key: cord-0717530-hbxan73y
authors: van Doremalen, Neeltje; Fischer, Robert J.; Schulz, Jonathan E.; Holbrook, Myndi G.; Smith, Brian J.; Lovaglio, Jamie; Petsch, Benjamin; Munster, Vincent J.
title: Immunogenicity of low dose prime-boost vaccination of mRNA vaccine CV07050101 in non-human primates
date: 2021-07-07
journal: bioRxiv
DOI: 10.1101/2021.07.07.451505
sha: e85fde7753f9a003e3d040dec3000a8a4269bb18
doc_id: 717530
cord_uid: hbxan73y

Many different vaccine candidates against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the etiological agent of COVID-19, are currently approved and under development. Vaccine platforms vary from mRNA vaccines to viral-vectored vaccines, and several candidates have been shown to produce humoral and cellular responses in small animal models, non-human primates and human volunteers. In this study, six non-human primates received a prime-boost intramuscular vaccination with 4 μg of mRNA vaccine candidate CV07050101, which encodes a pre-fusion stabilized spike (S) protein of SARS-CoV-2. Boost vaccination was performed 28 days post prime vaccination. As a control, six animals were similarly injected with PBS. Humoral and cellular immune responses were investigated at time of vaccination, and two weeks afterwards. No antibodies could be detected two and four weeks after prime vaccination. Two weeks after boost vaccination, binding but no neutralizing antibodies were detected in 4 out of 6 non-human primates. SARS-CoV-2 S protein specific T cell responses were detected in these 4 animals. In conclusion, prime-boost vaccination with 4 μg of vaccine candidate CV07050101 resulted in limited immune responses in 4 out of 6 non-human primates.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent 30 responsible for COVID-19. SARS-CoV-2 has spread worldwide and over 185 million cases have been 31 detected as of July 2021. The pandemic has resulted in an unprecedented research effort towards the 32 development of a SARS-CoV-2 vaccine and several vaccines against SARS-CoV-2 have now been 33

Preclinical assessment of SARS-CoV-2 vaccines in non-human primate models is advantageous 39 due to the close relatedness of non-human primates to humans, thereby resulting in a higher degree of 40 clinical translation than smaller animal models. Indeed, rhesus macaques have been successfully used to 41 study vaccines 14 . Inoculation of rhesus macaques with SARS-CoV-2 results in respiratory disease which 42 includes virus replication in upper and lower respiratory tract 15 . Two reports on the immune response of 43 SARS-CoV-2 mRNA vaccine candidates in non-human primates describe the induction of binding and 44 neutralizing antibodies, as well as antigen-specific T cell responses 9, 10 . 45 SARS-CoV-2 messenger RNA (mRNA) vaccines encoding the SARS-CoV-2 spike (S) protein 46 have a good safety and immunogenicity profile, both in non-human primates 9,10 and in humans 6, 7, 16 . Here, 47

we investigate the immunogenicity of another SARS-CoV-2 S mRNA vaccine, CV07050101, in non-48 human primates. CV07050101 is based on mRNA technology, RNActive ® , developed by CureVac for the 49 accelerated development of human vaccines 17-21 . The efficaciousness of this platform has been 50 demonstrated for a rabies vaccine in mice and humans 18, 22 . Moreover, mRNA vaccines have been 51 discussed as particular well suited to combat outbreak pathogens 23 . 52 53

In order to investigate the immunogenicity of mRNA vaccine CV07050101, we vaccinate six 55 rhesus macaques (all male) at 0 and 28 days via intramuscular injection, using 4 µg per dose. As a 56 control, six rhesus macaques were injected with an equal volume of sterile PBS ( Figure 1A ). No adverse 57 events were observed upon vaccination, and overall hematology and clinical chemistries were 58 unremarkable. No differences between the control and vaccinated groups were noted. No binding 59 antibodies could be detected 14 or 28 days post prime vaccination ( Figure 1B ). 14 days post boost 60 vaccination, low titers of spike-specific binding antibodies (reciprocal endpoint IgG titers of 400-800) 61 could be detected in 4 out of 6 animals ( Figure 1B ). Virus-specific neutralizing antibodies were not 62 and is also made available for use under a CC0 license.

