key: cord-0733437-ukm1wbh0
authors: Saunders, Kevin O.; Pardi, Norbert; Parks, Robert; Santra, Sampa; Mu, Zekun; Sutherland, Laura; Scearce, Richard; Barr, Maggie; Eaton, Amanda; Hernandez, Giovanna; Goodman, Derrick; Hogan, Michael J.; Tombacz, Istvan; Gordon, David N.; Rountree, R. Wes; Wang, Yunfei; Lewis, Mark G.; Pierson, Theodore C.; Barbosa, Chris; Tam, Ying; Shen, Xiaoying; Ferrari, Guido; Tomaras, Georgia D.; Montefiori, David C.; Weissman, Drew; Haynes, Barton F.
title: Lipid nanoparticle encapsulated nucleoside-modified mRNA vaccines elicit polyfunctional HIV-1 antibodies comparable to proteins in nonhuman primates
date: 2020-12-31
journal: bioRxiv
DOI: 10.1101/2020.12.30.424745
sha: 332283c04bcc1d576dfcf9457e9a9be5773beadf
doc_id: 733437
cord_uid: ukm1wbh0

Development of an effective AIDS vaccine remains a challenge. Nucleoside-modified mRNAs formulated in lipid nanoparticles (mRNA-LNP) have proved to be a potent mode of immunization against infectious diseases in preclinical studies, and are being tested for SARS-CoV-2 in humans. A critical question is how mRNA-LNP vaccine immunogenicity compares to that of traditional adjuvanted protein vaccines in primates. Here, we found that mRNA-LNP immunization compared to protein immunization elicited either the same or superior magnitude and breadth of HIV-1 Env-specific polyfunctional antibodies. Immunization with mRNA-LNP encoding Zika premembrane and envelope (prM-E) or HIV-1 Env gp160 induced durable neutralizing antibodies for at least 41 weeks. Doses of mRNA-LNP as low as 5 μg were immunogenic in macaques. Thus, mRNA-LNP can be used to rapidly generate single or multi-component vaccines, such as sequential vaccines needed to protect against HIV-1 infection. Such vaccines would be as or more immunogenic than adjuvanted recombinant protein vaccines in primates.

For mRNA to be effective at transducing cells in vivo, it must be protected from RNAses. One 54 method for protecting the mRNA from degradation has been its encapsulation in lipid 55 nanoparticles (LNP) 7, 8, 13 . Importantly, small interfering RNAs (siRNAs) in LNP have been 56 approved by the FDA for treatment of a genetic form of amyloidosis 14 . Thus, advances in 57 encapsulation and preventing mRNA immune sensing have made mRNA vaccines a feasible 58 vaccine platform. 59 mRNA vaccines are also attractive as a vaccine platform, because they can accept the 60 genes of various pathogen antigens, and they can be rapidly manufactured at scale. Theses two 61 aspects made mRNA vaccines a prime candidate for responding to the SARS-CoV-2 outbreak 62 15,16 . High levels of SARS-CoV-2 neutralizing antibodies, defined as higher than geometric 63 adjuvant formulation composed of monophosphoryl lipid A (a TLR-4 agonist) and QS21 110 (ALFQ) 39 . Additionally, two groups of macaques were immunized with nucleoside-modified 111 mRNA-LNP encoding different forms of A244 11 gp120. One group of macaques received 112 A244 11 gp120, whereas the other group received mRNAs encoding A244 11 gp120 with a 113 D368R mutation that disrupted the CD4 binding site (Fig. 1A) . The purpose of the CD4 binding 114 site knockout mutation was to determine whether Env binding to CD4 + T cells in vivo altered 115 immunogenicity of HIV-1 Env gp120. Macaques were immunized with either protein or mRNA-116 LNP at weeks 0 and 6 and antibody responses were followed for 18 total weeks (Fig. 1A) . 117

Binding nnAbs to Env immunogens have been associated with protection in macaques 118 from SHIV infection 40, 41 . Thus, we first determined serum binding IgG elicited by each of the 119 mRNA/LNP or protein groups. Regardless of site of immunization, gp120-specific IgG titers in 120 the macaque serum were detectable after a single immunization and continued to rise after 121 second immunization (Fig. 1B) . mRNA-LNP elicited nearly identical serum IgG antibody titers 122 as compared to macaques immunized with A244 11 gp120 formulated with ALFQ (Fig. 1B) . 123

