA and 5 μg Ma trix-M adjuv a n t (IM), 10i:jμg of A/ A W/SC/2021 HA with 5 μg Ma trix-M adjuv a n t (IN), or 100i:jμg of A/ A W/SC/2021 HA with 5 μg Ma trix-M adj uv an t (IN). Se rum w as c ollec t ed on S tudy Da y 34/35, t o ev aluate hemag glutinin inhibiti on and pseudovir us neutr alization. Br onchoalveola r la v ag e (BAL) samples w er e collec t ed fr om six of th e mice in the vaccine gr oups and all in placebo gr oup on S tudy Da y 34/35. Lung s w er e collec t ed fr om six of th e mice in t he IN t r e at ed gr o ups on Study Da y 34/35. Spleens w er e collec t ed fr om six of the mice in each tre a tme n t g r oup on Study Da y 34/35. Nonhuman Prima t e Study Design F or th e primary immuniza tion s eries, t e n rhesus mac aqu es ( Macaca mulatta , 4–11 y ear s old, w eighing 6–17 kg) w er e r andomiz ed into tw o t r e at men t gr oups (n=5 pe r gr oup) and v accinat ed by IM injecti on with tw o doses of quad riv ale n t nan opar t icle influenz a v accine (qNIV) f or 2023-20 24 season flu s tr ains (A/Darwin/6/2021 /H3N2, A/W es t Vir gini a/30/2022/H1N1, B/ Aus tria/1359417/2021, and B/Phuk et/3073/2013; 60 µg HA per s tr ai n) c o-f ormula t e d with 75 µg Ma tri x-M ad juv an t (IM) on Study Da y s 0 and 21. Animals w ere administ e r e d the fir s t bo ost er d ose of H5-MNP v accine (full-leng th HA without th e ΔKRRK mutation) on Study Da y 83 (8 w eek s aft er the se c ond dos e of qNIV) via IM or IN r outes and a se c ond dose of th e H5-MNP v accine on Study Da y 139 (8 w eek s aft er the fir s t d ose of H5- MNP). NHP s r eceiving IM boo s ter doses w er e giv e n 600i:jμg A/ A W/SC /2021 HA f or both doses ; NHP s r eceiving IN bo ost er d oses w e r e giv en 24 00i:jμg A/ A W/SC /2021 HA f or the fir s t dos e and 600i:jμg A/ A W/SC/2021 HA f or the sec ond d ose. J us t as f or the prim ary seri es, all booster doses w ere adminis tered with 75 µg Ma tri x-M adjuv an t . Serum w as collec t ed f or analy sis on Study Da y s 0, 35, 83, 97, 139, and 153. P eripher al bl ood mono nuclear cells (PBMCs) w er e c ollec ted on Study Da y s 0, 35, 83, 97, and 153. B ALs and nas al w ashes w e r e c ompleted on Study Da y s 35, 97, and 15 3. .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 November 21, 2024. ; https://doi.org/10.1101/2024.11.21.624712doi: bioRxiv preprint 19 Hemag glutinin Inhibitio n (HAI) Assa y HAI responses ag ainst influenz a A/ A W/SC/2021 w er e ev aluated in mouse and N HP serum samples, and mAb HAI activity ag ainst A/ A W/SC/2021, A/Color ado/18/2022, and A/Mink/Spain/3691-8 w er e also ev aluat ed . A 1% suspension of hor se r ed blood cells (RBC; Lampir e Biologi c al Labo r at ori es, Piper s ville, P A, USA) w as pr e pared in Dulbecc o 's pho sphat e-buf f er e d saline (DPBS). Serum samples w er e treated with r ecep t or-d es t r oying enz yme (RDE) f r om Vibrio cholerae (Denk a Seik en , Stamf or d, TX, USA) ov ernight at 37 °C, t o eliminat e n onspeci fic RBC hemag glutina ting activity . RDE w as inactiv at ed the n e x t da y by incuba tion at 56 °C f or 1 h. RDE -tr eat ed s er a or mAb were serially dilu t ed 2 -f old in D PBS (s t arting a t 1:10, 25 µL) in 96-w ell, V bot t om pl at e s and incubat ed with st and ar dized influenz a virus c oncentra tion (4 HA units in 25 μL ) f or 25 min. A t the e nd of the incubation, 1% suspension of h or se RBC (50 µL ) w er e added t o each well and th e plates w ere i ncubat ed a t 25 °C f or 45 min. HAI w as det e rmined by obser ving when the non-ag glutin at ed RBCs, in th e t ilt ed pl at e posi tion, st a rt t o run down f or ming a t ea r dr op butt on in th e sample w ells a nd in the n e g a tiv e contr ol wells. The HA I ti t er s were rec orded as th e r ecipr o c al of the high es t se rum dilutio n wher e HA I w as obser v ed (last w ell with a t ea r dr o p butt on). For a titer below th e assa y limit of det ec tion (L OD), a tit e r of <10 (s t ar ting diluti on) w as r epo rted and a v alu e “5” assigned t o the sample . P seudovirus Neutr aliz a tion The pseudovirus neutr al iz ation assa y w as dev eloped using a le n tivir al backbon e with dual repor t e r (lucZ and gr een fluo r esce n t p r o t ein) including the c ogn at e