key: cord-0985864-t2qrzcts
authors: He, Xuan; Chandrashekar, Abishek; Zahn, Roland; Wegmann, Frank; Yu, Jingyou; Mercado, Noe B.; McMahan, Katherine; Martinot, Amanda J.; Piedra-Mora, Cesar; Beecy, Sidney; Ducat, Sarah; Chamanza, Ronnie; Huber, Sietske Rosendahl; van der Fits, Leslie; Borducchi, Erica N.; Lifton, Michelle; Liu, Jinyan; Nampanya, Felix; Patel, Shivani; Peter, Lauren; Tostanoski, Lisa H.; Pessaint, Laurent; Van Ry, Alex; Finneyfrock, Brad; Velasco, Jason; Teow, Elyse; Brown, Renita; Cook, Anthony; Andersen, Hanne; Lewis, Mark G.; Schuitemaker, Hanneke; Barouch, Dan H.
title: Low-Dose Ad26.COV2.S Protection Against SARS-CoV-2 Challenge in Rhesus Macaques
date: 2021-01-27
journal: bioRxiv
DOI: 10.1101/2021.01.27.428380
sha: 0726d4ce97297571a0e234c018a11c0fa1ffb596
doc_id: 985864
cord_uid: t2qrzcts

We previously reported that a single immunization with an adenovirus serotype 26 (Ad26) vector-based vaccine expressing an optimized SARS-CoV-2 spike (Ad26.COV2.S) protected rhesus macaques against SARS-CoV-2 challenge. In this study, we evaluated the immunogenicity and protective efficacy of reduced doses of Ad26.COV2.S. 30 rhesus macaques were immunized once with 1×1011, 5×1010, 1.125×1010, or 2×109 vp Ad26.COV2.S or sham and were challenged with SARS-CoV-2 by the intranasal and intratracheal routes. Vaccine doses as low as 2×109 vp provided robust protection in bronchoalveolar lavage, whereas doses of 1.125×1010 vp were required for protection in nasal swabs. Activated memory B cells as well as binding and neutralizing antibody titers following vaccination correlated with protective efficacy. At suboptimal vaccine doses, viral breakthrough was observed but did not show evidence of virologic, immunologic, histopathologic, or clinical enhancement of disease compared with sham controls. These data demonstrate that a single immunization with a relatively low dose of Ad26.COV2.S effectively protected against SARS-CoV-2 challenge in rhesus macaques. Moreover, our findings show that a higher vaccine dose may be required for protection in the upper respiratory tract compared with the lower respiratory tract.

Immune correlates of protection against SARS-CoV-2 have yet to be defined in humans.

We recently reported that purified IgG from convalescent rhesus macaques protected naïve animals against SARS-CoV-2 challenge in a dose-dependent fashion and that cellular immune responses may also contribute to protection 1 . We previously demonstrated that an adenovirus serotype 26 (Ad26) vector 2 expressing a stabilized SARS-CoV-2 Spike 3,4 , termed Ad26.COV2.S, effectively protected rhesus macaques against SARS-CoV-2 infection and protected hamsters against severe SARS-CoV-2 disease 5,6 . In these studies, vaccine-elicited binding and neutralizing antibodies correlated with protection 5,6 . DNA vaccines, mRNA vaccines, ChAdOx1 vectors, and inactivated virus vaccines have also been reported to protect against SARS-CoV-2 challenge in macaques 7-11 .

In multiple SARS-CoV-2 vaccine studies in nonhuman primates, protection in the upper respiratory tract appeared less robust than protection in the lower respiratory tract [6] [7] [8] 11 . These data have raised the possibility that protection against asymptomatic infection may be more difficult to achieve than protection against severe pneumonia in humans. However, the role of vaccine dose in protection in the upper and lower respiratory tracts has not previously been defined. Moreover, suboptimal vaccine doses can be utilized to assess the theoretical concern of vaccine-associated enhanced respiratory disease (VAERD), although VAERD has not been reported to date in SARS-CoV-2 vaccine studies in animals or humans.

In this study, we assessed the immunogenicity and protective efficacy of a titration of Ad26.COV2.S dose levels to evaluate immune correlates of protection, to define the role of reduced vaccine doses in protecting different anatomic respiratory compartments, and to assess the possibility of VAERD. We observed that low doses of Ad26.COV2.S protected against Ad26.COV2.S challenge in the lower respiratory tract but that higher vaccine dose levels were required to protect in the upper respiratory tract. Suboptimal vaccine dose levels resulted in reduced protective efficacy, but no evidence of VAERD was observed.