Comparison of mRNA-LNP that encoded wildtype gp120 versus CD4 mutated gp120 showed 124 identical binding IgG elicitation (Fig. 1B) . Macaques that received A244 11 gp120 formulated 125 with Rehydragel generated the lowest group mean serum IgG binding titers to A244 11 gp120. 126

For these ELISA assays, the differences between the Rehydragel group and the other groups 127 were greatest after one immunization, but the small numbers of monkeys per group precluded 128 statistical analysis. Nonetheless, ALFQ adjuvant induced higher levels of A244 11 gp120 129 antibody compared to gp120 in Rehydragel (Fig. 1B) . The same group ranking was also 130 observed for binding IgG to a HIV-1 Env gp120 B.63521 from a non-vaccine matched HIV-1 131 introduction of mutations in the CD4 binding, glycan-V3, and glycan-V2 sites (Fig. 1B) . Thus, 133 mRNA-LNP gp120 vaccination produced comparable IgG titers in plasma as recombinant gp120 134 protein in the potent adjuvant ALFQ. 135

IgG responses to the second variable (V2) region of HIV-1 Env were a correlate of 136 reduced infection risk in the RV144 trial 24 . Similarly, K169 in the V2 region was a site of 137 immune pressure during the RV144 trial 42 . To assess whether nucleoside-modified mRNA-LNP 138 vaccination elicited comparable V2 region IgG antibodies in plasma, we tested serum IgG 139 binding to various minimal antigens that recapitulated the V2 region of HIV-1 Env. These 140 antigens included V1V2 proteins, a V2 peptide, and V2 scaffolded on gp70. Binding to each 141 antigen showed the same pattern as gp120 binding. Recombinant protein adjuvanted with AFLQ 142 and mRNA-LNP immunization elicited nearly identical plasma IgG responses (Fig. 2) . 143

Recombinant gp120 adjuvanted with Rehydragel elicited the weakest responses for all the 144 antigens tested, with group difference being largest after a single immunization (Fig. 2) . V1V2-145 specific binding IgG titers were comparable whether the V1V2 antigen matched the vaccine 146 strain or if it was from an unmatched virus 1086C ( Fig. 2A) . The first variable region (V1) was 147 not necessary for V2 binding, as plasma IgG bound to a short V2 only peptide, K178 (Fig. 2B) . 148

The antigen for which binding IgG correlated with reduced infection risk in the RV144 trial was 149 B.CaseA V1V2 scaffolded on gp70 24,43 . Each group of macaques generated binding IgG 150 antibodies to gp70 B.CaseA V1V2 protein (Fig. 2C) . The antibodies were specific for HIV-1 151 V1V2, as no antibodies were detected against the control gp70 scaffold presenting murine 152 leukemia virus V1V2 (Fig. 2C) . However, mutation of K169 had little effect on B.CaseA V1V2 153 binding IgG titers. Similarly, mutation of the K169 in the V2 region of A244 gp120 had little 154 effect on binding IgG titers (Fig. 1B) . Thus, V1V2 antibodies were not solely dependent on the 155 K169 at the site of immune pressure observed in the RV144 trial. 156

To determine the breadth of reactivity and epitope specificity of the first and second 157 variable (V1V2) region antibodies induced by each vaccine, we performed linear V1V2 peptide 158 arrays. Vaccination induced only low levels of V1-binding antibodies, but high titers of V2 159 antibodies across the different vaccines ( Fig. 3A-D and Supplementary Fig. 1 ). V2 antibody 160 binding magnitudes were similar for mRNA-LNP and protein formulated with ALFQ (Fig. 3) . 161

Each vaccine elicited antibodies capable of binding V2 peptides from clade AE and C (Fig. 3  162 and Supplementary Fig. 1) . Therefore, mRNA-LNP and adjuvanted protein vaccination elicited 163 V2 antibody responses that were similar in magnitude, breadth, and epitope specificity. 164

Next, we compared plasma antibody specificities induced by protein or mRNA-LNP 165 immunization at HIV-1 Env gp120 sites outside of the V1V2 site. mRNA-LNP induced similar 166 A244 gp120 linear epitope antibodies compared to protein formulated in adjuvant with two 167 exceptions. First, recombinant protein, but not mRNA-LNP, elicited plasma IgG to the C-168 terminal portion of the second constant (C2) region ( Fig. 3E-H and Supplementary Fig. 2) . 169