influenz a surf ace gly c o pr o t ei ns HA and NA f or A/ Americ an Wig eo n/South Car oli na/22/000345-001/2021 (A(H5N1)). E ach p seudovirus w as tr ans f ec t e d using the Je tOptimus (P olyplus, New Y or k, NY , USA) tr ans f ectio n r ea g e n t using HE K293T cells; har v es ted 48 h pos t tr an s f ec tion; ce n t rifug ed and fi lt e r ed using a 0.45-micr on filter . S er a fr o m NHP s or mice w er e tr e at ed with RDE prio r to he a t-inactiva ti on, as describe d abov e. S er a w e r e dilu te d t o a st ar ting diluti on of 1:20 in r educed se rum Opti-MEM (Gib c o/Thermo Fisher Sci entific, W altham , M A, USA) and seri ally dilut e d in a 96-w ell opaqu e plat e (R evvity , W altham, MA , USA). T o ev alua te neu tr a lizing activity of purified mouse monoclon al antibodi es, mAbs w er e dilu t ed t o a s t a rting concentra tio n of 5 µg /m L and serially diluted th r e e-f old t o de t ermin e t he 50% inhibit o ry c once n tra ti on (IC 50 ). C ommer cially a v ailable , char acteriz ed monoclo nal antibodi es (CR6261 (Absolut e A n tibody , Shirley , M A, US A), CR8020 (Absolut e An tib ody), and CR9114 (Cr ea tiv e Bi olabs, Shirley , NY , USA)) w er e included wit h ea ch run as w ell as an in tr a-pl at e c o n t r ol of pseudovirus only or cell only as the 0% t o 100% neutraliza tio n v alues, r esp ectiv ely , f or each plat e . After ser a o r monoclonal an tib odies w e r e dilu ted, each pseudovir us, diluted to t a r g e t 100,000–250,000 r el a tive luminescenc e units, w as adde d to all w ells of each pl a te e x cept f or th e cell .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 November 21, 2024. ; https://doi.org/10.1101/2024.11.21.624712doi: bioRxiv preprint 20 c ontr ol only . Plates w ere incubated at 37 °C and 5% C O 2 f or 1 h. F ollowing incuba ti on, 100 µL of a t 2.5 × 10 5 HEK-293 cells/m L , supplement ed wit h 1 µg /m L N-t os yl-L-phen ylalanine chlorometh yl k et on e-treat e d trypsin in HEK293 assa y diluen t cell medi a (Dulbecc o’ s Modified E agle M edium, 5 % hea t-inac tiv at ed f e t al bovine serum and 1% P enicillin/Str ep t o m y cin/Glut amine) w ere add ed to each well. Plat es w e r e then incubat ed a t 37 °C and 5% C O 2 f or 72 h. T hen, 50 µL of Brigh t Glo Lucif er as e (Pr omeg a, Madison, WI, USA) subs tr at e w as add ed to each w ell a nd incubat ed f or 5 min in the absenc e of ligh t. Plates w ere r e ad on ID3 Spectr aM ax® (Molecular Devices, Sunn yv ale, CA, USA). The pseu dovirus-only and cell-only c ontr ols ser v ed as the 0% and 100% neut r aliz ation v alues, r esp ectiv ely . Dat a were analy z ed using Gr aphP ad® Prism (La Jolla, CA, USA) and 50% pseudovirus neutr aliza tion ti t er s (pVN 50 ) w er e c alcul at ed using sigmoidal 4-par amet er cur v e fi t ting . An ti-A/ A W/SC/2021 HA IgG and IgA by ELIS A The HA pr o t ein IgG ELISA w as used t o de termine a n ti-A/ A W/SC/2021 HA IgG ti t er s in ser a, nasal w ash, and br onchoalveola r la v ag e (BAL) and HA pr otein IgA ELISA w as used t o de t e rmine an ti-A/ A W/SC/2021 HA IgA ti t e r s in nasal w ash and B AL. Bri e f ly , 96-w ell micr otit e r plat es (Thermo Fish er Scientific, R ochest er , N Y , USA) w ere c o at ed with 1.0 µg /m L of A/ A W/S C/2021 HA pr ot ein (No vavax ) . P l a te s we re w ashed with PBS-T and non-specific binding w as block ed with TBS St ar tblock bloc king buf f er (Thermo Fisher Scientific, R ochest er , NY , USA). Mo nk ey serum w as serially diluted 3-f old, s tarting with a 1:100 dilution f or th e IgG ELISA, and nasal w as h or B AL samples wer e s erially dilu t ed 2-f old, s tarting with a 1 :2 dilution f or both IgG and Ig A ELISA. Diluted samples w e r e add ed to the c o a ted plat es and incub at ed a t 25 °C f or 2 h. F ollowing incuba tion, pl at e s w er e w ash ed with PBS-T . F or th e IgG EL ISA, pl at es w e r e incubat ed with HRP-c onjug ated mouse a n ti-monk ey IgG or goat anti-mouse IgG (S outhe rn Bio t ech, Birmingham, AL , USA) f or 1 h. F or the IgA ELISA, mouse anti-monk ey IgA (Bio-Rad, Her cules, C A, USA) w as added t o th e plat es . F ollowing a 1-h incuba ti on, pl a tes w e r e w ashe d with PBS-T and incuba ted with HRP-c onjug a t e