key: cord-0757940-car394ou
authors: Chandrashekar, Abishek; Liu, Jinyan; Martinot, Amanda J.; McMahan, Katherine; Mercado, Noe B.; Peter, Lauren; Tostanoski, Lisa H.; Yu, Jingyou; Maliga, Zoltan; Nekorchuk, Michael; Busman-Sahay, Kathleen; Terry, Margaret; Wrijil, Linda M.; Ducat, Sarah; Martinez, David R.; Atyeo, Caroline; Fischinger, Stephanie; Burke, John S.; Slein, Matthew D.; Pessaint, Laurent; Van Ry, Alex; Greenhouse, Jack; Taylor, Tammy; Blade, Kelvin; Cook, Anthony; Finneyfrock, Brad; Brown, Renita; Teow, Elyse; Velasco, Jason; Zahn, Roland; Wegmann, Frank; Abbink, Peter; Bondzie, Esther A.; Dagotto, Gabriel; Gebre, Makda S.; He, Xuan; Jacob-Dolan, Catherine; Kordana, Nicole; Li, Zhenfeng; Lifton, Michelle A.; Mahrokhian, Shant H.; Maxfield, Lori F.; Nityanandam, Ramya; Nkolola, Joseph P.; Schmidt, Aaron G.; Miller, Andrew D.; Baric, Ralph S.; Alter, Galit; Sorger, Peter K.; Estes, Jacob D.; Andersen, Hanne; Lewis, Mark G.; Barouch, Dan H.
title: SARS-CoV-2 infection protects against rechallenge in rhesus macaques
date: 2020-05-20
journal: Science
DOI: 10.1126/science.abc4776
sha: 8cc1f70e6072a0b3da37792dd66a441c7faaeb45
doc_id: 757940
cord_uid: car394ou

An understanding of protective immunity to SARS-CoV-2 is critical for vaccine and public health strategies aimed at ending the global COVID-19 pandemic. A key unanswered question is whether infection with SARS-CoV-2 results in protective immunity against re-exposure. We developed a rhesus macaque model of SARS-CoV-2 infection and observed that macaques had high viral loads in the upper and lower respiratory tract, humoral and cellular immune responses, and pathologic evidence of viral pneumonia. Following initial viral clearance, animals were rechallenged with SARS-CoV-2 and showed 5 log(10) reductions in median viral loads in bronchoalveolar lavage and nasal mucosa compared with primary infection. Anamnestic immune responses following rechallenge suggested that protection was mediated by immunologic control. These data show that SARS-CoV-2 infection induced protective immunity against re-exposure in nonhuman primates.

*These authors contributed equally to this work. †Corresponding author. Email: dbarouch@bidmc.harvard.edu An understanding of protective immunity to SARS-CoV-2 is critical for vaccine and public health strategies aimed at ending the global COVID-19 pandemic. A key unanswered question is whether infection with SARS-CoV-2 results in protective immunity against re-exposure. We developed a rhesus macaque model of SARS-CoV-2 infection and observed that macaques had high viral loads in the upper and lower respiratory tract, humoral and cellular immune responses, and pathologic evidence of viral pneumonia. Following initial viral clearance, animals were rechallenged with SARS-CoV-2 and showed 5 log 10 reductions in median viral loads in bronchoalveolar lavage and nasal mucosa compared with primary infection. Anamnestic immune responses following rechallenge suggested that protection was mediated by immunologic control. These data show that SARS-CoV-2 infection induced protective immunity against reexposure in nonhuman primates. . S3 ), but fever, weight loss, respiratory distress, and mortality were not observed.

To help differentiate input challenge virus from newly replicating virus, we developed an RT-PCR assay to assess E gene subgenomic mRNA (sgmRNA). E gene sgmRNA reflects viral replication cellular intermediates that are not packaged into virions and thus represent putative replicating virus in cells (9) . Compared with total viral RNA ( Fig.  1B) , sgmRNA levels were lower in NS on day 1 with a median of 5.11 (range <1.70-5.94) log10 sgmRNA copies/swab, but then increased by day 2 to a median of 6.50 (range 4.16-7.81) log 10 sgmRNA copies/swab (Fig. 1C) .