Second, mRNA-LNP immunization elicited plasma IgG against the third constant region but 170 recombinant protein immunization did not ( Fig. 3E-H and Supplementary Fig. 2) . 171

To further compare polyclonal plasma IgG specificities, we assessed the ability of post-172 vaccination plasma to block the binding of gp120 monoclonal antibodies (mAbs) to HIV-1 Env. 173

Plasma from either adjuvanted protein-or mRNA-LNP-immunized macaques was added to HIV-174 1 Env protein, followed by the addition of biotinylated mAbs to determine plasma antibody 175 blocking of monoclonal antibody binding to Env. We examined blocking of non-neutralizing 176 effector antibodies CH58, which targets the V2 site of immune pressure identified in RV144 44 , 177 and A32 which defines an immunodominant ADCC gp120 site 45 that synergizes with V2 178 antibodies to mediate ADCC 46 (Fig. 4A) . Next, we determined whether plasma could block the 179 HIV-1 entry receptor CD4 from binding to Env (Fig. 4B) , or block V2-glycan bnAbs PG9 and 180 CH01 and N332 glycan-dependent bnAbs 2G12 and PGT125 Env binding (Fig. 4C,D) . Either 181 immunization with adjuvanted recombinant gp120 protein or mRNA-LNP elicited plasma 182 antibodies that blocked the binding of CH58, A32, CD4, CH01, 2G12, and PGT125 (Fig. 4) . 183

Comparison of mRNA-LNP-induced antibodies versus gp120 protein-induced plasma antibodies 184

showed that mRNA-LNP immunization elicited the same magnitude of blocking after 2 185 immunizations as protein adjuvanted with ALFQ (Fig. 4) . Blocking activity was lowest for 186 animals immunized with protein adjuvanted with Rehydragel (Fig. 4) . The blocking of CH01 187 binding to A244 most likely represented CH58-like antibody binding and not V2-glycan bnAb 188 binding, since CH01 binding can be blocked by CH58-like linear V2 antibodies (Fig. 4) . IgG from all four groups of macaques was able to bind to HIV-infected cells (Fig. 5A,B) . When 199 binding levels of IgG was quantified as mean fluorescence intensity of bound IgG or the 200 percentage of cells positive for HIV-1 protein p24 and plasma IgG, mRNA-LNP vaccination and 201 recombinant protein adjuvanted with ALFQ were not different (Fig. 5A,B) . In agreement with 202 the overall lower IgG titers, Rehydragel-adjuvanted protein gave the weakest cell binding 203 responses (Fig. 5A,B) . 204 ADCC activity was measured in a flow cytometry-based granzyme B assay. There was a 205 clear adjuvant effect between the two protein immunized group. ADCC titers were 206 approximately one order of magnitude higher when ALFQ was used as the adjuvant as compared 207

to Rehydragel. Similarly, mRNA-LNP immunization (mean±standard deviation = 208 22,916±22,102) induced activity lower than ALFQ-formulated recombinant protein, but 3-fold 209 higher than protein adjuvanted with Rehydragel (mean±standard deviation = 7,324±7,024) ( Fig.  210 5C). Elimination of CD4 binding to A244 11 gp120 had no effect on ADCC activity (Fig. 5C) . 211

Lastly, we compared ADCP activity of week 8 plasma antibodies from macaques administered 212 adjuvanted recombinant protein or mRNA-LNP. Median plasma ADCP activity against A244 213 gp120-coated beads was similar across all groups with A244 11 gp120 in Rehydragel eliciting a 214 wider range of responses (Fig. 5D ). In agreement with previous studies, nucleoside-modified 215 mRNA-LNP vaccination elicited antibody effector functions that have been shown previously to 216 correlate with reduced infection risk 24, 48 . 217

