key: cord-0877846-m2xqeuq5 authors: Rosati, Margherita; Agarwal, Mahesh; Hu, Xintao; Devasundaram, Santhi; Stellas, Dimitris; Chowdhury, Bhabadeb; Bear, Jenifer; Burns, Robert; Donohue, Duncan; Pessaint, Laurent; Andersen, Hanne; Lewis, Mark G.; Terpos, Evangelos; Dimopoulos, Meletios Athanasios; Wlodawer, Alexander; Mullins, James I.; Venzon, David. J.; Pavlakis, George N.; Felber, Barbara K title: Control of SARS-CoV-2 infection after Spike DNA or Spike DNA+Protein co-immunization in rhesus macaques date: 2021-06-11 journal: bioRxiv DOI: 10.1101/2021.06.11.448032 sha: 3bf1ac3e1223f770e47b6b3798c85ef4748202a8 doc_id: 877846 cord_uid: m2xqeuq5 The speed of development, versatility and efficacy of mRNA-based vaccines have been amply demonstrated in the case of SARS-CoV-2. DNA vaccines represent an important alternative since they induce both humoral and cellular immune responses in animal models and in human trials. We tested the immunogenicity and protective efficacy of DNA-based vaccine regimens expressing different prefusion-stabilized SARS-CoV-2 Spike antigens upon intramuscular injection followed by electroporation in rhesus macaques. Different Spike DNA vaccine regimens induced antibodies that potently neutralized SARS-CoV-2 in vitro and elicited robust T cell responses. The DNA-only vaccine regimens were compared to a regimen that included co- immunization of Spike DNA and protein in the same anatomical site, the latter of which showed significant higher antibody responses. All vaccine regimens led to control of SARS-CoV-2 intranasal/intratracheal challenge and absence of virus dissemination to the lower respiratory tract. Vaccine-induced binding and neutralizing antibody titers and antibody-dependent cellular phagocytosis inversely correlated with transient virus levels in the nasal mucosa. Importantly, the Spike DNA+Protein co-immunization regimen induced the highest binding and neutralizing antibodies and showed the strongest control against SARS-CoV-2 challenge in rhesus macaques. Author summary Anti-Spike neutralizing antibodies provide strong protection against SARS-CoV-2 infection in animal models, and correlate with protection in humans, supporting the notion that induction of strong humoral immunity is key to protection. We show induction of robust antibody and T cell responses by different Spike DNA-based vaccine regimens able to effectively mediate protection and to control SARS-CoV-2 infection in the rhesus macaque model. This study provides the opportunity to compare vaccines able to induce different humoral and cellular immune responses in an effort to develop durable immunity against the SARS-CoV-2. A vaccine regimen comprising simultaneous co-immunization of DNA and Protein at the same anatomical site showed best neutralizing abilities and was more effective than DNA alone in inducing protective immune responses and controlling SARS-CoV-2 infection. Thus, an expansion of the DNA vaccine regimen to include co-immunization with Spike protein may be of advantage also for SARS-CoV-2. 13 study revealed rank-ordered neutralization capability against a series of additional Spike variants 245 with distinct mutations in RBD. 246 Control of CoV-2 challenge in macaques 247 Five weeks after the last vaccination, groups G1 to G4 and 4 naïve control macaques were 248 challenged with SARS-CoV-2 (strain-2019-nCoV/USA-WA1/2020) via a combined intranasal 249 (IN) and intratracheal (IT) route. Virus loads (VL) were measured using the subgenomic (sg) 250 Nucleocapsid (N-sg) (Fig 4A) and Envelope (E-sg) (Fig 4B) PCR assays. VL were measured at 251 days 1 and 3 in nasal and in pharyngeal swabs, respectively, and on day 4 in bronchoalveolar 252 lavage (BAL) (Fig 4A and 4B) . Although both assays measured viral mRNA transcripts the N-253 sg assay was more sensitive. 254 All control animals became infected following challenge, with positive VL at day 1 and 255 increasing VL by day 3 in the nasal swabs (Fig 4A and 4B , left panels), positive VL in 256 pharyngeal swabs at day 1 and 3 (Fig 4A and 4B , middle panels) as well as in the lungs (BAL) 257 at day 4 (Fig 4A and 4B, right panels) . All control animals also developed anti-Spike antibodies 258 by 8-10 days post-infection (Fig 5A) . 259 In contrast to the controls, all vaccinated animals showed control of virus replication and lack 260 of dissemination to the lower respiratory tract. Indeed, all animals in DNA+Protein vaccine 261 group (G3) had significantly lower or undetectable VL, in all assays, tissues and sampling times 262 (Fig 4A and 4B, left panels) . On the other hand, in the DNA-only vaccine groups (G1, G2, G4), 263 virus replication was detected on day 1 in nasal swabs by the N-sg RNA VL assay. In contrast to 264 the control group, however, by day 3 viral loads were significantly reduced in the G1 and G2 265 groups. Interestingly, similar as in the control group, the G4 (2x DNA vaccinations) had high 266 viral loads in nasal swabs at day 1 that diminished only marginally by day 3. However, in 267 14 contrast to the control group, no viral RNA was detected at day 4 in the lung (BAL) in none of 268 these 3 vaccine groups, and in was only infrequently detected in the pharyngeal mucosa at days 1 269 or 3 (Fig 4A and 4B, middle and right panels) . Hence Together, anti-Spike antibody levels, neutralization ability and ability to mediate ADCP 287 effector function were predictive of challenge outcomes. Thus, DNA-based vaccines, either 288 alone or as part of the protein co-immunization regimen were able to control SARS-CoV-2 289 infection in the rhesus macaque model. 