key: cord-319337-w9zyshzb
authors: Yu, Jingyou; Tostanoski, Lisa H.; Peter, Lauren; Mercado, Noe B.; McMahan, Katherine; Mahrokhian, Shant H.; Nkolola, Joseph P.; Liu, Jinyan; Li, Zhenfeng; Chandrashekar, Abishek; Martinez, David R.; Loos, Carolin; Atyeo, Caroline; Fischinger, Stephanie; Burke, John S.; Slein, Matthew D.; Chen, Yuezhou; Zuiani, Adam; N. Lelis, Felipe J.; Travers, Meghan; Habibi, Shaghayegh; Pessaint, Laurent; Van Ry, Alex; Blade, Kelvin; Brown, Renita; Cook, Anthony; Finneyfrock, Brad; Dodson, Alan; Teow, Elyse; Velasco, Jason; Zahn, Roland; Wegmann, Frank; Bondzie, Esther A.; Dagotto, Gabriel; Gebre, Makda S.; He, Xuan; Jacob-Dolan, Catherine; Kirilova, Marinela; Kordana, Nicole; Lin, Zijin; Maxfield, Lori F.; Nampanya, Felix; Nityanandam, Ramya; Ventura, John D.; Wan, Huahua; Cai, Yongfei; Chen, Bing; Schmidt, Aaron G.; Wesemann, Duane R.; Baric, Ralph S.; Alter, Galit; Andersen, Hanne; Lewis, Mark G.; Barouch, Dan H.
title: DNA vaccine protection against SARS-CoV-2 in rhesus macaques
date: 2020-05-20
journal: Science
DOI: 10.1126/science.abc6284
sha: 
doc_id: 319337
cord_uid: w9zyshzb

The global COVID-19 pandemic caused by the SARS-CoV-2 virus has made the development of a vaccine a top biomedical priority. In this study, we developed a series of DNA vaccine candidates expressing different forms of the SARS-CoV-2 Spike (S) protein and evaluated them in 35 rhesus macaques. Vaccinated animals developed humoral and cellular immune responses, including neutralizing antibody titers comparable to those found in convalescent humans and macaques infected with SARS-CoV-2. Following vaccination, all animals were challenged with SARS-CoV-2, and the vaccine encoding the full-length S protein resulted in >3.1 and >3.7 log(10) reductions in median viral loads in bronchoalveolar lavage and nasal mucosa, respectively, as compared with sham controls. Vaccine-elicited neutralizing antibody titers correlated with protective efficacy, suggesting an immune correlate of protection. These data demonstrate vaccine protection against SARS-CoV-2 in nonhuman primates.

(Page numbers not final at time of first release) 2 and neutralizing antibodies (NAbs) using both a pseudovirus neutralization assay (10) (Fig. 2B ) and a live virus neutralization assay (14, 15) (Fig. 2C) . Two animals had binding antibodies at baseline by ELISA, which we speculate might reflect cross-reactivity of other natural primate coronaviruses. NAb titers measured by the pseudovirus neutralization assay correlated with NAb titers measured by the live virus neutralization assay (P < 0.0001, R = 0.8052, two-sided Spearman rankcorrelation test; fig. S1 ). Moreover, NAb titers in the vaccinated macaques (median titer 74; median titer in the S and S.dCT groups 170) were comparable in magnitude to NAb titers in a cohort of 9 convalescent macaques (median titer 106) and a cohort of 27 convalescent humans (median titer 93) who had recovered from SARS-CoV-2 infection (Fig. 2D ).

S-specific and RBD-specific antibodies in the vaccinated macaques included diverse subclasses and effector functions, including antibody-dependent neutrophil phagocytosis (ADNP), antibody-dependent complement deposition (ADCD), antibody-dependent monocyte cellular phagocytosis (ADCP), and antibody-dependent NK cell activation (IFN-γ secretion, CD107a degranulation, and MIP-1β expression) (16) (Fig. 2E) . A trend toward higher ADCD responses was observed in the S and S.dCT groups, whereas higher NK cell activation was observed in the RBD and S.dTM.PP groups. A principal component analysis of the functional and biophysical antibody features showed overlap of the different vaccine groups, with more distinct profiles in the S and RBD groups (Fig. 2E) .