d goa t a n ti-mouse IgG (Southern Bio t ech, Bi rmingham, AL , USA) 1 h. Subseque n tly , all plat es w e r e w ash ed with PBS-T and incubat ed with 3,3 ’ ,5,5’- t etrameth ylbenzidi n e (TMB) per o xidase subs tr at e (Sigma, St . Louis, MO , USA) unt il r eacti ons w ere st opp ed with TMB st op solution (ScyT ek Labor at ori es, Inc . Log an, UT). Pla tes w ere r ead a t OD 450 nm with a SpectraMax Plus pla te reade r (Molecular Devices, Sunn yv ale, CA , USA). E C50 an ti-HA IgG t iter s w e r e calculat e d by 4-par amet er fitting using SoftMax Pr o 6.5.1 GxP softw a r e . F o r an IgG ti ter below th e assa y low er limit of det ecti on (L OD), a titer of <100 (s t a rting dilu tion) w as r epo r t ed an d a v alue of “50” assigned. The OD v alue a t a 1 :2 dilution w as used t o r ep r ese n t IgA r esp onse in na sal w ash and B AL. Cellular Assa ys .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 November 21, 2024. ; https://doi.org/10.1101/2024.11.21.624712doi: bioRxiv preprint 21 F or th e in tracellula r cyt okin e s taining ass a y of NHP PBMCs, the cells w er e thawed and r ested at 37 °C ov ernight. The cells w e r e then stimulate d with Influenz a HA pr o tein of the homol ogous A(H5N1) s tr ain. Cells w er e lab eled with huma n/NHP an ti bodies B V650-c onjug at ed a n ti-CD3 (Clone SP34-2, 1:10), APC- H7–c onjug at ed anti-CD4 ( Clone L200, 1:10), APC-c onjug a t ed anti-CD8 (Clone RP A-T8, 1:10), and the y ellow LIVE/DE AD® dy e (1:300) f or surf ac e s t ai ning; B V421-c onjug at ed a n ti –IL-2 (Clone MQ1-17H12, 1:25), P erCP- Cy5.5–c onjug a ted anti– IFN- γ (Clone 4S. B3, 1:10), and PE-cy7-c onjug at ed a n ti –TNF-α (Clone Mab11, 1:50) (BD Biosciences, Fr anklin Lak es, N J , USA) f or in tr acell ular st aining . A ll s t ain ed samples w e r e acquir ed using an LSR-F or tessa™ flow cyt omet e r or S ymphon y A3 (Becton Dickinson, San Jos e, CA, USA) and the d at a w e r e an aly z ed with FlowJo softw ar e ver sion 10 (T r ee S t a r Inc., Ashla nd, OR, USA). F or intr acellul ar cytokine st aining (ICCS) assa y of mouse spleen cells, th e single ce ll suspension w as pr epa r ed a nd cultured in a 96-w ell U bo tt om pl a te at 2×10 6 cells per well. The cell s w er e stimulated with Influenz a HA pr o tein of the homol ogous A(H5N1) s tr ain. The pl at e w as incub at ed 6 h a t 370i:j°C in the pr esenc e of BD GolgiPlug™ and BD GolgiSt op™ (BD Biosciences) f or the la s t 40i:jh o f incuba tion . Cells w ere labeled with muri ne a n tibod ies ag ainst CD3 (B V650 ), CD4 (APC -H7), CD8 (FIT C ), C D44 (Ale x a Fluor 700), and CD62L (PE ) (BD Pharming en, CA) and y ellow LIVE/ DE AD® dy e. Aft er fixation with Cyt ofix/Cyt op erm (BD Biosciences), cells w er e incub at ed wi th P erCP-Cy 5.5–c onjug at ed a n ti –IF N-γ, BV421-c onjug a t ed a n ti – IL-2, PE-C y7–c onjug a t e d anti–TNF-α, and APC-c onjug a t ed a n ti –IL-4 (BD Biosciences). F or ICCS assa y of mouse lung cells, murine lungs w e r e min ced and tre a ted with c oll ag en ase type I at 370i:j°C f or 1 h. A single-cell suspension w as prepared, t he n the cells w e r e stimulat e d in the sam e manner as sple en cells. Cells w er e lab eled with muri ne a n tibod ie s ag ains t CD3 (B V650), CD4 (APC -H7), C D 8 (B V711), C D11a (FIT C), CD44 (Ale x a Fluor 700), and CD62L (P E) (BD Pharming en, CA) and y ellow LI VE/DE AD® dy e. Aft er fix a tio n with Cyt ofix/Cyt o perm (BD Biosciences), cells w ere incubated with P erCP-Cy5.5–c onjug at ed an ti –IF N-γ, B V421-c onjug at ed anti–IL-2, PE-C y7–c onjug a ted anti–T NF-α, and APC-c onjug at ed a n ti –IL-4 (BD Biosciences). All s t ained sampl es wer e acqui r ed using an LSR-F o rtessa or a F ACS ymphon y™ flow cyt ome t er (Bec t on Dickinson, San J ose, C A) and the d a ta w ere analy zed with FlowJo softw a r e v er si on X v10 (T r ee St ar Inc., Ashl and, O R, USA). Da ta shown w ere g a ted on CD44 hi CD62L low eff e c to r C D 4 + 0i:jT cell population (spleen cells) and CD11a + CD44 hi lung r eside n t CD4 + 0i:jT cell population (l ung cells). Mouse Monoclonal An tibody G ene r a tion T o g ener ate monoclonal a n tib odies ag ain s t A(H5N1) HA, f emale B ALB/ c mice ( M. musculus; n = 6) w ere immuniz ed with 5 µg A/ A W/S C/2021 HA and 5 µg Ma trix-M a djuv an t i n tr amuscul arly on Study Da y s 0 and 14. T w o mice with th e highest A/ A W /SC/2021 HA an tibody titer s w e r e sel ecte d and in traperi t o neal fusion boos ted with 5 µg A/ A W/SC/2021 HA on Study Da y 29 (no adjuv an t). Splee ns w er e har vest ed on .