key: cord-0686059-i969tu6a
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 Heerden, Marjolein; 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: 2021-06-01
journal: Cell
DOI: 10.1016/j.cell.2021.05.040
sha: 5f723db624c63dec35a6e8157d9254a5e2fdd34f
doc_id: 686059
cord_uid: i969tu6a

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. To evaluate reduced doses of Ad26.COV2.S, 30 rhesus macaques were immunized once with 1x1011, 5x1010, 1.125x1010, or 2x109 vp Ad26.COV2.S or sham and were challenged with SARS-CoV-2. Vaccine doses as low as 2x109 vp provided robust protection in bronchoalveolar lavage, whereas doses of 1.125x1010 vp were required for protection in nasal swabs. Activated memory B cells and binding or neutralizing antibody titers following vaccination correlated with protective efficacy. At suboptimal vaccine doses, viral breakthrough was observed but did not show enhancement of disease. These data demonstrate that a single immunization with relatively low dose of Ad26.COV2.S effectively protected against SARS-CoV-2 challenge in rhesus macaques, although a higher vaccine dose may be required for protection in the upper 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 . We previously demonstrated that an adenovirus serotype 26 (Ad26) vector (Abbink et al., 2007) expressing a stabilized SARS-CoV-2 Spike (Bos et al., 2020; Wrapp et al., 2020) , termed Ad26.COV2.S, effectively protected rhesus macaques against SARS-CoV-2 infection and protected hamsters against severe SARS-CoV-2 disease Tostanoski et al., 2020) . In these studies, vaccine-elicited binding and neutralizing antibodies correlated with protection Tostanoski et al., 2020) . DNA vaccines, mRNA vaccines, ChAdOx1 vectors, and inactivated virus vaccines have also been reported to protect against SARS-CoV-2 challenge in macaques Gao et al., 2020; van Doremalen et al., 2020; Wang et al., 2020; Yu et al., 2020) .

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 Mercado et al., 2020; van Doremalen et al., 2020; Yu et al., 2020) . 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 J o u r n a l P r e -p r o o f 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 Yang et al., 2004; Yu et al., 2020) 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 J o u r n a l P r e -p r o o f kinetics and lower magnitude NAb responses (Fig. 1B) . NAb responses at week 6 were 2.5-fold lower against the B.1.1.7 variant but were 3.4-fold lower against the B.1.351 variant (Fig. S2) .

IFN-γ 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 .

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. S3) , suggesting activated memory B cells (Koutsakos et al., 2018; Neumann et al., 2015; Titanji et al., 2010) . RBD-specific activated memory B cells expanded following vaccination in a dose-dependent fashion, with robust responses in animals that received 1x10 11 vp but marginal responses in animals that received 2x10 9 vp (Fig. 3) . The frequency of RBDspecific 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, two-sided Spearman rank-correlation tests) and moderately correlated with IFN-γ ELISPOT responses (P=0.0063, R=0.5310) (Fig. S4 ).

We challenged all animals at week 6 with 1.0x10 5 TCID50 SARS-CoV-2 by the intranasal (IN) and intratracheal (IT) routes McMahan et al., 2020; Mercado et al., 2020; Yu et al., 2020) . We assessed viral loads in bronchoalveolar lavage (BAL) J o u r n a l P r e -p r o o f and nasal swabs (NS) by RT-PCR specific for subgenomic mRNA (sgRNA), which is believed to measure replicating virus Wolfel et al., 2020) . All 10 sham controls were infected and showed a mean peak of 4.45 (range 3.2-6.5) log 10 sgRNA copies/ml in BAL (Fig. 4A) . In contrast, vaccinated animals demonstrated no detectable virus in BAL (limit of quantitation 1.69 log 10 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) log 10 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.

The log 10 ELISA and NAb titers at week 6 inversely correlated with peak log 10 sgRNA in 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 J o u r n a l P r e -p r o o f 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. 7A , Table S1 ). We previously reported histopathologic evidence of viral pneumonia in rhesus macaques on day 2 and day 4 following SARS-CoV-2 infection . 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. S5 , Table S1 ). 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. S6) . 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 ( Fig. 7B ; Table S1 ). 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 Tostanoski et al., 2020) . 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 vectorbased vaccines, which have consistently shown superior protection in the lungs than in the nasal cavity Mercado et al., 2020; van Doremalen et al., 2020; Yu et al., 2020) .

These findings suggest that future work evaluating mucosal immune responses in these anatomic compartments is warranted. Future work also will need to evaluate receptor density in the upper and lower respiratory tract.