We immunized 30 adult male and female rhesus macaques with a single dose of 1x10 11 , 5x10 10 , 1.125x10 10 , or 2x10 9 viral particles (vp) Ad26.COV2.S (N=5/group) or sham (N=10) at

week 0 (Fig. S1) . We observed induction of RBD-specific binding antibodies by ELISA in animals that received the 1x10 11 , 5x10 10 , and 1.125x10 10 vp doses by week 2 and in animals that received the 2x10 9 vp dose by week 4 (Fig. 1A) . Neutralizing antibody (NAb) responses were assessed using a pseudovirus neutralization assay 11-13 and were observed in the majority of animals in the three higher dose groups by week 2, with increasing titers through week 6 ( Fig.   1B) . NAb titers remained low in the 2x10 9 vp group at weeks 2 and 4 but became detectable in all animals by week 6, suggesting slower kinetics and lower magnitude NAb responses (Fig.   1B) .

IFN-g ELISPOT assays using pooled S peptides demonstrated T cell responses in the majority of vaccinated animals that received the 1x10 11 , 5x10 10 , and 1.125x10 10 vp doses at week 4, although there was a trend for lower responses with lower vaccine doses ( Fig. 2A) . In animals that received the 2x10 9 vp dose, only 2 of 5 animals developed detectable ELISPOT responses ( Fig. 2A) . IL-4 ELISPOT responses were undetectable (Fig. 2B) , suggesting induction of Th1biased responses and consistent with prior findings 6 .

We next monitored B cell responses following vaccination by multiparameter flow cytometry. SARS-CoV-2 RBD-specific IgG+ B cells were detected in peripheral blood by week 2 following vaccination and generally expressed the activation marker CD95 and the memory marker CD27 (Fig. S2) , suggesting activated memory B cells 14-16 . RBD-specific activated memory B cells expanded following vaccination in a dose-dependent fashion, with robust responses in all animals that received 1x10 11 vp but marginal responses in animals that received 2x10 9 vp (Fig. 3) . The frequency of RBD-specific activated memory B cells strongly correlated with NAb and ELISA titers (P<0.0001, R=0.7997 and P<0.0001, R=0.8851, respectively, twosided Spearman rank-correlation tests) and moderately correlated with IFN-γ ELISPOT responses (P=0.0063, R=0.5310) (Fig. S3 ).

We challenged all animals at week 6 with 1.0x10 5 TCID50 SARS-CoV-2 by the intranasal (IN) and intratracheal (IT) routes 1,6,11,13 . We assessed viral loads in bronchoalveolar lavage (BAL) and nasal swabs (NS) by RT-PCR specific for subgenomic mRNA (sgRNA), which is believed to measure replicating virus 13,17 . All 10 sham controls were infected and showed a mean peak of 4.45 (range 3.2-6.5) log10 sgRNA copies/ml in BAL (Fig. 4A) . In contrast, vaccinated animals demonstrated no detectable virus in BAL (limit of quantitation 1.69 log10 sgRNA copies/ml), with the exception of one animal in the 2x10 9 vp group and one animal in the 1.125x10 10 vp group (Fig. 4A) . Similarly, all sham controls showed a mean peak of 5.68 (range 3.8-6.9) log10 sgRNA in NS (Fig. 4B ). In contrast with limited viral breakthroughs in the BAL, 80% (4 of 5) of animals in the 2x10 9 vp group, 40% (2 of 5) of animals in the 1.125x10 10 vp group, and 20% (1 of 5) of animals in the 5x10 10 vp group showed viral breakthroughs in NS (Fig. 4B) . These data suggest that a higher vaccine dose may be required for protection in the upper respiratory tract compared with protection in the lower respiratory tract. Suboptimal vaccine dose levels led to loss of protection in NS but did not result in enhanced virus replication compared with the sham controls. P=0.0001, R=-0.6936 and P=0.0014, R=-0.6039, respectively, for day 28 responses; two-sided Spearman rank-correlation tests; Fig. 6A ). In addition, RBD-and S-specific activated memory B cells were higher in completely protected animals compared with partially protected or nonprotected animals (P=0.0006 and P=0.0005, two-sided Mann-Whitney tests; Fig. 6B ). Taken together, these data show that both memory B cell responses and binding and neutralizing antibody titers correlated with protection against SARS-CoV-2 in rhesus macaques.