While the goal of A244 11 gp120 immunization was to induce non-neutralizing effector 218 functions like those seen in the RV144 trial, we compared elicitation of neutralizing antibodies 219 by each of the vaccines. We selected for testing the tier 1 HIV-1 strain CRF_01AE 92TH023, 220 against which A244 has consistently induced neutralizing antibodies 24,44 . We found that there 221 was no significant difference among the different vaccination regimens for induction of HIV-1 222 92TH023 neutralizing antibodies (Supplementary Fig. 3) . These studies showed that for immunization with gp120 monomers aiming to elicit potentially protective non-neutralizing V2 224 antibodies, nucleoside-modified mRNA-LNP vaccination was superior to recombinant protein 225 formulated with aluminum hydroxide and comparable to recombinant protein formulated with a 226 more complex TLR-4/QS21/liposomal adjuvant. A316W mutations 51 . The immunogenicity of these two sets of mRNA-LNP was compared to 238 that of 100 g of the same set of CH505 Envs as soluble gp140 SOSIP proteins formulated in the 239 TLR-4 adjuvant, GLA-SE (Fig. 6A) . Four rhesus macaques were immunized every four weeks 240 with either set of immunogens (Fig. 6A) , and binding antibody and neutralizing antibodies were 241

All three vaccines were immunogenic in macaques. With regard to induction of binding 243 titers of gp120 antibodies, mRNA-LNP encoding sequential CH505 gp160s induced higher 244 gp120 titers than did either of the mRNA-LNP encoding soluble gp140 SOSIP trimers or soluble 245 gp140 SOSIP trimers proteins (Fig. 6B) . These binding titers rose dramatically after two immunizations with gp160 mRNA-LNP, but rose gradually over the course of five 247 immunizations in macaques immunized with SOSIP gp140s (Fig. 6B) . Plasma IgG binding titers 248 to gp120 were equivalent between the mRNA-LNP and the adjuvanted soluble gp140 SOSIP 249 trimer protein-immunized animals (Fig. 6B) . Soluble gp120 exposes non-neutralizing epitopes, 250 thus we examined whether the high titers of gp120 antibodies in macaques immunized with 251 gp160 mRNA-LNP were due to antibodies targeting these sites. Indeed, the gp160 mRNA-LNP 252 induced very high titers of antibodies to linear CH505 TF V2 peptides, whereas titers in the 253 gp140 SOSIP trimer groups showed no or a slight increase from baseline (Fig. 6C) . Linear V3 254 epitopes were also highly immunogenic in gp160 mRNA-LNP-immunized macaques, and to a 255 lesser extent was immunogenic in soluble gp140 SOSIP trimer protein immunized macaques 256 (Fig. 6D) . Interestingly, V3 antibodies were not elicited by gp140 SOSIP trimer mRNA-LNP, 257 suggesting a difference in V3 exposure when the trimer was expressed in vivo or potential effects 258 of the adjuvant. To examine the induction of gp120 antibodies that may target conserved 259 neutralizing epitopes, we assessed the amount of gp120 antibody binding that was dependent on 260 the CD4 binding site. We mutated the CD4 binding site with a deletion of the isoleucine at 261 position 371 and determined the decrease in plasma IgG binding to CH505 TF gp120. Both 262 groups of macaques that received SOSIP trimers as either protein gp140s or mRNA-LNP were 263 superior to the mRNA-LNP encoding the gp160s (Fig. 6E) . Immunization with gp140 SOSIP 264 trimers elicited differential binding between the mutant and wildtype gp120 that rapidly receded 265 after each immunization, but was maintained for 16 weeks once immunizations were stopped 266 ( Fig. 6E) . Similar to linear V2 and V3 peptide antibodies, gp160 mRNA-LNP induced high 267 levels of gp41 antibodies whereas gp140 SOSIP trimers delivered as mRNA-LNP or adjuvanted 268 recombinant protein had levels close to baseline (Fig. 6F) . Thus, gp160 mRNA-LNP 269 immunization elicited higher titers of undesired antibodies targeting non-neutralizing gp120 and 270 gp41 epitopes and lower titers of desired CD4 binding site antibodies than SOSIP trimer- Among these variants of the M5 gp160 envelope, adding H66A and A582T reduced binding by 284 non-neutralizing antibodies against V3 and gp41, while leaving trimer-specific (PGT145) or 285 timer-preferring (PG9 and CH01) antibody binding unchanged (Fig. 6G) . Thus, mRNA 286 immunization with stabilized HIV-1 envelope gp160 is one potential approach to improve the 287 elicitation of neutralizing antibodies, while not engaging the B cell receptor of B cells that 288 produce antibodies that cannot bind native, fusion-competent HIV-1 envelope. 289