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 November 21, 2024. ; https://doi.org/10.1101/2024.11.21.624712doi: bioRxiv preprint 22 Study Da y 33, f ollow ed by splenocyt e c oll ection, isol a ti on of IgG-expressing cells by neg a tiv e sel ection , and h ybridoma fusion 45 . F our hund r ed a nd thir t e en h ybridoma supe rnat a n ts were scr een ed by A(H5N1) A/ A W/SC/2021 HA IgG ELISA (described abov e), and appr oximately 185 positiv e c lones w ere isol a ted. T w o h ybridomas with A(H5N1) pseudovirus neutr alizi ng activity w ere selec t ed f or subcloning , e xpansio n, purific ation, an d char ac t erization. R eal-Time Competition Bin ning P oly clonal ser a c ollec t ed on S tudy Da y s 35, 97, or 153 fr om NHP s immuniz ed as described abov e wer e t ested in c ompet ition binni ng assa y with monoclonal antibodi es and Competi tiv e An tib ody E quiv alent (CAE) lev els ag ains t A/ Americ an Wi g eon/ South Car olin a/22/000345-001/2021 (A( H5N1)) w er e measu r ed using BioLa y er Interf e r omet ry (BLI) on an Oct et R H96 ins trum ent (Sar t orius AG , G erman y). Assa y s w ere set up on Greine r Bio 96-w ell black plate s and AR2G bios ensor s (Sar t orius A G, G er man y) w er e pr eso ak ed f or a t lea s t 10 min in WFI wa ter a t 25 °C. Upon assa y s t a rt, A R2G sens or s w e r e eq uilibr at ed in W FI w ater f or 60 s t o es tablish a baseli ne, f ollow ed by biosensor activ ation in EDC/ s-NHS f or 300 s. A/ Americ an Wig eon/Sou th Car olin a/22/000345-001/ 2021 (A(H5N1)) an tig en a t 6 .37 µg /mL , d ilut ed in 10 mM sodium aceta te pH 6.0 (≈100 nM), w as th en c ouple d to the bios ensor f or 600 s, f ollow ed by a 300-s quenching s tep in eth anolamine t o bl ock non-specific binding t o th e sensor . Anot h er baselin e in 1x kinetics buf f er w as est a blished f or 60 s aft e r antig en immobilization and qu enchin g. Next, sens or s w e r e e xpos ed to poly clonal ser um samples (dilut ed in 1 x kinetics buf f e r) a t v arying c onc en trations f or 600 s. A D0 sensor f or each gr oup w as used as a n eg a tive c o n trol sensor while a posi tiv e co n trol w ell c o nt ained 5 µg /mL of each c ompeting antibody (CR6261, NVX.73.2, or NVX.361 .4) spik ed in to 1:50 D0 pool in 1x kinetics buf f er . Aft e r a bri e f 60-s w ash s t e p in 1x kinetics buf f er , sens or s w e r e immer sed in 5 µg /mL of each of the c ompe ting antibodi es f or a 600 -s associa tion/ c ompe ting an t ibody s te p. Da ta analy sis w as c ompleted using Octet An aly sis Studio so ftw ar e 12.2 (Sar t o rius AG, Go t tingen, G er man y) by normalizing the v alues t o St udy Da y 0, as a per cent age of the positive c o n trol v alue. Epit ope Mapping of HA-A/ A W/SC/2021 with molecular modeling and Se quen ce Analysis A molecular mod el of A/ A W/SC/2021 HA w as g enerat ed by the op en-sour ce , templat e-base d softw are SWISS-MODEL (h t tps://pubmed.ncbi .nlm .nih. gov/29788355/ ), and each pr omoter w as modeled main taining the H A0 s tr ucture as seen wi th int act trimer s in TEM dat a ( T able S1). HA-neutralizing mAb molecular models were g ene r at e d using AlphaF old software (h ttps:// alphaf old.eb i.ac.uk/). K nown PDBs (with mor e than 80% seque nce ide n ti ty) w er e us ed as templates (PDB ID: 6A0Z, 6 E3H, 5DUP , 2IB X, 6E7H) t o map th e epi t op es in head r egion . R esu lting models f or an ti g en-a n tib ody c omplex es wer e a naly z ed an d .