key: cord-0989152-jyzk4rtr authors: Cunha, Luis Eduardo R.; Stolet, Adilson A.; Strauch, Marcelo A.; Pereira, Victor A. R.; Dumard, Carlos H.; Gomes, Andre M. O.; Souza, Patrícia N. C.; Fonseca, Juliana G.; Pontes, Francisco E.; Meirelles, Leonardo G. R.; Albuquerque, José W. M.; Sacramento, Carolina Q.; Fintelman-Rodrigues, Natalia; Lima, Tulio M.; Alvim, Renata G. F.; Marsili, Federico F.; Caldeira, Marcella Moreira; Higa, Luisa M.; Monteiro, Fábio L.; Zingali, Russolina B.; de Oliveira, Guilherme A. P.; Souza, Thiago M. L.; Tanuri, Amilcar; Oliveira, Andréa C.; Guedes, Herbert L. M.; Castilho, Leda R.; Silva, Jerson L. title: Potent neutralizing equine antibodies raised against recombinant SARS-CoV-2 spike protein for COVID-19 passive immunization therapy date: 2020-11-11 journal: bioRxiv DOI: 10.1101/2020.08.17.254375 sha: 730c312515bb3c9fec30270db475d4ad3a1d2d05 doc_id: 989152 cord_uid: jyzk4rtr We used the trimeric spike (S) glycoprotein (residues 1-1208) in the prefusion conformation to immunize horses for production of hyperimmune globulins against SARS-CoV-2. Serum antibody titers measured by anti-spike ELISA were above 1:1,000,000, and neutralizing antibody titer was 1:14,604 (average PRNT90), which is 140-fold higher than the average neutralizing titer of plasma from three convalescent COVID-19 patients analyzed for comparison. Using the same technology routinely used for industrial production of other horse hyperimmune products, plasma from immunized animals was pepsin digested to remove the Fc portion and purified, yielding a F(ab’)2 preparation with PRNT90 titers 150-fold higher than the neutralizing titers in human convalescent plasma. Repeating the hyperimmunization in a second group of horses confirmed the very high neutralizing titers in serum and in a GMP clinical F(ab’)2 lot. Virus-neutralizing activity in samples from mice that received the F(ab’)2 preparation was detected even three days after injection, indicating an appropriate half-life for therapeutic intervention. These results supported the design of a clinical trial (identifier NCT04573855) to evaluate safety and efficacy of this horse F(ab’)2 preparation. The pandemic caused by SARS-CoV-2, the etiological agent of COVID- 19 , is an urgent health problem worldwide, especially in the Americas (https://covid19.who.int/). Consequences for human health and for the global economy have been devastating. Considering the absence of approved antiviral treatments and vaccines and the uncertainties regarding antibody responses in individuals infected by SARS-CoV-2 1-4 , the COVID-19 scenario seems to be still far from a solution. Passive immunization using plasma from COVID-19 convalescent patients has been used as an alternative therapy 5, 6 . However, the heterogeneous neutralizing titers in different convalescent donors and the simultaneous transfer of other plasma components are drawbacks that hinder its wide use. The development of virus-neutralizing purified hyperimmune globulins produced in horses or llamas may be an approach to treat SARS-CoV-2 infection. The use of llamas 7 to develop passive immunization therapies is still experimental and limited by animal availability. On the other hand, hyperimmune serum, immunoglobulins or IgG fragments produced in horses have been used to treat many diseases, such as rabies, tetanus and snake envenomation, among others 8, 9 . Brazil, such as many other countries, has a large established capacity to produce equine hyperimmune globulin preparations for a range of indications, which makes the production of such products against SARS-CoV-2 highly feasible. Previous works with other related betacoronaviruses reported that equine hyperimmune sera resulted in neutralizing antibodies against SARS-CoV 10 and MERS-CoV 11 . In these works, immunization was performed using virus or virus-like particles, using complete Freund's adjuvant (CFA) in the first immunization and incomplete Freund's adjuvant (IFA) in the subsequent immunizations. More recently, the recombinant receptor-binding domain (RBD) of the SARS-CoV-2 S protein was shown to stimulate antibody production in mice and equines 12, 13 . In the present work, we also used a recombinant antigen to immunize horses. However, the antigen chosen was the trimeric version of the complete ectodomain of the spike protein, comprising both the S1 (responsible for receptor binding) and the S2 (responsible for fusion to the cell membrane) domains. Moreover, the chosen antigen was produced in house using a gene construct that yields the protein in a stabilized, trimeric prefusion conformation 14 , with the aim of maximizing the formation of high-quality neutralizing antibodies. Our strategy may be easily reproduced in any part of the world and could be rapidly tested as a therapy for COVID-19. Equine immunization is a well-known and easily scalable technology proven for generating high titers of neutralizing antibodies, thus showing advantages over other strategies, such as using convalescent human plasma. In this study, we demonstrated extremely high neutralizing titers obtained by means of equine immunization. The final purified