We next evaluated SARS-CoV-2-specific humoral and cellular immune responses in these animals. All 9 macaques developed binding antibody responses to the SARS-CoV-2 Spike (S) protein by ELISA ( Fig. 2A) and neutralizing antibody (NAb) responses using both a pseudovirus neutralization assay (10) (Fig. 2B ) and a live virus neutralization assay (11, 12) (Fig. 2C ). NAb titers of approximately 100 were observed in all animals on day 35 regardless of dose group (range 83-197 by the pseudovirus neutralization assay and 35-326 by the live virus neutralization assay). Antibody responses of multiple subclasses were observed against the receptor binding domain (RBD), the prefusion S ectodomain (S), and the nucleocapsid (N), and antibodies exhibited diverse effector functions, including antibody-dependent complement deposition (ADCD), antibody-dependent cellular phagocytosis (ADCP), antibody-dependent neutrophil phagocytosis (ADNP), and antibody-dependent NK cell degranulation (NK CD107a) and cytokine secretion (NK MIP1β, NK IFNγ) (13) (Fig. 2D ). Cellular immune responses to pooled S peptides were observed in the majority of animals by IFN-γ ELISPOT assays on day 35, with a trend toward lower responses in the lower dose groups (Fig. 2E) . Intracellular cytokine staining assays demonstrated induction of both S-specific CD8+ and CD4+ T cell responses (Fig.  2F ).

Only limited pathology data from SARS-CoV-2 infected humans are currently available. To assess the pathologic char-acteristics of SARS-CoV-2 infection in rhesus macaques, we inoculated 4 animals with 1.1 × 10 5 PFU virus by the IN and IT routes as above and necropsied them on day 2 (N = 2) and day 4 (N = 2) following challenge. Multiple regions of the upper respiratory tract, lower respiratory tract, gastrointestinal tract, lymph nodes, and other organs were harvested for virologic and pathologic analyses. High levels of viral RNA were observed in all nasal mucosa, pharynx, trachea, and lung tissues, and lower levels of virus were found in the gastrointestinal tract, liver, and kidney ( fig. S4 ). Viral RNA was readily detected in paratracheal lymph nodes but was only sporadically found in distal lymph nodes and spleen ( fig. S4 ).

Upper airway mucosae, trachea, and lungs were paraformaldehyde fixed, paraffin embeded, and evaluated by histopathology. On day 2 following challenge, both necropsied animals demonstrated multifocal regions of inflammation and evidence of viral pneumonia, including expansion of alveolar septae with mononuclear cell infiltrates, consolidation, and edema (Fig. 3, A and B) . Regions with edema also contained numerous polymorphonuclear cells, predominantly neutrophils. Terminal bronchiolar epithelium was necrotic and sloughed with clumps of epithelial cells detected within airways and distally within alveolar spaces (Fig. 3 , C and D) with formation of occasional bronchiolar epithelial syncytial cells (Fig. 3E ). Hyaline membranes were occasionally observed within alveolar septa, consistent with damage to type I and type II pneumocytes (Fig. 3F ). Diffusely reactive alveolar macrophages filled alveoli, and some were multinucleated and labeled positive for nucleocapsid by immunohistochemistry (Fig. 3G ). Alveolar lining cells (pneumocytes) also prominently labeled positive for nucleocapsid (Fig. 3H ).