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 November 21, 2024. ; https://doi.org/10.1101/2024.11.21.624712doi: bioRxiv preprint 23 dock ed int o EM densi ty maps using Chim er aX 46 and figur es w er e generat ed . Critical r esides withi n >5Å w er e p r edic t e d using Ellipr o 47 and labele d on 3D model r eprese n ta ti ons using Chimer aX 46 . St a ti s tical Analysis Geome tric mean ti ter (GMT) and 95% c onfidence in ter v al (95% CI) w er e c alculate d and plott ed , and s ta tistic al analy ses wer e p erf ormed using Gr aphP ad Prism® 10.2.2 softw are (Gr ap hP ad Softw ar e, La J olla, CA, USA). The Mann –Whi tney U T es t (two-t ailed) w as used t o compare dif f e r ence s betw een tw o gr oups and the K rusk al –W allis multipl e c ompa ris ons t est with Dunn’ s c omp arison w as use d t o c omp ar e dif f erences among th r ee groups. p v alues ≤ 0.05 w ere c onsid er e d s ta ti s tically signific an t . ADDI TIONAL I NFORM A TI ON R egis tered and pr o prie t a ry names, e. g., t r ademark s , used he r ein a r e no t to be c on sider ed un pr o t ec t ed by law ev en when not specific ally mark ed as such. Funding: This s tudy w as funded by No vavax , I n c . The sponso r w as in v olv ed in conc eptualiza ti on, design, dat a c oll ection an d analy sis. The autho r s w er e r esponsib le f or the final d ecision to publish and pr eparation of the manusc ript. A CKN O WLEDGE MENTS Autho r Con tri butio ns: Conceptuali z ation: N.P ., F .D ., G.S . In v e s tiga tion : N.P ., A .R ., J .F . T ., Z.L., M. G .-X., H.Z., B.Z., K. J., D . J . , X.B. , R.K. , T .K., G .S. Dat a An aly sis: N.P . , M.H, J.F . T . W riting— original d r aft: A. R. , M.M ., M.H ., A.M . G, J.F . T ., G.S. W ri ting—r eview and ed iting: M.H ., A .M.G. , N.P ., J .F . T . , M. G .-X., H. Z., K. J ., F .D ., G .S. St atistic al A naly sis- M.H. Visualization: A. R., M .M. , M.H ., A .M.G., N.P . Pr ojec t Admi nis tra tion : M.G.-X. Da t a A v ail ability: The d at asets generat ed during and/ or analy zed during th e current s tudy are a v ailabl e fr om the c or r esp onding auth or on reaso nable request. Competing Int e r ests: All au thor s a r e cur ren t o r f ormer employ e es of No vavax , Inc. , and ma y hold s t ock in N o vavax , I n c . .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 November 21, 2024. ; https://doi.org/10.1101/2024.11.21.624712doi: bioRxiv preprint 2 4 FIGURE S Figur e 1. Fi gur e 1: Cha r act eriz a tion of the r eco m b in a n t Infl uenz a A/ Americ an Wi g eon/South C ar olin a/22-000345- 001/2021 (A/ A W/S C/2021) hemag glut inin (HA) an tig en. (A) Linear diagram of the full-length HA from A(H5N1) clade 2.3.4.4b A/ A W/SC/2021 with the polybasic cleavage site deletion ΔKRRK. The diagram shows the HA construct from N- to C-terminus including the globular head region containing the signal peptide (SP , tan), and the stalk/stem region containing the fusion peptide (FP , red), transmembrane domain (TMD, yellow), and cytoplasmic 4 .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 November 21, 2024. ; https://doi.org/10.1101/2024.11.21.624712doi: bioRxiv preprint 25 t ail (CT , whit e) s t ruc tur al el emen ts. (B) R educ ed SDS- P AGE gel wi th C o om ass ie blu e s t a inin g of pu rifi ed r e co mbinan t A( H5N1) A / A W /S C/2021 HA (l ef t) and w es t ern bl ot (righ t) using an an ti-H5N 1 HA pr im ary an tibody t o c onfi rm the iden ti ty of the main pr ot ein pr oduct. (C) R epr e sen t a tiv e ele ct r on mi cr og r aphs of neg a tiv e s t a ined A(H5N1) A/ A W/ SC /2021 HA p r ot eins as pr e-f usion HA-t rim er s (y el l ow ar r o ws) anch or ed in det e r gen t (PS-80; whit e ar r o w s) mi cell es. Neg a tiv e s t aining TE M-2D c l ass a v er age i mag es of A/ A W/ SC /2021 HA nan opa rti cle s sh own wi th one o r tw o HA-t ri me r s ass oc ia t ed with d et e r gen t m ic elle. (D) R epr e sen t a ti on of a 3D-r e c o ns truct ed m odel of A/ A W/ SC /2021 HA f r om c ry oEM m ap a t 4.2 Å (l eft). A mol ecul ar mode l f o r A/ A W /SC /202 1 HA w a s g ene r a t ed us ing PDB ID 6HJR as a t empla t e and supe rimp os e d in t o EM densi ty t o r epr e sen t an in t ac t ful l-l eng th A/ A W /S C/2021 HA nanopa rti cle (f r on t v iew on the l eft and t op v iew on the righ t). .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 November 21, 2024. ; https://doi.org/10.1101/2024.11.21.624712doi: bioRxiv preprint 26 T able 1. P article siz e and therm os t ability of r eco mbinan t A(H5N1) HA nanopartic les Infl uenz a s tr ain Dyna m ic l igh t sca t t ering Diff er en ti al sc ann ing c alor im etry Z-a v g dia met er (nm) PDI T m (ºC) ΔH (kca l3i77mo l −1 ) T m1 T m2 ΔH 1 ΔH 2 A(H5N1) A/ A W/ SC /2021 HA 34.78 ± 0.28 0.16 ± 0.01 45.55 52.10 72.3 598.0 ΔH, c hange in enthalpy; PDI, po ly dispe r sity inde x; T m melt ing t emper atur e ; Z-avg, Z-a ver age .