F(ab')2 preparation had an average PRNT90 neutralizing titer 150-fold higher than that of human convalescent plasma from three patients in Brazil. A GMP clinical lot has been produced for use in a Phase 1 clinical trial (identifier NCT04573855). We produced trimers of the spike protein in the prefusion conformation 14 by stably expressing the gene construct in mammalian cells as described previously 15 . A quality check of the spike protein was performed by SDS-PAGE, and its trimeric prefusion conformation was verified by size-exclusion chromatography (Fig. S1 ). Initially, five horses were immunized with six subcutaneous injections of S protein, with an interval of one week between inoculations. No adverse effects or animal suffering were observed for any of the five horses that received the protein injections. Anti-spike IgG measured by enzyme-linked immunosorbent assay (ELISA) in weekly serum samples showed that one week after the first immunization anti-SARS-CoV-2 IgG was not yet detectable but that IgG titers increased progressively after successive immunizations (Fig. 1A) . Four out of the five immunized horses produced similar amounts of specific antibodies, but one did not show a strong response (Fig. 2 ). Despite the low-responding horse (#835), the average IgG titer for all five horses reached 1,180,980 after 42 days, i.e., one week after the sixth immunization ( Fig. 1B) , indicating that the trimeric S protein is a good immunogen to induce the production of specific anti-SARS-CoV-2 antibodies. We first evaluated the in vitro neutralizing activity against SARS-CoV-2 of equine sera collected after four to six immunizations (days 28 to 49 after the first immunization). PRNT50 titers (average values for all five horses) seemed to achieve a plateau of approximately 1:23,000 after the fourth immunization, whereas the more stringent PRTN90 titers (average values for all five horses) still showed an ascendant trend over time, reaching an average PRNT90 titer of 1:14,604 on day 49. Due to the low-responding horse (#835), the standard deviations were high ( Fig. 3 ; Table S1 ). Plasma from all five horses was then pooled, digested with pepsin to cleave off the Fc portion and precipitated with ammonium sulfate for purification, resulting in a concentrate of F(ab´)2 fragments containing approximately 90 mg/mL total protein. The F(ab')2 concentrate maintained the capacity to recognize the trimeric S protein, displaying an ELISA titer of 1:1,000,000 (Fig. 4) . In order to confirm if F(ab')2 fragments also maintained their capacity to recognize SARS-CoV-2 proteins in cell culture, we prepared a F(ab')2-FITC conjugate, which was shown to specifically bind to SARS-CoV-2 infected Vero E6 cells ( Figure S2 ). F(ab')2 neutralizing titers were also very high, achieving a PRNT50 of 1:32,000 and a PRNT90 of 1:16,000 ( Fig. 3 and Table S1 ). We further compared the neutralizing titers of the equine samples to the neutralizing titers determined for plasma from three convalescent COVID-19 patients. Interestingly, the average neutralizing titers of equine serum (from day 49) were 78-and 138-fold higher than the average human convalescent plasma titers in terms of PRNT50 and PRNT90, respectively. Regarding the F(ab')2 concentrate, the neutralizing titers were 107-fold (for PRNT50) and 151-fold (for PRNT90) higher than the average human convalescent plasma titer. These data show the great potential of using equine immunoglobulins in the treatment of COVID-19. In order to evaluate sustainability of equine antibody production and to enable scale-up to industrial manufacturing, the first group of five horses was reimmunized, and a second group of five horses was immunized using the same protocol as the first group ( concentrate lot and of the plasma pool used as raw material for its production. Antibody titers were in the range of 1:1,000,000 as obtained for the pilot lot produced from the first bleeding of the first horse group (Fig. 4 ). Neutralizing activity in the GMP API (Table S1 ) was 1:65,556 as PRNT50 and 1:16,384 as PRNT90, showing that the well-established routine GMP process implemented at IVB leads to higher F(ab')2 yields than the pilot process using laboratory equipment. This API was further processed (dilution in water-forinjection, fill and finish) for use in the Phase I/II clinical trials of the anti-COVID-19 F(ab')2 concentrate. To test the clearance of the neutralizing activity, mice were injected with different doses (10, 25 or 50 µg) of the pilot lot of equine F(ab')2 concentrate. After 72 h, blood was collected, and neutralizing activity in mice plasma was determined (Fig. 6 ). The data clearly shows that high neutralizing titers are maintained for at least 3 days after injection, with PRNT50 and PRNT90 titers for the highest dose (50 µg) being comparable to human convalescent plasma (Table S1 ). This result indicates that the F(ab')2 fragments would have a sufficient half-life to reach different organs, including the lungs, to exert its antiviral activity. In the setting of a pandemic, when no vaccines and no specific treatments are available, passive immunotherapies using