Multifocal clusters of virus infected cells were present throughout the lung parenchyma, as detected by immunohistochemistry and in situ RNA hybridization (RNAscope) (14, 15) To further characterize infected tissues, we performed cyclic immunofluorescence (CyCIF) imaging, a method for multiplex immunophenotyping of paraformaldehyde fixed tissue specimens (16) . Tissues were stained for nucleocapsid (SARS-N), pan-cytokeratin (to identify epithelial cells), Iba-1 (ionized calcium binding adaptor as a pan-macrophage marker), CD68 (monocyte/macrophage marker), and CD206 (macrophage marker), in addition to a panel of markers to identify other immune cells and anatomical structures (table S1), and counterstaining for DNA to label all nuclei. Foci of virus infected cells were randomly dispersed throughout the lung and were variably associated with inflammatory infiltrates (Fig. 4 , A to D). Some areas of parenchymal consolidation and inflammation contained little to no virus ( Fig. 4A, arrows, and fig. S8 ). Virus infected cells frequently co-stained with pan-cytokeratin (Fig. 4 , E to H), suggesting that they were alveolar epithelial cells (pneumocytes). Uninfected Iba-1+ CD68+ CD206+ activated macrophages were also frequently detected adjacent to virally infected epithelial cells (Fig. 4 , E and I to K). These data demonstrate that SARS-CoV-2 induced multifocal areas of acute inflammation and viral pneumonia involving infected pneumocytes, ciliated bronchial epithelial cells, and likely other cell types.

On day 35 following initial viral infection (Figs. 1 and 2), we rechallenged all 9 rhesus macaques with the same doses of SARS-CoV-2 that were utilized for the primary infection, namely 1.1 × 10 6 PFU (Group 1; N = 3), 1.1 × 10 5 PFU (Group 2; N = 3), or 1.1 × 10 4 PFU (Group 3; N = 3). We included 3 naïve animals as positive controls in the rechallenge experiment. Very limited viral RNA was observed in BAL on day 1 following rechallenge in two Group 1 animals and in one Group 2 animal, with no viral RNA detected at subsequent timepoints (Fig. 5A ). In contrast, high levels of viral RNA were observed in the concurrently challenged naïve animals (Fig. 5A) , as expected. Median peak viral loads in BAL were >5.1 log10 lower following rechallenge as compared with the primary challenge (P < 0.0001, two-sided Mann-Whitney test; Fig. 5B ). Viral RNA following rechallenge was higher in NS compared with BAL, but exhibited dose dependence and rapid decline (Fig. 5C) , and median peak viral loads in NS were still >1.7 log 10 lower following rechallenge as compared with the primary challenge (P = 0.0011, two-sided Mann-Whitney test; Fig. 5D ).

We speculated that the majority of virus detected in NS following rechallenge was input challenge virus, and we therefore assessed sgmRNA levels in NS following rechallenge. Low but detectable levels of sgmRNA were still observed in 4 of 9 animals in NS on day 1 following rechallenge, but sgmRNA levels declined quickly (Fig. 5E) , and median peak sgmRNA levels in NS were >4.8 log10 lower following rechallenge as compared with the primary challenge (P = 0.0003, two-sided Mann-Whitney test; Fig. 5F ). Consistent with these data, plaque assays in BAL and NS samples following rechallenge showed no recoverable virus and were lower than following primary infection (P = 0.009 and 0.002, respectively, two-sided Mann-Whitney tests; fig.  S9 ). Moreover, little or no clinical disease was observed in the animals following rechallenge (fig. S10) .

Following SARS-CoV-2 rechallenge, animals exhibited rapid anamnestic immune responses, including increased virus-specific ELISA titers (P = 0.0034, two-sided Mann-Whitney test), pseudovirus NAb titers (P = 0.0003), and live virus NAb titers (P = 0.0003) as well as a trend toward increased IFN-γ ELISPOT responses (P = 0.1837) by day 7 after rechallenge ( Fig. 6 ). In particular, NAb titers were markedly higher on day 14 following rechallenge compared with day 14 following primary challenge (P < 0.0001, two-sided Mann-Whitney test) (fig. S11). All animals developed anamnestic antibody responses following rechallenge, regardless of the presence or absence of residual viral RNA or sgmRNA in BAL or NS, and thus we speculate that the protective efficacy against rechallenge was mediated by rapid immunologic control.