against Zika prM-E and HIV-1 Env. 291

in the RV144 trial, protective antibodies fell dramatically over the first 42 weeks after 293 vaccination 24 . Thus, if immunization with mRNA-LNP induced durable antibody responses it 294 would benefit their use as a vaccine platform for many different infectious diseases. We assessed 295 the durability of antibody responses using neutralization assays of the tier 1 CH505 w4.3 virus, 296 since antibodies capable of neutralization of the tier 2 CH505 transmitted founder (TF) virus 297

were not elicited (Supplementary Tables 1-3 ). CH505 w4.3 is an early virus isolate that is 298 identical to the tier 2 CH505 TF virus with the exception of a W680G mutation that makes it 299 highly sensitive to HIV antibody neutralization 49,56,57 . Longitudinal comparative analyses of 300 neutralization of the CH505 w4.3 virus showed that the gp160-encoding mRNA-LNP elicited 301 higher titers of neutralizing antibodies than the gp140 SOSIP trimer-encoding mRNA-LNP or 302 the gp140 SOSIP trimer proteins in GLA/SE 58 (Fig. 7A) . Notably, neutralization of the CH505 303 w4.3 virus was still detectable in all three groups 12 weeks after the last immunization ( Fig. 7A) . 304

There were also sporadic low levels of neutralization of CH505 or 426C viruses that were 305 modified to be highly sensitive to CD4 binding site antibodies (Supplementary Tables 1-3) . 306

While these results indicated that durable neutralizing antibodies were elicited by each 307 vaccine regimen, the downward trend of the neutralization titers in these macaques and the 308 A244-immunized macaques raised the question of how long would neutralizing antibodies 309 persist at detectable levels. A subset of the macaques used in this HIV-1 study were previously 310 administered 50 g of mRNA-LNP encoding ZIKV pre-membrane and envelope (prM-E). These 311 macaques generated protective anti-Zika neutralizing antibody responses 1 . Using these 312 macaques that were administered both HIV and Zika mRNA-LNP vaccines, we determined 313 serum neutralizing antibodies titers over 52 weeks (Fig. 7B) . We found that neutralizing antibodies against ZIKV were maintained until the last timepoint of follow-up 52 weeks after 315 immunization. Neutralizing antibodies to HIV-1 persisted for the duration of the 41 weeks of 316 follow-up. During these 41 weeks, HIV-1 titers initially fell approximately 10-fold after the last 317 immunization but plateaued at ~1:100 titer (Fig. 7B) . In contrast, Zika neutralizing antibody 318 levels were maintained at the same level for the 52 weeks after being detected at week 2 ( Fig.  319   7B ). Similar patterns were observed for plasma binding IgG titers to CH505 gp120 and Zika 320

prM-E (Fig. 7C) . Taken together, nucleoside-modified mRNA-LNP immunizations induced 321 durable serum neutralizing antibodies for both HIV-1 and Zika viruses. 322

Neutralizing antibody titers are dependent on mRNA-LNP dose. In our previous ZIKV 323 vaccine studies in nonhuman primates, we administered 50, 200 or 600 g of mRNA-LNP 1 . 324

Given the potent elicitation of neutralizing antibodies by mRNA administered at each of these 325 doses, we sought to determine whether the mRNA dose could be further decreased. We 326 immunized four macaques intramuscularly with either 50, 20, or 5 g of mRNA-LNP encoding 327

Zika prM-E (Fig. 8A ) and compared titers of binding IgG and neutralizing antibodies. Fifity and 328 twenty microgram doses of mRNA-LNP elicited similar titers of binding IgG and neutralizing 329 antibodies ( Fig. 8B and C) . Titers of ZIKV binding IgG and neutralizing antibodies were 330 substantially decreased when the mRNA-LNP dose was lowered to 5 g, but were detectable 331 with administration of this single administration of a small amount of mRNA-LNP in macaques. 332

The route of immunization was not critical as administering 50 g of mRNA-LNP either 333 intramuscularly or intradermally elicited comparable Zika envelope binding IgG and Zika 334 neutralizing antibodies ( Fig. 8B and C) . Thus, the mRNA-LNP dose could be lowered from 50 335 to 20 g in macaques without detrimental effects to antibody responses. Thus, more 336 immunizations can be performed with each preparation of mRNA-LNP by reducing the dose by 337 60 percent. 338