290 Post-challenge immune response analysis 291 Analysis of post challenge anti-Spike immune responses showed, as noted above, that anti-Spike 292 antibodies were present by day 10 post challenge in all control animals (Fig 5A) . In contrast, no 293 anamnestic anti-Spike antibody response were found in vaccine groups G1-G3 (Fig 5B) . Only 294 two macaques in G4, which also had the highest and most persistent VL in nasal swabs among 295 the vaccinees (up to day 3; see Fig 4A) , developed an anamnestic anti-Spike antibody response 296 by day 8 -10 ( Fig 5B) . 297 Spike-specific T cell responses were also monitored 2 and 4 weeks after the last vaccination 298 (challenge days -21, and -7) and post-challenge (day 10) (Fig 5C and 5D ). The size of the Spike-299 specific total IFN-g + memory T cell population did not significantly change in the vaccinees over 300 time (Fig 5C) . Analysis of the proliferative capacity of Spike-specific CD4 + memory T cells 301 (Ki67 expression) showed the expected contraction after the last vaccination (Fig 5D) , followed 302 by very limited proliferation after challenge that was mor pronounced in G4. Thus, the 303 anamnestic humoral and cellular responses to virus challenge in the two animals in G4 is likely 304 associated with the slightly longer persistence of virus in the upper respiratory tract. The size of 305 the CD8 + T cell population did not change upon challenge within the timeframe measured (day 306 10) and T cell responses could not be correlated with virus loads (day 1). Spike-specific T cell 307 responses were not detected in the control group during the 10-day post-infection monitoring 308 period. 309 Together, our data showed induction of robust antibody and T cell responses by the DNA-310 only and the DNA+Protein vaccines regimens able to effectively mediate protection and to 311 control SARS-CoV-2 infection in the rhesus macaque model. The best vaccination efficacy was 312 achieved in animals that received the DNA+Protein co-immunization regimen. 313 In this report, two types of SARS-CoV-2 vaccination protocols were explored, DNA-only and 315 simultaneous co-immunization of DNA and Protein at the same anatomical site. Immunogenicity 316 was evaluated for DNA vaccines expressing prefusion stabilized Spike proteins differing in the 317 presence or absence of the furin cleavage site and C-terminal region in mice and rhesus 318 macaques. The DNA+Protein regimen combined Spike-DF to maximize the prefusion structure 319 together with soluble GLA-SE adjuvanted Spike-RBD protein. Each vaccine regimens induced 320 antibodies able to neutralize SARS-CoV-2 in vitro and in vivo and mounted robust protection 321 against SARS-CoV-2 replication in the lower respiratory tract of the macaques. Importantly, 322 DNA+Protein co-immunization regimen induced the highest magnitude of binding and 323 neutralizing antibodies and afforded an improved virologic control in the upper respiratory tract. 324 The results of SARS-CoV-2 DNA+Protein co-immunization were consistent with our 325 previous findings from HIV/SIV DNA+Protein co-immunization studies, where co-326 immunization in the same site provided the highest antibody responses, while also inducing T 327 cell responses [28] [29] [30] [31] [32] [33] . Here, DNA+Protein immunization induced the most robust anti-Spike 328 antibody responses, evident after a single vaccination, and these levels remained higher 329 throughout the study, also consistent with previously reported in HIV/SIV vaccine studies [28-330 33] . During the completion of our report, Li et al [51] reported a vaccine regimen in which DNA 331 and protein were administered simultaneously (but in separate anatomical sites). They found 332 better protection in this group compared to DNA or protein only vaccines. These data are in 333 overall agreement with our current study and support the inclusion of both vaccine components 334 starting with the priming vaccination. Importantly, however, we have previously shown that 335 administration of HIV-Env DNA and protein in the same sites resulted in significant differences 336 in antibody magnitude quality, and superior protective immunity from SHIV infection versus 337 DNA and protein administered in contralateral sites [28] . 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