Individuals who recover from certain viral infections typically develop virus-specific antibody responses that provide robust protective immunity against re-exposure, but some viruses do not generate protective natural immunity, such as HIV-1 (17) . Human challenge studies for the common cold coronavirus 229E have suggested that there may be partial natural immunity (18) . However, there is currently no data whether humans who have recovered from SARS-CoV-2 infection are protected from re-exposure (World Health Organization, Scientific Brief, April 24, 2020; https:// www.who.int/news-room/commentaries/detail/immunitypassports-in-the-context-of-covid-19). This is a critical issue with profound implications for vaccine development, public health strategies, antibody-based therapeutics, and epidemiologic modeling of herd immunity. In this study, we demonstrate that SARS-CoV-2 infection in rhesus macaques provided protective efficacy against SARS-CoV-2 rechallenge.

We developed a rhesus macaque model of SARS-CoV-2 infection that recapitulates many aspects of human SARS-CoV-2 infection, including high levels of viral replication in the upper and lower respiratory tract (Fig. 1) (19) . However, neither nonhuman primate model led to respiratory failure or mortality, and thus further research will be required to develop a nonhuman primate model of severe COVID-19 disease.

SARS-CoV-2 infection in rhesus macaques led to humoral and cellular immune responses (Fig. 2) and provided protection against rechallenge (Fig. 5) . Residual low levels of subgenomic mRNA in nasal swabs in a subset of animals (Fig. 5 ) and anamnestic immune responses in all animals (Fig. 6) following SARS-CoV-2 rechallenge suggest that protection was mediated by immunologic control and likely was not sterilizing.

Given the near-complete protection in all animals following SARS-CoV-2 rechallenge, we were unable to determine immune correlates of protection in this study. SARS-CoV-2 infection in rhesus monkeys resulted in the induction of neutralizing antibody titers of approximately 100 by both a pseudovirus neutralization assay and a live virus neutralization assay, but the relative importance of neutralizing antibodies, other functional antibodies, cellular immunity, and innate immunity to protective efficacy against SARS-CoV-2 remains to be determined. Moreover, additional research will be required to define the durability of natural immunity.

In summary, SARS-CoV-2 infection in rhesus macaques induced humoral and cellular immune responses and provided protective efficacy against SARS-CoV-2 rechallenge. These data raise the possibility that immunologic approaches to the prevention and treatment of SARS-CoV-2 infection may in fact be possible. However, it is critical to emphasize that there are important differences between SARS-CoV-2 infection in macaques and humans, with many parameters still yet to be defined in both species, and thus our data should be interpreted cautiously. Rigorous clinical studies will be required to determine whether SARS-CoV-2 infection effectively protects against SARS-CoV-2 re-exposure in humans. International (CC BY 4.0) license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. To view a copy of this license, visit https://creativecommons.org/licenses/by/4.0/. This license does not apply to figures/photos/artwork or other content included in the article that is credited to a third party; obtain authorization from the rights holder before using such material.

science.sciencemag.org/cgi/content/full/science.abc4776/DC1 Materials and Methods Table S1 Figs. S1 to S11 

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China Novel Coronavirus Investigating and Research Team, A Novel Coronavirus from Patients with Pneumonia in China

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Clinical features of patients infected with 2019 novel coronavirus in Wuhan

A familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: A study of a family cluster

Virological assessment of hospitalized patients with COVID-2019

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

Reverse genetics with a full-length infectious cDNA of the Middle East respiratory syndrome coronavirus

Reverse genetics with a full-length infectious cDNA of severe acute respiratory syndrome coronavirus

Dissecting Polyclonal Vaccine-Induced Humoral Immunity against HIV Using Systems Serology

Impact of early cART in the gut during acute HIV infection

Defining HIV and SIV Reservoirs in Lymphoid Tissues

Highly multiplexed immunofluorescence imaging of human tissues and tumors using t-CyCIF and conventional optical microscopes

HIV-1 superinfection despite broad CD8 + T-cell responses containing replication of the primary virus

The time course of the immune response to experimental coronavirus infection of man

Cwiak for generous advice, assistance, and reagents. Funding: We acknowledge support from the Ragon Institute of MGH, MIT, and Harvard, Mark and Lisa Schwartz Foundation

led the clinical care of the animals and performed the virologic assays. R.Z. and F.W. participated in study design and interpretation of data

is an inventor on patent application WO 2017/184733 A1 submitted by Massachusetts General Hospital that covers systems serology