was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105

The copyright holder for this preprint (which this version posted July 7, 2021. ; https://doi.org/10. 1101 detected in animals at any time post boost vaccination ( Figure 1C ). No SARS-CoV-2 spike-specific T cell 63 responses were detected 14 days post prime vaccination but were detected in the same 4 out of 6 animals 64 at 14 days post boost vaccination ( Figure 1D ). The detection of specific T cell responses correlated with 65 the detection of spike-specific binding antibodies. 66 67

Here, we show that prime-boost vaccination of rhesus macaques with 4 µg of CV07050101 69 results in the induction of binding antibodies in some, but not all vaccinated animals. This contrasts with 70 other studies with mRNA vaccines, in which a prime-boost vaccination elicits a robust humoral and 71 cellular response in all animals. Using a prime-boost regimen of 10 µg of mRNA-1273, which encodes a 72 prefusion-stabilized S protein utilizing modified mRNA, S-specific binding antibodies were detected in 73 all animals, whereas neutralizing antibodies were detected in 7 out of 8 animals 9 . Likewise, a prime-boost 74 vaccination using 30 µg of vaccine candidate BNT162b2, which also encodes a prefusion-stabilized S 75 protein, elicits binding and neutralizing antibodies in 6 out of 6 animals. Moreover, binding and 76 neutralizing antibodies were detected in all animals 21 days post prime only vaccination with 30 µg of 77 BNT162b2 10 . A recently released preprint investigating CV07050101 showed that vaccination of rhesus 78 macaques with 8 µg of mRNA elicits binding and neutralizing antibodies, whereas a dose of 0.5 µg of 79 mRNA did not 23 . 80

One important difference between these studies is the amount of mRNA used to vaccinate 81 animals. We used 4 µg of mRNA per vaccination, whereas the other doses which elicited an immune 82 response used between 8 µg and 100 µg per vaccination 9,10,24 . Using a dose of 10 µg mRNA-1273 vaccine 83 resulted in no detectable neutralizing antibodies in 1 out of 8 animals on the day of challenge, but 100 µg 84 of mRNA-1273 resulted in neutralizing antibodies in all animals 9 , suggesting a dose-dependent response 85 to the vaccine. Likewise, whereas 8 µg of CV07050101 induced an immune response, 0.5 µg did not 23 . 86 and is also made available for use under a CC0 license.

was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105

The copyright holder for this preprint (which this version posted July 7, 2021. ; https://doi.org/10. 1101 Compared to the limited immunogenicity in non-human primates we observed here, robust 87 SARS-CoV-2 neutralizing titers were observed in Balb/c mice immunized with the CV07050101 vaccine 88 after prime-boost regimen. Challenge studies in hamsters, which were performed at a later stage, utilized 89 a 10 µg prime-boost regimen of CV07050101 vaccine and a challenge dose of 10 2 TCID50 SARS-CoV-2 90 and provided protection of the lower respiratory tract 24 . 91

As the elicited immune response was low or absent in the vaccinated rhesus macaques, we 92 decided not to challenge the animals. In rhesus macaques, neutralizing antibodies are a correlate of 93 protection 25 . The presence of neutralizing antibodies in humans correlates with immunity against SARS-94

CoV-2 26 . Since we did not detect neutralizing antibodies, we hypothesize that these animals would not 95 have been protected. 96

Since CV07050101 is now assessed in clinical trial studies, we compared the available 97 immunogenicity results. The 12 µg high dose vaccine prime-boost regime was able to induce neutralizing 98 antibody titers comparable to non-hospitalized individuals, whereas the 2-8 µg doses induced neutralizing 99 titers that were lower in clinical trial participants. However, virus neutralizing antibodies could be 100 detected in 66% of human volunteers given 4 µg of CV07050101 16 , in contrast to no neutralizing 101 antibodies in serum from NHPs vaccinated with the same dose. It has been hypothesized that a difference 102 in the lubricant used in syringes can decrease integrity of the mRNA vaccine, which may explain the low 103 immunogenicity detected in this NHP study (B.P. personal communication). 104

In conclusion, we show that prime-boost vaccination of rhesus macaques with 4 µg of 105 CV07050101 does not elicit a uniform nor robust immune response. However, vaccination using 8 µg of 106 the same vaccine was protective in a NHP challenge study demonstrating protection against SARS-CoV-2 107 infection by CV07050101 vaccination 23 . 108 109 and is also made available for use under a CC0 license.

was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105

The copyright holder for this preprint (which this version posted July 7, 2021. assay; open red circle = animal negative in ELISA assay; blue triangles = control animals; grey triangles 117 = convalescent human sera; dotted line = lower limit of detection. 118 and is also made available for use under a CC0 license.

was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105

The copyright holder for this preprint (which this version posted July 7, 2021. 