Previous studies have suggested that vaccine-elicited binding and neutralizing antibodies correlated with protection against SARS-CoV-2 in rhesus macaques and that adoptive transfer of purified IgG from convalescent macaques protected against SARS-CoV-2 in this model . The present data are consistent with these prior observations, and both RBD-specific activated memory B cells and binding and J o u r n a l P r e -p r o o f 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 enhanced viral replication or increased pathology in the lungs of vaccinated animals compared with sham controls, although other mechanisms may also contribute to enhanced disease.

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 mild disease or asymptomatic infection in humans. However, these findings in macaques will need to be evaluated in humans. To this end, two phase 3 trials are currently in progress to determine the safety and efficacy of Ad26.COV2.S in humans, including against variants of concern. Ad26.COV.2 has recently received FDA emergency use authorization for prevention of COVID-19 disease in humans.

A key limitation of our study is that experiments in macaques may not fully model human vaccine immunogenicity and efficacy. Our study is also limited by the small number of animals per group and the lack of correlations between mucosal immune responses and protective efficacy. Lesions reported included 1) inflammation interstitial/septal thickening 2) infiltrate, macrophage 3) alveolar infiltrate, mononuclear 4) perivascular infiltrate, macrophage 5) bronchiolar type II pneumocyte hyperplasia 6) BALT hyperplasia 7) inflammation, bronchiolar/peribronchiolar infiltrate 8) neutrophils, bronchiolar/alveolar and 9) infiltrate, eosinophils. Lesions such as focal fibrosis and syncytia were reported but not included in scoring. Edema, alveolar flooding was excluded from scoring since animals received terminal bronchoalveolar lavages. Each feature J o u r n a l P r e -p r o o f assessed was assigned a score of 0= no significant findings; 1=minimal; 2= mild; 3=moderate; 4=marked/severe. Eight representative samples from cranial, middle, and caudal lung lobes from the left and right lungs were evaluated from each animal and were scored independently. Scores were added for all lesions across all lung lobes for each animal for a maximum possible score of 288 for each monkey. 

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Dr. Dan Barouch (dbarouch@bidmc.harvard.edu).

This study did not generate new unique reagents.

There is no dataset/code associated with the paper.

Animals and study design. 30 outbred Indian-origin adult male (10) and female (20) rhesus macaques (Macaca mulatta) were randomly allocated to groups. Animals were 5-8 kg.

All animals were housed at Bioqual, Inc. (Rockville, MD). Animals received a single immunization of 1x10 11 , 5x10 10 , 1.125x10 10 , or 2x10 9 viral particles (vp) Ad26.COV2.S (Janssen; N=5/group) or sham (N=10) by the intramuscular route without adjuvant at week 0. At week 6, all animals were challenged with 1.0x10 5 TCID50 (1.2x10 8 RNA copies, 1.1x10 4 PFU) SARS-CoV-2, which was derived from USA-WA1/2020 (NR-52281; BEI Resources) and deep sequenced . Virus was administered as 1 ml by the intranasal (IN) route ( of SuperScript VILO Master Mix, and 25ul of RNA template. The cycling conditions for reverse transcription were, 25ºC for 10 Minutes, 42ºC for 1 Hour then 85ºC for 5 Minutes. cDNA was stored at 4ºC until RT-PCR assays were performed. A Taqman custom gene expression assay (ThermoFisher Scientific) was designed using the sequences targeting the E gene sgRNA (Wolfel et al., 2020) . The sequences for the custom assay were as follows, forward to 110%, and slope −3.1 < x > −3.6. Analysis was performed on the QuantStudio Real-Time PCR J o u r n a l P r e -p r o o f Software (Life Technologies). Standard curves were used to calculate subgenomic RNA copies per ml or per swab; the quantitative assay sensitivity was 50 copies per ml or per swab.

Enzyme-linked immunosorbent assay (ELISA). Binding antibodies were assessed by ELISA essentially as described Yu et al., 2020) . Briefly, 96-well plates were coated with 1 µg/ml SARS-CoV-2 spike (S) or receptor binding domain (RBD) protein in 1X DPBS and incubated at 4°C overnight. After incubation, plates were washed once with wash buffer (0.05% Tween 20 in 1 X DPBS) and blocked with 350 µL Casein block/well for 2-3 h at room temperature. After incubation, block solution was discarded and plates were blotted dry. Serial dilutions of heat-inactivated serum diluted in casein block were added to wells and plates were incubated for 1 h at room temperature, prior to three further washes and a 1 h incubation with a 1:1000 dilution of anti-macaque IgG HRP (NIH NHP Reagent Program) at room temperature in the dark. Plates were then washed three times, and 100 µL of SeraCare KPL TMB SureBlue Start solution was added to each well; plate development was halted by the addition of 100 µL SeraCare KPL TMB Stop solution per well. The absorbance at 450nm was recorded using a VersaMax or Omega microplate reader. ELISA endpoint titers were defined as the highest reciprocal serum dilution that yielded an absorbance > 0.2. Log10 endpoint titers are reported.