We also observed cellular immune responses to pooled S peptides in the majority of vaccinated animals by IFN-γ ELISPOT assays at week 5 (Fig. 3A) . Intracellular cytokine staining assays at week 5 demonstrated induction of S-specific IFN-γ+ CD4+ and CD8+ T cell responses, with lower responses induced by the shorter S1 and RBD immunogens (Fig. 3B ). S-specific IL-4+ CD4+ and CD8+ T cell responses were marginal (Fig. 3C ), suggesting induction of Th1-biased cellular immune responses.

At week 6, which was 3 weeks after the boost immunization, all animals were challenged with 1.2 × 10 8 VP (1.1 × 10 4 PFU) SARS-CoV-2, administered as 1 ml by the intranasal (IN) route and 1 ml by the intratracheal (IT) route. Following challenge, we assessed viral RNA levels by RT-PCR (17) in bronchoalveolar lavage (BAL) and nasal swabs (NS). Viral RNA was negative in plasma, and animals exhibited only mild clinical symptoms. High levels of viral RNA were observed in the sham controls with a median peak of 6.46 (range 4.81-7.99) log10 RNA copies/ml in BAL and a median peak of 6.82 (range 5.96-7.96) log 10 RNA copies/swab in NS ( fig. S2 ). Lower levels of viral RNA were observed in the vaccine groups (figs. S3 and S4), including 1.92 and 2.16 log 10 reductions of median peak viral RNA in BAL and NS, respectively, in S vaccinated animals compared with sham controls (P = 0.02 and P = 0.04, two-sided Mann-Whitney tests) ( fig. S5 ). Viral RNA assays were confirmed by PFU assays, which similarly showed lower infectious virus titers in S vaccinated animals compared with sham controls (P = 0.04, two-sided Mann-Whitney test) ( fig. S5) .

We speculated that a substantial fraction of viral RNA in BAL and NS following challenge represented input challenge virus, and thus we also assessed levels of subgenomic mRNA (sgmRNA), which is believed to reflect viral replication cellular intermediates that are not packaged into virions and thus putative replicating virus in cells (18) . High levels of sgmRNA were observed in the sham controls (Fig. 4A ) with a median peak of 5.35 (range 3.97-6.95) log10 sgmRNA copies/ml in BAL and a median peak of 6.40 (range 4.91-7.01) log 10 sgmRNA copies/swab in NS. Peak viral loads occurred variably on day 1-4 following challenge. Markedly lower levels of sgmRNA were observed in the vaccine groups (Fig. 4 , B and C), including >3.1 and >3.7 log 10 decreases of median peak sgmRNA in BAL and NS, respectively, in S vaccinated animals compared with sham controls (P = 0.03 and P = 0.01, two-sided Mann-Whitney tests) (Fig. 4D ). Reduced levels of sgmRNA were also observed in other vaccine groups, including S.dCT, S1, RBD, and S.dTM.PP, although minimal to no protection was seen in the S.dTM group, confirming the importance of prefusion ectodomain stabilization, as reported previously (13) . Protection was generally more robust in BAL compared with NS, particularly for the less immunogenic constructs. A total of 8 of 25 vaccinated animals exhibited no detectable sgmRNA in BAL and NS at any timepoint following challenge.