12 male rhesus macaques between 3-5 years old were screened for SARS-CoV-2 status by ELISA, and 137 when found to be negative for prior exposure were sorted by body weight, and then divided into two 138 groups of six animals, resulting in near equal contribution of body weights. Group 1 (vaccine) was 139 vaccinated with 4 µg of mRNA vaccine CV07050101 in sterile PBS at 0 and 28 days, group 2 (control) 140 was vaccinated with sterile PBS at 0 and 28 days via intramuscular injection, using Monoject 1 mL 141

Tuberculin syringes (Covidien, 25G x 5/8"). Blood samples were obtained before vaccination and 14 days 142 and is also made available for use under a CC0 license.

was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105

The copyright holder for this preprint (which this version posted July 7, 2021. ; https://doi.org/10.1101/2021.07.07.451505 doi: bioRxiv preprint after each vaccination. Hematology analysis was completed on a ProCyte DX (IDEXX Laboratories, 143

Westbrook, ME, USA) and the following parameters were evaluated: red blood cells (RBC), hemoglobin 144 (Hb), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean 145 corpuscular hemoglobin concentration (MCHC), red cell distribution weight (RDW), platelets, mean 146 platelet volume (MPV), white blood cells (WBC), neutrophil count (abs and %), lymphocyte count (abs 147 and %), monocyte count (abs and %), eosinophil count (abs and %), and basophil count (abs and %). 148

Serum chemistries were completed on a VetScan VS2 Chemistry Analyzer (Abaxis, Union City, CA) and 149 the following parameters were evaluated: glucose, blood urea nitrogen (BUN), creatinine, calcium, 150 albumin, total protein, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline 151 phosphatase (ALP), total bilirubin, globulin, sodium, potassium, chloride, and total carbon. 152

A plasmid encoding the prefusion stabilized SARS-CoV-2 spike protein with a T4 fibritin trimerization 154 motif was obtained from the Vaccine Research Centre, Bethesda, USA and expressed in-house. Maxisorp 155 plates (Nunc) were coated overnight at 4 °C with 100 ng/well spike protein in PBS. Plates were blocked 156 with 100 µl of casein in PBS (Thermo Fisher) for 1hr at RT. Serum serially diluted 2x in casein in PBS 157 was incubated at RT for 1hr. Antibodies were detected using affinity-purified polyclonal antibody 158 peroxidase-labeled goat-anti-monkey IgG (Seracare, 074-11-021) in casein and TMB 2-component 159 peroxidase substrate (Seracare, 5120-0047), developed for 5-10 min, and reaction was stopped using stop 160 solution (Seracare, 5150-0021) and read at 450 nm. All wells were washed 3x with PBST 0.1% tween in 161 between steps. Threshold for positivity was set at 3x OD value of negative control (serum obtained from 162 non-human primates prior to start of the experiment) or 0.2, whichever one was higher. 163

PBMCs were isolated from ethylene diamine tetraaceticacid (EDTA) whole blood using LeucosepTM 165 tubes (Greiner Bio-one International GmbH) and Histopaque®-1077 density gradient cell separation 166 and is also made available for use under a CC0 license.

was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105

The copyright holder for this preprint (which this version posted July 7, 2021. ; https://doi.org/10.1101/2021.07.07.451505 doi: bioRxiv preprint medium (Sigma-Aldrich) according to the manufacturers' instructions. IFN-γ ELISpot assay of PBMCs 167 was performed using the ImmunoSpot® Human IFN-γ Single-Color Enzymatic ELISpot Assay Kit 168 according to the manufacturer's protocol (Cellular Technology Limited). PBMCs were plated at a 169 concentration of 300,000 cells per well and were stimulated with two contiguous peptide pools spanning 170 the length of the SARS-CoV-2 S protein sequence at a concentration of 2 µg/mL per peptide 171 (Mimotopes). Imaging was performed using the CTL ImmunoSpot® Software (Cellular Technology 172 Limited). Spot forming units (SFU) were hand counted and calculated per 10 6 PBMCs as summed across 173 the peptide pools for each animal. 174