convalescent human plasma or animal-derived hyperimmune globulins usually represent the first specific antiviral therapies to become available. In previous outbreaks caused by other viruses, such as SARS-CoV, MERS-CoV, Ebola and avian influenza virus, horse immunization to produce hyperimmune globulins was evaluated 10, 11, 16, 17 . In addition to their application in emerging and reemerging infectious diseases, heterologous polyclonal antibody therapies are also useful to treat longstanding neglected tropical diseases 18 . Equine antivenom products are routinely produced in both high-and low-income countries 16 Table S1 ). As mentioned above, convalescent plasma is another therapeutic alternative that quickly becomes available in the setting of outbreaks. For comparison, we evaluated the neutralization titer of three human convalescent plasma samples. We found that the neutralization titers of the equine F(ab')2 concentrates were two orders of magnitude higher than those of human For the production of the bench-scale pilot lot, plasma processing to produce F(ab')2 was initiated by adding 3 L of water and 15 mL of 90% phenol solution to 2 L of horse plasma in a reactor. The solution was homogenized for 10 min, and the pH was adjusted to 4.3. Under agitation, 1.25 g/L pepsin was added, the pH was adjusted to 3.2, and the sample remained under agitation for 10 min. The sample was then stirred at 37°C, and the pH was adjusted to 4.2 with sodium hydroxide. Under constant agitation, sodium pyrophosphate decahydrate (12.6 mM) and toluene (10 µM) were added. Later, ammonium sulfate was added at 12% (m/v), and the solution was incubated at 55°C for 1 h. To separate the F(ab')2 fragments, the Fc portion was precipitated and subsequently filtered under pressure at constant agitation. F(ab')2 was recovered from the liquid phase. To subsequently precipitate F(ab')2 from the liquid phase, ammonium sulfate was added at 19% (m/v), and a second precipitation was performed under constant agitation and alkaline pH. Subsequently, the solution was diafiltered using a 30-kDa tangential ultrafiltration system until ammonium sulfate became undetectable in the retentate. The samples were isotonized with 15 mM NaCl, and 90% phenol solution was added to a final concentration of 0.3% (v/v). After sterile filtration, the F(ab´)2 product was stored at 4°C. The industrial scale GMP-compliant lot was produced according to an analogous process (but scaled up to start from 160 L of plasma), using the industrial GMP facility of IVB in Rio de Janeiro state, Brazil. In brief, polystyrene high-adsorption 96-well microplates (ThermoFisher, USA) were coated with 500 ng/well recombinant SARS-CoV-2 S protein (100 µL/well at 5 µg/mL) in carbonate-bicarbonate buffer (pH 9.6) overnight at room temperature and then blocked with 3% BSA (Sigma, USA) in PBS for 2 h at 37°C. In order to investigate if neutralizing activity against SARS-CoV-2 can be observed in animals treated with the fragments, equine F(ab')2 (pilot lot) was injected by the intraperitoneal route and neutralizing titers were measured in the plasma of mice. 11 Balb/C mice were divided into 4 groups, which received different doses of the antibody fragments: 50 µg (n = 3); 25 µg (n = 3); 10µg (n = 3) and negative control (saline) (n = 2). The total volume injected was 200 µL per mouse. Blood was collected from the retro-orbital plexus 72 hours after inoculation, and then the animals were euthanized by saturation with isoflurane. The neutralization titer in mice plasma was determined by the plate reduction neutralization test (PRNT). Statistical significance was calculated using Graphpad Prism® 7 software, by one-way ANOVA and Tukey post-hoc test to the confidence levels indicated in Figure S2 . Samples of human convalescent plasma collected at the State Hematology Institute Hemorio followed a protocol approved by the local ethics committee (CEP Hemorio; approval #4008095), as described previously 15 . Mice were injected via intraperitoneal inoculation with equine F(ab´)2 (50 µg, 25 µg or 10 µg total protein concentration), and blood was collected 72 h after injection. Neutralizing titers were determined by PRNT assay. The PRNT50 and PRNT90 results are shown in panels A and B, respectively. Data are shown as the mean ± SD; *=p<0.05; **=p<0.01 according to one-way ANOVA and Tukey's post hoc test. Recombinant prefusion trimeric S protein is used to inoculate horses and to produce hyperimmune F(ab`)2 concentrate. The equine antibody preparation presented a much higher capacity to neutralize a SARS-CoV-2 isolate than human convalescent plasma. One advantage of using the full-length recombinant spike trimer is the production of antibodies against different antigenic segments of the viral protein. This strategy may result in more efficient neutralizing capacity than that of antibodies produced against isolated fragments of the spike protein, such as the RBD. 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The results are shown as the mean ± standard error for analytical duplicates. The negative control is a pool of preimmune sera collected from all 5 horses of Group 1.