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 November 21, 2024. ; https://doi.org/10.1101/2024.11.21.624712doi: bioRxiv preprint 2 7 Figur e 2. Fi gur e 2: Structur al and bi oph y sic al cha r act e riz a tion o f A(H5N1) A/ A W/SC/2021 HA –Ma t rix nan opa rticle (M NP) f orma tion. NS-TEM 3D reconstructions of (A) an A(H5N1) A/ A W/SC/2021 HA nanoparticle and (B) a Matrix-M nanoparticle. (C) High-pressure siz e-ex clusion chromatography (HP-SEC) analysis of a mixture of 30 µg /mL A/ A W/SC/2021 HA nanoparticles with 150 µg/mL Matrix-M at time 0 and aft er incubation at room temperature f or 2–12 h (hr), showing the percentage of H5-MNP f ormation as % bound across time. (D , E) NS-TEM landscape charact erizing H5-MNPs mixed at an HA:Matrix-M molar ratio of approximately 40:1. In (D) we show a micrograph of H5N1-nanoparticle (NP) dis tribution at time 0 (T0). In (E) we show a micrograph of H5N1-MNP f orma tion (yello w 7 w .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 November 21, 2024. ; https://doi.org/10.1101/2024.11.21.624712doi: bioRxiv preprint 28 ar r o w s) a t t im e 24 h (T24hr) ( ma gen t a ar r o w s). The whit e a rr o ws r epr esen t H5 tr im er s , y e llo w ar r ow r epr esen ts a Ma tr ix-M cag e, and purple a rr o ws r epr es en t H5-MNP f or ma ti ons. (F) A 3D m odel of an H5 -MNP , with Ma t rix- M shown in g r a y s cal e and H5 tr im er s (PDB 6 HJR sho wn in blue) d ock ed t o the Ma t rix- M v e rt e x t o mi mi c the MNP for m ati o n f r om 2 D c l as s i f ic a t io n . .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 November 21, 2024. ; https://doi.org/10.1101/2024.11.21.624712doi: bioRxiv preprint 2 9 Figur e 3. Fi gur e 3: A/ A W/SC/2021 (A/ A W) H5-MN P v ac c ine eli c its ser oc on v erting an tibod y- and cell-me dia t ed immune r esponses in mi c e when ad mini s t er ed IM or IN. (A) Mice (n=3-10/group) were immuniz ed on Study Days 0 and 21 with a two-dose series of 1kiµFμg A/ A W/SC/2021 HA with 5kiµFμg Matrix-M adjuvant intramuscularly (IM), 1kiµFμg A/ A W/SC/2021 HA with 5kiµFμg Ma trix-M adjuvant intranasally (IN), or 10kiµFμg A/ A W/SC/2021 HA with 5kiµFμg Matrix-M adjuvant (IN). Hemagglutinin inhibiting (HAI) antibody titers (B) and pseudovirus neutralizing tit ers (C) against A/ A W/SC/2021 were det ermined in serum collect ed on Study Day 34 or 35 (2 weeks aft er completion o f 9 f .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 November 21, 2024. ; https://doi.org/10.1101/2024.11.21.624712doi: bioRxiv preprint 30 the pr im ary se rie s). (D) An ti-A/ A W IgA and Ig G tit e r s w er e det er min ed in br on cho alv eo la r la v age (B AL) f luid on Study Da y 34 o r 35. P olyfuncti onal T riple Th1 + cyt okine ((I FN-MiJÅ, I L-2, TN F-α) CD4 + T c ell r e s ponses w er e ev alua t ed in (E) spleen t issue and (F ) lung t issue co lle ct ed on S tudy Da y 34 /35 fr o m gr oups tha t r e ce iv ed the in tr an asa l H5- MNP v accin e. S y mbol s r epr e sen t indiv idual da t a p oin ts, bar s r epr e sen t g r oup ge om et ri c me an t it er s , e r r o r b ar s r epr es en t 95% c onf idence in t e r v al s, and the ho riz on t al dashed lin e r epr e sen ts the ass a y l imi t of qua n tifica ti on ( L O Q ) o r l im it of d et e cti on ( L OD) or the se r o c on v er ting thr e shold tit e r of 1:40. Dif f e r enc es bet we en gr ou ps wer e ev alua t ed b y K rusk al– W a llis mul tipl e c omp ari sons t es t or Mann–Wh itney U T es t. * p ≤ 0.05; **pMiJÅ≤MiJÅ0.0 05 ; ***pMiJÅ<MiJÅ0.0005. Only s t a tis ti ca lly signifi can t dif f er ence s ar e indi ca t ed in the figur e. .