VeroE6 cells were maintained in Dulbecco's modified Eagle's media (DMEM) supplemented with 10% 176 fetal bovine serum (Gibco), 1 mM L-glutamine, 50 U/mL streptomycin and 50 ug/mL penicillin. Sera 177 were heat-inactivated (30 min, 56 °C), two-fold serial dilutions were prepared in DMEM supplemented 178 with 2% fetal bovine serum (Gibco), 1 mM L-glutamine, 50 U/mL streptomycin and 50 ug/mL penicillin 179 and 100 TCID50 of SARS-CoV-2 was added. After 1hr incubation at 37 °C and 5% CO2, virus:serum 180 mixture was added to VeroE6 cells and incubated at 37 °C and 5% CO2. At 6 dpi, cytopathic effect was 181 scored. The virus neutralization titer was expressed as the reciprocal value of the highest dilution of the 182 serum which still inhibited 100% of virus replication. A positive control standardized against the NIBSC 183 serum control 20/130 was used in all VN assays. 184

Phase 1-2 Trial of a SARS-CoV-2 Recombinant Spike Protein 186 Nanoparticle Vaccine

Immunogenicity and Safety of a SARS-CoV-2 Inactivated Vaccine in 188

Healthy Adults Aged 18-59 years: Report of the Randomized, Double-blind, and Placebo-controlled 189 Phase 2 Clinical Trial

Safety and immunogenicity of the ChAdOx1 nCoV-19 191 vaccine against SARS-CoV-2: a preliminary report of a phase 1/2, single-blind, randomised controlled 192 trial

Safety and immunogenicity of an rAd26 and rAd5 194 vector-based heterologous prime-boost COVID-19 vaccine in two formulations: two open, non-195 randomised phase 1/2 studies from Russia

Safety, tolerability, and immunogenicity of a recombinant 197 adenovirus type-5 vectored COVID-19 vaccine: a dose-escalation, open-label, non-randomised, first-in-198 human trial

An mRNA Vaccine against SARS-CoV-2 -Preliminary 200 Report

Phase 1/2 study of COVID-19 RNA vaccine BNT162b1 in 202 adults

ChAdOx1 nCoV-19 vaccine prevents SARS-CoV-2 204 pneumonia in rhesus macaques

Evaluation of the mRNA-1273 Vaccine against SARS-CoV-2 206 in Nonhuman Primates

A prefusion SARS-CoV-2 spike RNA vaccine is highly 208 immunogenic and prevents lung infection in non-human primates

Single-shot Ad26 vaccine protects against SARS-CoV-2 in 210 rhesus macaques

Evaluation of the immunogenicity of prime-boost 212 vaccination with the replication-deficient viral vectored COVID-19 vaccine candidate ChAdOx1 nCoV-19. 213

SARS-CoV-2 mRNA vaccine design enabled by prototype 215 pathogen preparedness

Animal models for COVID-19

Respiratory disease in rhesus macaques 219 inoculated with SARS-CoV-2

Phase 1 224 Assessment of the Safety and Immunogenicity of an mRNA-Lipid Nanoparticle Vaccine Candidate 225 Against SARS-CoV-2 in Human Volunteers

RNActive(R) Technology: Generation and 227 Testing of Stable and Immunogenic mRNA Vaccines

Unmodified mRNA in LNPs constitutes a competitive 229 technology for prophylactic vaccines

Messenger RNA-based vaccines with dual 231 activity induce balanced TLR-7 dependent adaptive immune responses and provide antitumor activity

Protective efficacy of in vitro synthesized, specific mRNA 234 vaccines against influenza A virus infection

An mRNA Vaccine Encoding Rabies Virus Glycoprotein Induces 236 Protection against Lethal Infection in Mice and Correlates of Protection in Adult and Newborn Pigs

Advances in RNA Vaccines for Preventive Indications: A Case 239 Study of A Vaccine Against Rabies. Vaccines (Basel)

New Vaccine Technologies to Combat Outbreak 241

mRNA-based SARS-CoV-2 243 vaccine candidate CVnCoV induces high levels of virus-neutralising antibodies and mediates protection 244 in rodents

Correlates of protection against SARS-CoV-2 in rhesus 246 macaques

Neutralizing Antibodies Correlate with Protection 248 from SARS-CoV-2 in Humans during a Fishery Vessel Outbreak with a High Attack Rate

We thank Olubukola Abiona

Les Shupert, and Marissa Woods for their assistance during this study

Author Contributions. N.v.D and V.M. designed the studies

National 259 Institutes of Health (NIH) (1ZIAAI001179-01). Competing interests. B.P. is an employee of CureVac 260