Here, we demonstrate in 28 rhesus macaques that immunization with nucleoside-modified 340 mRNA-LNP (n=16) was equal to or superior to the immunogenicity of adjuvanted Env protein. There are currently two ongoing efficacy trials that test the protective capacity of non-376 neutralizing HIV-1 antibodies with viral vector prime, Env protein boosts to determine if the 42 377 month efficacy of ALVAC/gp120 can be replicated. In addition, the just-completed HVTN 702 378 trial (NCT02968849) that uses an ALVAC with a clade C Env insert as prime and ALVAC-C + 379 a bivalent C gp120 Env boost failed to confer any protection from HIV-1 infection in South 380

Africa in an area of high HIV-1 infection rates. The ongoing HVTN 705 trial (NCT03060629) 381

tests the efficacy of an adenovirus (Ad) 26 vector containing mosaic HIV-1 genes as a prime and 382 the same Ad26 + a clade C gp140 Env as a boost, and the HVTN 706 trial (NCT03964415) tests 383 the efficacy of Ad26 mosaic HIV as a prime and Ad26 + a bivalent clade C gp140 Env + a 384 mosaic gp140 Env as boost 65 . The correlate of decreased transmission risk in macaque studies 385 of the A26, Ad26 + gp140 boost SHIV challenge studies did not include neutralization breadth 386 of antibodies, but instead included clade C Env ELISA binding antibodies and interferon gamma 387

Env ELISPOT values 65 . Thus, these latter two vaccines will test whether non-neutralizing 388 antibody effector functions can mediate significant protection in humans. 389

The ease and cost-effectiveness of mRNA-LNP production compared to the production 390 While mRNA vaccination is promising, there are still improvements that can be made for 397

HIV mRNA-LNP vaccination. Specifically, for bnAb induction by mRNAs, the near-native Env 398 trimers will need to be stabilized such that they will be able to be produced by the transfected 399 cell in a well-folded state. This challenge is not unique to mRNA as DNA vaccination has also 400 faced similar roadblocks 67 . This aspect of genetic vaccination differs from recombinant proteins 401 that can be purified prior to immunization and recognition by the immune system. However, 402

there is a plethora of available mutations for stabilizing HIV-1 envelope, and each envelope has 403 different intrinsic stability that can be further augmented. We investigated only two potential sets 404 and found H66A and A587T reduced non-neutralizing antibody binding to envelope in vitro. The Env (pUC-TEV-CH505w136.B18chim.6R.SOSIP.664v4.1 Env-A101); gp120 HIV-1 Envs: 503 A244delta11 gp120 Env (pUC-TEV-A244delta11 gp120 Env-A101), A244delta11 K368R 504 gp120 Env (pUC-TEV-A244delta11 K368R gp120 Env-A101). mRNAs were transcribed to 505 contain 101 nucleotide-long poly(A) tails based on the DNA-encoded poly(A) tail sequence. 506

One-methylpseudouridine (m1Ψ)-5'-triphosphate (TriLink) instead of UTP was used to generate 507 modified nucleoside-containing mRNA. RNAs were capped using the m7G capping kit with 2'-508 O-methyltransferase (ScriptCap, CellScript) to obtain a type 1 cap. mRNA was purified by Fast 509