The variability in protective efficacy in this study facilitated an analysis of immune correlates of protection. The log10 pseudovirus NAb titer at week 5 inversely correlated with peak log 10 sgmRNA in both BAL (P < 0.0001, R = −0.6877, two-sided Spearman rank-correlation test) and NS (P = 0.0199, R = −0.4162) (Fig. 5A) . Similarly, the log 10 live virus NAb titer at week 5 inversely correlated with peak log 10 sgmRNA levels in both BAL (P < 0.0001, R = −0.7702) and NS (P = 0.1006, R = −0.3360) (Fig. 5B) . These data suggest that vaccine-elicited serum NAb titers may be immune correlates of protection against SARS-CoV-2 challenge. We speculate that correlations were more robust with viral loads in BAL compared with viral loads in NS due to intrinsic variability of collecting swabs. The log10 ELISA titer at week 5 also inversely correlated with peak log 10 In addition to NAb titers, S-and RBD-specific ADCD responses inversely correlated with peak log 10 sgmRNA levels in BAL (Fig. 5C, top panel) . Two orthogonal unbiased machine learning approaches were then utilized to define minimal combined correlates of protection. A nonlinear random forest regression analysis and a linear partial least squares regression analysis showed that utilizing two features improved the correlations with protection, such as RBD-specific FcγR2a-1 binding with ADCD responses, or NAb titers with RBD-specific IgG2 responses (Fig. 5C, bottom left panel) . Moreover, NAb titers correlated with most antibody effector functions, except for antibody-mediated NK cell activation (Fig. 5C, bottom right panel) . Taken together, these data suggest a primary role of NAbs in protecting against SARS-CoV-2, supported by certain innate immune effector functions such as ADCD. Finally, we compared antibody parameters in the vaccinated animals that were completely protected (defined as no detectable sgmRNA following challenge) with the vaccinated animals that were partially protected (defined as detectable sgmRNA following challenge). Completely protected animals showed higher log10 NAb titers (P = 0.0004, twosided Mann-Whitney test), RBD-specific ADCD responses (P = 0.0001), S-specific RBD responses (P = 0.0010), and RBDspecific ADCP responses (P = 0.0005) compared with partially protected animals (Fig. 5D ). S13 ) on day 14 after challenge. These data suggest that vaccine protection was probably not sterilizing, including in the 8 of 25 animals that had no detectable sgmRNA in BAL and NS at any timepoint following challenge, but rather was likely mediated by rapid virologic control following challenge.

A safe and effective SARS-CoV-2 vaccine may be required to end the global COVID-19 pandemic. Several vaccine candidates have initiated clinical testing, and many others are in preclinical development (19, 20) . However, very little is currently known about immune correlates of protection and protective efficacy of candidate SARS-CoV-2 vaccines in animal models. In this study, we generated a series of prototype DNA vaccines expressing various S immunogens and assessed protective efficacy against intranasal and intratracheal SARS-CoV-2 challenge in rhesus macaques. We demonstrate vaccine protection with substantial >3.1 and >3.7 log10 reductions in median viral loads in BAL and NS, respectively, in S immunized animals compared with sham controls. Protection was likely not sterilizing but instead appeared to be mediated by rapid immunologic control following challenge.

Our data extend previous studies on SARS and MERS vaccine protection in mice, ferrets, and macaques (10, (21) (22) (23) (24) . Phase 1 clinical studies for SARS and MERS vaccine candidates have also been conducted (25) , but these vaccines have not been tested for efficacy in humans. Our data suggest that vaccine protection against SARS-CoV-2 in macaques is feasible. We observed a dramatic reduction of viral replication in both the upper respiratory tract and the lower respiratory tract with the optimal vaccines. In contrast, the less immunogenic vaccines, such as S.dTM, showed partial protection in BAL but essentially no protection in NS. These data suggest that it may be easier to protect against lower respiratory tract disease compared with upper respiratory tract disease. In the present study, optimal protection was achieved with the full-length S immunogen in both the upper and lower respiratory tracts, and reduced protection was observed with soluble constructs and smaller fragments. Our study did not address the question of whether emerging mutations in the SARS-CoV-2 S sequence may mediate escape from NAb responses induced by immunogens designed from the Wuhan/WIV04/2019 sequence.

Further research will need to address the important questions of the durability of protective immunity and the optimal vaccine platforms for a SARS-CoV-2 vaccine for humans (26) . Although our data are restricted to DNA vaccines, we believe that our findings should be generalizable to other gene-based vaccines as well, including RNA vaccines and recombinant vector-based vaccines. Additional research should also evaluate vaccine immunogenicity and protective efficacy in older animals. Further studies will also need to address the question of enhanced respiratory disease, which may result from antibody-dependent enhancement (27) (28) (29) . Although our study was not designed to address safety issues, it is worth noting that the DNA vaccines induced Th1 rather than Th2 responses, and we did not observe enhanced clinical disease even with the suboptimal vaccine constructs that failed to protect.