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 November 21, 2024. ; https://doi.org/10.1101/2024.11.21.624712doi: bioRxiv preprint 3 1 Figur e 4. Figur e 4: A/ A W/SC/2021 (A/ A W) H5-MN P v ac c ine eli c its ser oc on v erting an tibod y- and cell-me dia t ed immune r esponses when adminis t er ed I M or IN in NHPs primed with qNIV . (A) Rhesus macaques (nonhuman primates; NHPs) were immuniz ed by a two-dose quadrivalent nanoparticle seasonal influenza vaccine (qNIV ; 60 µg HA per strain) with 75 µg Matrix-M adjuvant (IM) on Study Days 0 and 21 (n=10). NHPs (n=5/group) were then administ ered two sequential boos ter doses of H5-MNP vaccine (either two IM 60 μg doses of HA or one IN dose of 240 μg HA f ollowed by one IN dose of 60 μg HA; all with 75 μg Matrix-M adjuvant) on Study Days 83 and 139. Hemagglutinin inhibiting (HAI) antibody titers (B) and pseudovirus neutralizing antibody titers (C ) ag ainst A/ A W/SC/2021 were evaluated in sera collect ed on Study Days indicated on the X axis. (D) T riple Th1 + CD4 + T cell responses were evaluated in PBMCs collected on Study Days indicat ed on the X axis. Colored symbols represent individual data points and bars represent group geometric mean tit ers . Error bars represent 95% confidence inter vals, and the horiz ontal dashed line represents the assay LOD/L OQ or the seroconverting threshold tit er of 1:40. Differences between groups were evaluated by Mann–Whitney U T est (two-tailed). Only statistically significant diff erences are indicated in the figure. *pkiµF< 0.05; **pkiµF≤kiµF0.005 ; ***pkiµF<kiµF0.0005. 1 .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 November 21, 2024. ; https://doi.org/10.1101/2024.11.21.624712doi: bioRxiv preprint 32 T able 2. Char act eriz a ti on of A/ A W/SC/202 1 HA Monoclonal An tibodies mAb, mon ocl onal antibo dy; RBS , r eceptor b ind ing sit e; V E, ves tigial es t e r ase A(H5N1) Virus Str ai ns A(H5N1) mA b NVX.361.4 (RBS) NVX.73.2 (VE subdom ain) Pseudovirus Neutralization (IC 50 , ng/mL) A/ A W/ SC /22/000345-001/2021 HA 3.571 42.8 A/Co lo r ado /18/2022 HA 8.497 79.5 A/Mink/ Spain /3691-8 HA 10.53 39.53 Hemagglutination Inhibition Endpoint Titers (ng/mL) A/ A W/ SC /22/000345-001/2021 HA 250 >500 A/Co lo r ado /18/2022 HA 250 >500 A/Mink/ Spain /3691-8 HA 250 >500 .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 November 21, 2024. ; https://doi.org/10.1101/2024.11.21.624712doi: bioRxiv preprint 3 3 Figur e 5. Fi gur e 5: M odel of A(H5N1) A / A W/SC /2021 HA a nd its ves tigi al es t er ase (VE) and r ece pt or bi ndin g s it e (R BS) neutr a lizin g epit opes wher e Nov a va x m o noclona l an tibodies bin d . The in silico molecular model of A(H5N1) A/ A W/SC/2021 HA with mapping where neutralizing mAbs interact with the protein. (A) HA-antigen mo deled with NVX.73.2 mAb (LC-light chain in blue and HC-heavy chain in purple) showing its interaction with the vestigial esterase (VE) subdomain (cyan) of A(H5N1) HA. V HC is the heavy chain variable domain and V LC is the variable light chain variable domain. The modeled 73.2 mAb represents binding of critical residues in the VE subdomain (green spheres). (B) HA-antigen modeled with NVX.361.4 mAb (Fab-variable heavy chain in yellow and light chain in cyan) 3 .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 November 21, 2024. ; https://doi.org/10.1101/2024.11.21.624712doi: bioRxiv preprint 34 show ing it s in t e r a cti on with the r e cept or bin ding sit e (RBS) of HA ( in purpl e). The m ode le d 361.4 mAb r epr es en ts binding of cr iti c al r es idues of the RBS in the head r egi on (cy an and o r ang e sphe r es ) dir ec tl y with HC- CDR2 and 3 (y ello w). (C ) C o mpet itiv e an tibody equiv al en t (CAE) f o r mAbs CR6261 (l eft ), N VX.73.2 ( mid dle), and NVX.361.4 (righ t) in NH P s er a c oll ect ed on S tudy Da y s in dica t ed on the X a x es (bo th IM- and IN- im mu niz ed gr oups). C ol or ed s ymbo ls r epr esen t individual anim al da t a p oi n ts and ba r s r epr esen t g r oup ge o me tr ic m ea n tit e r s . E rror ba r s r epr e sen t 95% c onf idence in t e r v al s, and the hori z on t al dash ed line r epr es en ts the ass a y L OD . Dif f e r enc es am ong gr oups w er e ev alua t ed by a K rusk a l-W alli s t e s t. Only s t a tis ti c ally signifi c an t dif f e r enc es ar e indi ca t ed in the figur e. *pMiJÅ< 0.05; * *pMiJÅ≤MiJÅ0.005 ; ***pMiJÅ<MiJÅ0.0005. .