Protein Liquid Chromatography (FPLC) (Akta Purifier, GE Healthcare), as described 74 . All Plasma competition ELISAs. Plasma competition assays were performed as described 540 previously 78,79 . In brief, NuncSorp plates were coated with HIV-1 Env, washed and blocked as 541 stated above for direct ELISAs. After blocking was complete, nonhuman primate plasma was 542 diluted in SuperBlock at a 1:50 dilution and incubated in triplicate wells for 90 min. Non-543 biotinylated monoclonal antibodies were incubated with the Env in triplicate as positive controls 544 for blocking. To determine relative binding no plasma or no antibody was added to a group of 545 wells scattered throughout the plate. After 90 min the non-biotinylated antibody or plasma was 546 washed away and biotinylated monoclonal antibodies was incubated in the wells for 1 h at 547 subsaturating concentrations. Specifically, biotinylated monoclonal antibody concentrations used 548 for binding to A244 11 gp120 were: 0.04 g/mL of CH58, 0.1 g/mL of A32, 1.9 g/mL of 549 2G12, 0.2 g/mL of PGT125, 1.2 g/mL of CH01, 1g/mL of PG9. Biotinylated CH01 and 550 PG9 concentrations used for binding to 9021 gp140 and B.6240 11 gp120 were 1.5 and 0.125 551 g/mL respectively. For CD4 blocking assays, soluble CD4 was added followed by biotinylated 552 anti-CD4 monoclonal antibody. Each well was washed and binding of biotinylated monoclonal 553 antibodies was determined with a 1:30000 dilution of HRP-conjugated streptavidin 554 (ThermoFisher). HRP was detected with TMB and stopped with 1% HCl. The absorbance at 555 450 nm of each well was read with a Spectramax plate reader (Molecular Devices). Binding of 556 the biotinylated monoclonal antibody to HIV-1 Env in the absence of plasma was compared to in 557 the presence of plasma to calculate percent inhibition of binding. Based on historical negative 558 controls, assays were considered valid if the positive control antibodies blocked greater than 20% 559 of the biotinylated antibody binding. Technologies GmbH (Germany) by printing a library of peptides onto epoxy glass slides 564 (PolyAn GmbH, Germany). The library contains overlapping peptides (15-mers overlapping by 12) covering 5 full-length gp160 consensus sequences (clade A, B, C, D, and group M). V3 566 peptide binding breadth was analyzed for a library of V3 peptides (15-mers overlapping by 12) 567 for 7 consensus sequences (clade A, B, C, D, CRF1, and CRF2, and group M) and 6 vaccine 568 strains (MN, A244, TH023, TV-1, ZM651, 1086C) . Three identical subarrays were blocked for 1 569 h, followed by a 2-h incubation with monoclonal antibody, and a subsequent 45-min incubation 570 with anti-monkey IgG conjugated with AF647 (Jackson ImmunoResearch, PA). Slides were 571

washed before the addition of monoclonal antibody or anti-monkey IgG. Unbound anti-monkey 572

IgG conjugated with AF647 was washed away, and array slides were scanned at a wavelength of 573 635 nm using an InnoScan 710 scanner (InnopSys, Denmark) and images were analyzed using 574 Magpix V8.1.1. 575

GranToxiLux (GTL) 81 and tested against subtype AE HIV-1 recombinant A244 ∆11 gp120-577 coated cells. NHP plasma were incubated with human PBMC as source of effector cells 82 and 578 gp120-coated target CEM.NKR.CCR5 cells 83 and ADCC was quantified as net percent granzyme 579 B activity, which is the percent of target cells positive for GranToxiLux (GTL) detected by flow 580 cytometry. For each subject at each timepoint, percent granzyme B activity was measured at six 581 dilution levels: 50, 250, 1250, 6250, 31,250 and 156,250 for each antigen. Peak activity less than 582 0% was set to 0%. A positive response was defined as peak activity greater than or equal to 8%. 583

The RSV-specific monoclonal antibody Palivizumab and a cocktail of HIV-1 monoclonal Abs 584 (A32, 2G12, CH44, and 7B2) were used as negative and positive control, respectively. The 585 potency of ADCC responses was evaluated by calculating the Area Under the Curve using the 586 non-parametric trapezoidal method. Antibody-dependent phagocytosis. Phagocytosis was measured as stated previously 85, 86 . 605

Briefly, recombinant A244 11 gp120 was incubated with 1 μM fluorescent beads overnight at 606 4°C while rotating. The beads were subsequently washed twice with 0.1% BSA/PBS to remove 607 unbound gp120. gp120-coated beads were incubated with prevaccination and postvaccination 608 plasma for 2 h at 37°C. As a positive control HIVIG was incubated with the beads. As a negative 609 control monoclonal antibody CH65 was incubated with the beads, since this antibody reacts with influenza hemagglutinin and not HIV-1 Env. THP-1 or monocytes were incubated with anti-CD4 611 antibody SK3 (Biolegend) and then added to the bead/plasma mixture. The mixture was 612 spinoculated for 1h at 4C followed by another 1h incubation at 37°C 87 . The cells were washed 613 and fixed and fluorescence due to bead internalization was measured by flow cytometry. Positive for 5 days at 37°C in 5% CO2. After incubation, the overlay was aspirated, cells were fixed and 647 stained with 0.5% crystal violet (Sigma) in 25% methanol, 75% deionized water, rinsed with 648 deionized water, and plaques inspected. Neutralization titers (EC50) were calculated as the 649 reciprocal dilution of sera required for 50% neutralization of infection. EC50 titers below the 650 limit of detection are reported as half of the limit of detection. 651