On day 10 following challenge, animals were necropsied, and lung tissues were assessed by histopathology and immunohistochemistry. We observed focal to locally extensive SARS-CoV-2 associated pathological lesions in sham controls (Fig. 7, Table 1 ). We previously reported histopathologic evidence of viral pneumonia in rhesus macaques on day 2 and day 4 following SARS-CoV-2 infection 13 . On day 10, lungs in sham controls still showed evidence of multifocal interstitial pneumonia with bronchoepithelial syncytia, perivascular mononuclear infiltrates, type II pneumocyte hyperplasia, rare thrombosis, and focal edema and consolidation ( Fig. S4, Table 1 ). RNAscope demonstrated in situ hybridization for viral RNA, immunohistochemistry showed staining for SARS nucleocapsid, and infiltrates included Iba-1+ macrophages, CD3+ T cells, and CD20+ B cells (Fig. S5) . In contrast, vaccinated animals showed minimal histopathologic changes, consistent with background lung pathology, although several animals that received the 2x10 9 vp dose showed evidence of mild inflammation (Figs. 7, 8; Table 1 ). No evidence of VAERD was observed in animals that received high or suboptimal doses of Ad26.COV2.S, including animals that showed breakthrough viral replication in BAL and/or NS.

In this study, we demonstrate that low doses of the Ad26.COV2.S vaccine protected rhesus macaques against SARS-CoV-2 challenge, although higher vaccine doses were required to protect in the upper respiratory tract as compared with the lower respiratory tract. Both activated memory B cells and binding and neutralizing antibody titers correlated with protective efficacy. Suboptimal vaccine dose levels led to viral breakthroughs in the upper respiratory tract, but no virologic, immunologic, histopathologic, or clinical evidence of VAERD was observed.

These data confirm and extend prior studies in which we showed that single-shot immunization with Ad26.COV2.S effectively protected against SARS-CoV-2 infection in rhesus macaques and against severe clinical disease in hamsters 5,6 . In the present study, we showed that Ad26.COV2.S doses as low as 2x10 9 vp protected the lower respiratory tract, whereas doses of 1.125x10 10 vp were required to protect the upper respiratory tract. This anatomic-specific difference in protective efficacy is consistent with multiple SARS-CoV-2 vaccine studies in nonhuman primates using DNA, RNA, and Ad vector-based vaccines, which have consistently shown superior protection in the lungs than in the nasal cavity [6] [7] [8] 11 . These findings suggest that future work evaluating mucosal immune responses in these anatomic compartments is warranted.

Previous studies have suggested that vaccine-elicited binding and neutralizing antibodies correlated with protection against SARS-CoV-2 in rhesus macaques 6, 11 and that adoptive transfer of purified IgG from convalescent macaques protected against SARS-CoV-2 in this model 1 . The present data are consistent with these prior observations, and both RBD-specific activated memory B cells and binding and neutralizing antibody responses correlated with protective efficacy. Moreover, lower vaccine doses led to diminished antibody responses and reduced protective efficacy, further suggesting the importance of humoral immunity in protection against SARS-CoV-2. Suboptimal vaccine dose levels led to viral breakthroughs but did not result in evidence of enhanced viral replication or increased histopathologic pathology in the lungs of vaccinated animals compared with sham controls.

In summary, our data demonstrate that a single immunization of relatively low doses Ad26.COV2.S protects against SARS-CoV-2 challenge in rhesus macaques. Low dose vaccination led to viral breakthrough in the upper respiratory tract prior to the lower respiratory tract, raising the possibility that SARS-CoV-2 vaccines may protect against severe pneumonia more effectively than against asymptomatic upper respiratory tract infection in humans. Two phase 3 trials are currently in progress to determine the safety and efficacy of Ad26. COV2 Pseudovirus neutralization assay. The SARS-CoV-2 pseudoviruses expressing a luciferase reporter gene were generated in an approach similar to as described previously 11-13 .

Briefly, the packaging construct psPAX2 (AIDS Resource and Reagent Program), luciferase reporter plasmid pLenti-CMV Puro-Luc (Addgene), and spike protein expressing pcDNA3.1-SARS-CoV-2 SΔCT were co-transfected into HEK293T cells with calcium phosphate. The supernatants containing the pseudotype viruses were collected 48 h post-transfection; pseudotype viruses were purified by filtration with 0.45 µm filter. To determine the neutralization activity of the antisera from vaccinated animals, HEK293T-hACE2 cells were seeded in 96-well tissue culture plates at a density of 1.75 x 10 4 cells/well overnight. Two-fold serial dilutions of heat inactivated serum samples were prepared and mixed with 50 µL of pseudovirus. The mixture was incubated at 37 o C for 1 h before adding to HEK293T-hACE2 cells. After 48 h, cells were lysed in Steady-Glo Luciferase Assay (Promega) according to the manufacturer's instructions.