We identified serum NAb titers, as measured by two independent assays (pseudovirus neutralization and live virus neutralization), as a significant correlate of protection in this study against both lower respiratory tract disease as well as upper respiratory tract disease. It is likely that protection in both anatomic compartments will be necessary for pandemic control, although protection in the upper respiratory tract may be more difficult to achieve. If this NAb correlate proves generalizable across multiple vaccine studies in both NHPs and humans, then this parameter would be a simple and useful benchmark for clinical development of SARS-CoV-2 vaccines. Innate immune effector functions such as ADCD may also contribute to protective efficacy. In summary, we Correlations of (A) pseudovirus NAb titers and (B) live NAb titers prior to challenge with log peak sgmRNA copies/ml in BAL or log peak sgmRNA copies/swab in nasal swabs following challenge. Red lines reflect the best-fit relationship between these variables. P and R values reflect two-sided Spearman rank-correlation tests. (C) The heat map (top panel) shows the Spearman and Pearson correlations between antibody features and log 10 peak sgmRNA copies/ml in BAL (*q < 0.05, **q < 0.01, ***q < 0.001 with Benjamini-Hochberg correction for multiple testing). The bar graph (bottom left panel) shows the rank of the Pearson correlation between cross-validated model predictions and data using the most predictive combination or individual antibody features for partial least square regression (PLSR) and random forest regression (RFR). The correlation heatmap (bottom right panel) represents pairwise Pearson correlations between features across all animals. (D) The heat map (top panel) shows the difference in the means of the z-scored features between the completely protected and partially protected animals (**q < 0.01 with Benjamini-Hochberg correction for multiple testing). The dot plots show differences in log 10 NAb titers, RBD-specific ADCD responses, S-specific ADCD responses, and RBD-specific ADCP responses between the completely protected and partially protected animals. Pvalues indicate two-sided Mann-Whitney tests.

A new coronavirus associated with human respiratory disease in China

A pneumonia outbreak associated with a new coronavirus of probable bat origin

Washington State 2019-nCoV Case Investigation Team, First Case of 2019 Novel Coronavirus in the United States

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

Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: A descriptive study

Clinical features of patients infected with 2019 novel coronavirus in Wuhan

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

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

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

Pre-fusion structure of a human coronavirus spike protein

Immunogenicity and structures of a rationally designed prefusion MERS-CoV spike antigen

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

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

Lack of therapeutic efficacy of an antibody to α4β7 in SIVmac251-infected rhesus macaques

Virological assessment of hospitalized patients with COVID-2019

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

A strategic approach to COVID-19 vaccine R&D

A recombinant VSV-vectored MERS-CoV vaccine induces neutralizing antibody and T cell responses in rhesus monkeys after single dose immunization

A synthetic consensus anti-spike protein DNA vaccine induces protective immunity against Middle East respiratory syndrome coronavirus in nonhuman primates

Adenovirus-based vaccine prevents pneumonia in ferrets challenged with the SARS coronavirus and stimulates robust immune responses in macaques

Immunogenicity, safety, and protective efficacy of an inactivated SARS-associated coronavirus vaccine in rhesus monkeys

VRC 301 Study Team, A SARS DNA vaccine induces neutralizing antibody and cellular immune responses in healthy adults in a Phase I clinical trial

Durability and correlates of vaccine protection against Zika virus in rhesus monkeys

Immunization with SARS coronavirus vaccines leads to pulmonary immunopathology on challenge with the SARS virus

Anti-spike IgG causes severe acute lung injury by skewing macrophage responses during acute SARS-CoV infection

We also acknowledge a Burroughs Wellcome Fund Postdoctoral Enrichment Program Award to D.R.M. Author contributions: D.H.B. designed the study

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. SUPPLEMENTARY MATERIALS science.sciencemag.org/cgi/content/full/science.abc6284/DC1 Materials and Methods Figs. S1 to S13 References