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 November 21, 2024. ; https://doi.org/10.1101/2024.11.21.624712doi: bioRxiv preprint 3 5 SUPPLEMENT AL M A TERIAL Supple m en t al F ig ur e S1: A / A W/SC/2021 (A / A W) H5- MNP v accine-i nduced ind ividu al c y t okine CD4 + T cel l r esponses in mice a nd NHP s . Th1 cyt okine (IFN-kiµF, IL-2, TNF-α) and Th2 cytokine (IL-4) responses were analyz ed in mouse spleen eff ect or CD4 + T cells (A) and lung resident CD4+ T cells (B) on Study Day 34/35 (n=3-6 per group). In 5 .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 November 21, 2024. ; https://doi.org/10.1101/2024.11.21.624712doi: bioRxiv preprint 36 (A), as t e risk s indi ca t e dif f e r enc es fr om the pl acebo-t r ea t ed g r oup as no dif f er enc es am on g v acc ina t ed gr oups wer e s t a tis ti ca lly signifi can t. (C) Th1 cyt okine (I FN- MiJÅ, IL-2, TNF- α) r esp onses wer e ev alua t ed in N HP cir cula ting CD4 + T cell s ( PBMC s) on Study Da y 97 and 153 (n=5 per g r oup). C ol or ed s ymbols r epr es en t indivi dual da t a p oin ts and b a r s r epr e sen t gr oup ge om et ri c me an tit er s. E rr o r bar s r epr e sen t 95% c onf idence in t e r v al s. Dif f er enc es b et w een g r oups we r e ev alua t ed by (A) K rusk al –W alli s t es t or (B, C ) Mann–Whi tney U T es t (tw o-t ai led). On ly s t a tis t ic ally signifi c an t dif f er en ces ar e indi ca t ed in the f igur e. *pMiJÅ< 0 .05; **pMiJÅ≤MiJÅ0.005 . .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 November 21, 2024. ; https://doi.org/10.1101/2024.11.21.624712doi: bioRxiv preprint 3 7 Supple m en t al F ig ur e S2 : H5-M NP v accine in duces an ti-A / A W/SC /2021 (A/ A W) I gA and I gG r esponses in NHP se r a and m uc osa. Anti-A/ A W IgG titers from NHP s (n=10 after primary series, n = 5 aft er boosters) were analyz ed in (A) serum collect ed on Study Days indicated on the X axis. Mucosal anti-A/ A W IgA and IgG titers from NHP s were 7 .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 November 21, 2024. ; https://doi.org/10.1101/2024.11.21.624712doi: bioRxiv preprint 38 analyz ed in (B ,D) nasal w ash and (C,E) B AL s a mples co lle ct ed on s tudy da y s indi ca t ed on t he X a xi s. C ol or ed s ymbo ls r epr esen t individual da t a p oin ts and bar s r epr esen t gr oup ge o me tr ic m ean tit e r s . Err or ba r s r epr esen t 95% c onfiden ce in t er v als. Dif f er ence s bet we en gr oups we r e ev a lua t ed b y K rusk al –W alli s t es t. Only s t a tis t i cal ly signifi can t dif f er en ces a r e indi ca t ed in the fig ur e. *pMiJÅ< 0.05; * *pMiJÅ≤MiJÅ0.005 ; ***pMiJÅ<MiJÅ0.0005 ; ****pMiJÅ<MiJÅ0.00005. .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 November 21, 2024. ; https://doi.org/10.1101/2024.11.21.624712doi: bioRxiv preprint 3 9 Supple m en t al F ig ur e S3: Structur a l ch ar act eriz a tion o f m A b N VX.73.2 and 361.4. (A ) 2D class average images from negative st aining TEM and (B ) cart oon representative schema tics of mAb NVX.73.2 binding to HA in diff erent o rientatio ns. ( C ) A 3D reconstruct ed map representing mAb NVX.73.2 (orange) bound to the lower region of the H A head (vestigial esterase domain) (D ) 2D class average from NS-TEM f or A/ A W/SC-HA and mAb NVX.361.4 and ( E ) cart oon representation of mAb NVX.361.4 binding to HA in various orientations. (E ) A 3D reconstructed map representing mAb NVX.361.4 (green) binding to the HA head (recept or binding site). Abbreviation: Fab, Fragment antibody binding. 9 A .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 November 21, 2024. ; https://doi.org/10.1101/2024.11.21.624712doi: bioRxiv preprint 40 Supplemental Table S1: EM da taset sta ti s tics f or H5N1-HA and F abs or H5 N1 HA- Ma t rix-M complex es Dataset H5N1-HA + NVX.361.4 Fab H5N1-HA + NVX.73.2 Fab H5N1-HA + Matrix-M (NS-TEM) Microscope FEI Glacios FEI Glacios FEI ARCTICA Detector FEI Falcon 4i FEI Falcon 4i Gatan K3 Raw pixel size, Å 1.538 1.538 0.888 No frames 40 55 36 Energy, kV 200 200 200 Magnification 91000x 91000x 45000x Exposure, s 10.4 10.4 2.5 Total dose, e/Ų 36.9 39.8 41.6 Defocus, µm 0.5-3.2 0.5-3.2 1-2 Micrographs Total 1100 800 1452 Accepted Micrographs 990 752 359 Total Particles 66000 253588 30000 .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. 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