SARS-CoV-2 neutralization titers were defined as the sample dilution at which a 50% reduction in RLU was observed relative to the average of the virus control wells.

with mouse anti-human IFN-γ monoclonal antibody from BD Pharmingen at a concentration of 5 µg/well overnight at 4°C, and assays were performed as described 11, 13 . Plates were washed with DPBS containing 0.25% Tween 20, and blocked with R10 media (RPMI with 11% FBS and 1.1% penicillin-streptomycin) for 1 h at 37°C. The Spike 1 and Spike 2 peptide pools contain 15 amino acid peptides overlapping by 11 amino acids that span the protein sequence and reflect the N-and C-terminal halves of the protein, respectively. Spike 1 and Spike 2 peptide pools were prepared at a concentration of 2 µg/well, and 200,000 cells/well were added. The peptides and cells were incubated for 18-24 h at 37°C. All steps following this incubation were performed at room temperature. The plates were washed with coulter buffer and incubated for 2 h with Rabbit polyclonal anti-human IFN-γ Biotin from U-Cytech (1 µg/mL). The plates are washed a second time and incubated for 2 h with Streptavidin-alkaline phosphatase antibody from Southern Biotechnology (1 µg/mL). The final wash was followed by the addition of Nitor-blue Tetrazolium Chloride/5-bromo-4-chloro 3 'indolyl phosphate p-toludine salt (NBT/BCIP chromagen) substrate solution for 7 min. The chromagen was discarded and the plates were washed with water and dried in a dim place for 24 h. Plates were scanned and counted on a Cellular Technologies Limited Immunospot Analyzer.

were washed and blocked. The assay was then performed as described above except the development time with NBT/BCIP chromagen substrate solution was 12 min. Histopathology and immunohistochemistry. At time of fixation, lungs were suffused with 10% formalin to expand the alveoli. All tissues were fixed in 10% formalin and blocks sectioned at 5 µm. Slides were baked for 30-60 min at 65 degrees then deparaffinized in xylene and rehydrated through a series of graded ethanol to distilled water. Heat induced epitope retrieval (HIER) was performed using a pressure cooker on steam setting for 25 minutes in citrate buffer (Thermo; AP-9003-500) followed by treatment with 3% hydrogen peroxide. Slides were then rinsed in distilled water and protein blocked (BioCare, BE965H) for 15 min followed by rinses in 1x phosphate buffered saline. Primary rabbit anti-SARS-nucleoprotein antibody (Novus; NB100-56576) diluted 1:250, rabbit anti-Iba-1 antibody (Wako; 019-19741) diluted 1:250; rabbit anti-CD3 antibody (Sigma, SAB5500057) diluted 1:300, rabbit anti-CD20

(Invitrogen PA5-16701) diluted 1:750 followed by rabbit Mach-2 HRP-Polymer (BioCare;

RHRP520L) for 30 minutes then counterstained with hematoxylin followed by bluing using 0.25% ammonia water. Labeling was performed on a Biocare IntelliPATH autostainer. All antibodies were incubated for 60 min at room temperature. Tissue pathology was assessed independently by two board-certified veterinary pathologists (AJM, RC).

Ad26.COV2.S Sham Dose (vp) 1x10 11 5x10 10 1.125x10 10 2x10 9 NA N 5 5 5 5 10 Median total histopathology score (range) 10 (9-14) 11 (9-14) 11 (8-12) 12 (9-23) 30 (16-45) Average total histopathology score (range) 11 (9-14) 11 (9) (10) (11) (12) (13) (14) 10 ( Eight lung tissue sections representing one section from each of 7 lung lobes, and two sections from the left cranial lobe, were processed histologically and evaluated microscopically for the histopathological findings listed in the table. The listed histopathological findings were graded on a scale of 0-5, with grade 0 representing no findings, grade 1 minimal histological change, 2: mild, 3: moderate, 4: marked and 5: severe/massive histological change. Histopathology grades were tallied for all lung sections, and average scores for each group were calculated. Total histopathology scores were calculated by adding up scores for the individual pathology parameters. BALT: bronchus-associated lymphoid tissue NAb ELISA 

No 310043) as a negative control. In brief, after slides were deparaffinized in xylene and rehydrated through a series of graded ethanol to distilled water, retrieval was performed for 30 min in ACD P2 retrieval buffer

322337) diluted 1:10 in PBS for 20 min at 40°C. Slides were then incubated with 3% H2O2 in PBS for 10 minutes at room temperature. Slides were developed using the RNAscope® 2.5 HD Detection Reagents-RED (ACD Cat

Analysis of virologic and immunologic data was performed using

Comparison of data between groups was performed using two-sided Mann-Whitney tests. Correlations were assessed by two-sided Spearman rank-correlation tests

We thank M. Gebre, K. Verrington, E. Hoffman, L. Wrijil, T. Hayes, and K. Bauer for generous advice, assistance, and reagents. This project was funded in part by the Department of