Pseudovirus neutralization assay. The SARS-CoV-2 pseudoviruses expressing a luciferase reporter gene were generated in an approach similar to as described previously Yang et al., 2004; Yu et al., 2021; Yu et al., 2020) . neutralization titers were defined as the sample dilution at which a 50% reduction in relative light unit (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 Yu et al., 2020) . 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-IRF4 (clone 3E4, PE-Cy7). After staining, cells were washed and fixed by 2% paraformaldehyde. All data were acquired on a BD FACSymphony flow cytometer. Subsequent analyses were performed using FlowJo software (BD Bioscience, v.9.9.6). For analyses, in singlet gate, dead cells were excluded by Aqua dye and CD45 was used as a 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 J o u r n a l P r e -p r o o f

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

ELISPOT assay. Precoated monoclonal antibody IL-4 ELISPOT plates

Fresh PBMCs were stained with Aqua live/dead dye for 20

with 2% FBS/DPBS buffer, and cells were suspended in 2% FBS/DPBS buffer with

Fc Block (BD) for 10 min, followed by staining with monoclonal antibodies against CD45 (clone D058-1283, BUV805), CD3 (clone SP34.2 , APC-Cy7), CD7 (clone M-T701, Alexa700)

CD123 (clone 6H6, Alexa700), CD11c (clone 3.9, Alexa700), CD20 (clone 2H7, PE-Cy5), IgA (goat polyclonal antibodies, APC), IgG (clone G18-145, BUV737), IgM (clone G20-127, BUV395), IgD (goat polyclonal antibodies, PE), CD80 (clone L307.4, BV786), CD95 (clone DX2, BV711), CD27 (clone M-T271, BUV563), CD21 (clone B-ly4, BV605), CD14 (clone M5E2, BV570), CD138 (clone DL-101, PE-CF594), and staining with SARS-CoV-2 antigens including biotinylated SARS-CoV-2 RBD proteins (Sino Biological) and full-length SARS

After staining, cells were washed twice with 2% FBS/DPBS buffer, followed by incubation with BV650 streptavidin (BD Pharmingen) for 10min

For intracellular staining, cells were permeabilized using Caltag Fix & Perm (ThermoFisher Scientific), then stained with monoclonal antibodies against Ki67 (clone B56, PerCP-cy5.5) and J o u r n a l P r e

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

2020) using SARS-CoV2 anti-sense specific probe v-nCoV2019-S (ACD Cat. No. 848561) targeting the positive-sense viral RNA and DapB (ACD Cat.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

322000) at 95-98°C, followed by treatment with protease III (ACD Cat. No. 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

Correlations were assessed by two-sided Spearman rank-correlation tests

P-values of less than 0.05 were considered significant. Graphical Abstract was generated using

Comparative seroprevalence and immunogenicity of six rare serotype recombinant adenovirus vaccine vectors from subgroups B and D

Ad26 vector-based COVID-19 vaccine encoding a prefusion-stabilized SARS-CoV-2 Spike immunogen induces potent humoral and cellular immune responses

SARS-CoV-2 infection protects against rechallenge in rhesus macaques

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

Development of an inactivated vaccine candidate for SARS-CoV-2

Circulating TFH cells, serological memory, and tissue compartmentalization shape human influenza-specific B cell immunity

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

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

Characterization of B and plasma cells in blood, bone marrow, and secondary lymphoid organs of rhesus macaques by multicolor flow cytometry

Acute depletion of activated memory B cells involves the PD-1 pathway in rapidly progressing SIV-infected macaques

Ad26 vaccine protects against SARS-CoV-2 severe clinical disease in hamsters

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

Development of an Inactivated Vaccine Candidate, BBIBP-CorV, with Potent Protection against SARS-CoV-2

Virological assessment of hospitalized patients with COVID-2019

Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation

A DNA vaccine induces SARS coronavirus neutralization and protective immunity in mice

Deletion of the SARS-CoV-2 Spike Cytoplasmic Tail Increases Infectivity in Pseudovirus Neutralization Assays

DNA vaccine protection against SARS-CoV-2 in rhesus macaques

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