Zika reporter virus particle (RVP) neutralization assay. RVP neutralization assays were 652 performed as described elsewhere 1 . RVPs were diluted to ensure antibody excess at informative 653 portions of the antibody dose response curve and incubated for 1 h at 37°C with serial dilutions 654 of heat-inactivated macaque sera to allow for steady-state binding. Serum-RVP mixtures were 655 subsequently mixed with Raji-DCSIGNR cells at 37°C. Each serum sample was tested in to 656 technical replicates. Every assay was repeated in two biologically-independent assays. GFP-657 positive infected cells were detected by flow cytometry 24-48 hr later. The EC50 was estimated 658 using a non-linear regression with a variable slope (GraphPad Prism). The initial dilution of sera 659 (based on the final volume of RVPs, cells, and sera) was set as the limit of quantification of the 660 assay. EC50 titers below the limit of quantification were reported as a titer half the limit of 661 quantification. A244 11 gp120/Rehydragel A244 11 gp120/ALFQ A244 11 gp120 mRNA-LNP A244.CD4KO 11 gp120 mRNA-LNP 

Immunization group Immunization group A244 11 gp120/Rehydragel A244 11 gp120/ALFQ A244 11 gp120 mRNA-LNP A244.CD4KO 11 gp120 mRNA-LNP 

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Formulation (ALF) family of vaccine adjuvants. Expert review of vaccines 19

Vaccine protection against acquisition of neutralization-resistant 783

SIV challenges in rhesus monkeys

Pentavalent HIV-1 vaccine protects against simian-human 785 immunodeficiency virus challenge

Increased HIV-1 vaccine efficacy against viruses with genetic 788 signatures in Env V2

Vaccine-induced IgG antibodies to V1V2 regions of multiple HIV-790 1 subtypes correlate with decreased risk of HIV-1 infection

Vaccine induction of antibodies against a structurally heterogeneous 793 site of immune pressure within HIV-1 envelope protein variable regions 1 and 2

Epitope specificity of human immunodeficiency virus-1 antibody 796 dependent cellular cytotoxicity [ADCC] responses

HIV-1 vaccine-induced C1 and V2 Env-specific antibodies synergize for

Dendritic cells pulsed with 836 protein antigens in vitro can prime antigen-specific, MHC-restricted T cells in situ. The 837

Dendritic 839 cells as antigen presenting cells in vivo

Systemic RNA delivery to dendritic cells exploits antiviral defence for 842 cancer immunotherapy

HIV-1 Vaccines Based on Antibody Identification

Human circulating PD-1+CXCR3-CXCR5+ memory Tfh cells are highly 847 functional and correlate with broadly neutralizing HIV antibody responses

Risk behaviour and time as covariates for efficacy of the HIV vaccine 850 regimen ALVAC-HIV (vCP1521) and AIDSVAX B/E: a post-hoc analysis of the Thai 851 phase 3 efficacy trial RV 144

Evaluation of a mosaic HIV-1 vaccine in a multicentre, randomised

/2a clinical trial (APPROACH) and in rhesus 855 monkeys (NHP 13-19)

Race: Challenges and Opportunities in Vaccine Formulation

Rational Design of DNA-Expressed Stabilized Native-Like HIV-1

Vaccine Induction of Heterologous Tier 2 HIV-1 Neutralizing 864

Soluble Prefusion Closed DS-SOSIP.664-Env Trimers of Diverse 867

HIV-1 Strains

Quantification of the Impact of the HIV-1-Glycan Shield on Antibody 869

Disruption of the HIV-1 Envelope allosteric network blocks CD4-871 induced rearrangements

In vitro transcription of long RNA 874 containing modified nucleosides

Sequence-engineered mRNA Without Chemical Nucleoside 877

Modifications Enables an Effective Protein Therapy in Large Animals

Analysis of a clonal lineage of HIV-1 envelope V2/V3 928 conformational epitope-specific broadly neutralizing antibodies and their inferred 929 unmutated common ancestors