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

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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 &gt;3.1 and &gt;3.7 log10 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.The COVID-19 pandemic has made the development of a safe, effective, and deployable vaccine a critical global priority (–). However, our understanding of immune correlates of protection to SARS-CoV-2 is currently very limited but is essential for the development of SARS-CoV-2 vaccines and other immunotherapeutic interventions. To facilitate the preclinical evaluation of vaccine candidates, we recently developed a rhesus macaque model of SARS-CoV-2 infection (). In the present study, we constructed a set of prototype DNA vaccines expressing various forms of the SARS-CoV-2 Spike (S) protein and assessed their immunogenicity and protective efficacy against SARS-CoV-2 viral challenge in rhesus macaques.We produced a series of prototype DNA vaccines expressing six variants of the SARS-CoV-2 S protein: 1) full-length (S), 2) deletion of the cytoplasmic tail (S.dCT) (), 3) deletion of the transmembrane domain and cytoplasmic tail reflecting the soluble ectodomain (S.dTM) (), 4) S1 domain with a foldon trimerization tag (S1), 5) receptor-binding domain with a foldon trimerization tag (RBD), and 6) a prefusion stabilized soluble ectodomain with deletion of the furin cleavage site, two proline mutations, and a foldon trimerization tag (S.dTM.PP) (–) (). Western blot analyses confirmed expression in cell lysates for all the constructs and in culture supernatants for the soluble S.dTM and S.dTM.PP constructs (). Proteolytic cleavage of the secreted protein was noted for S.dTM but not S.dTM.PP, presumably due to mutation of the furin cleavage site in S.dTM.PP.We immunized 35 adult rhesus macaques (6-12 years old) with DNA vaccines in the following groups: S (N = 4), S.dCT (N = 4), S.dTM (N = 4), S1 (N = 4), RBD (N = 4), S.dTM.PP (N = 5), and sham controls (N = 10). Animals received 5 mg DNA vaccines by the intramuscular route without adjuvant at week 0 and week 3. After the boost immunization at week 5, we observed S-specific binding antibodies by ELISA () and neutralizing antibodies (NAbs) using both a pseudovirus neutralization assay () () and a live virus neutralization assay (, ) (). 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 &lt; 0.0001, R = 0.8052, two-sided Spearman rank-correlation 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 ().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) () (). 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 ().We also observed cellular immune responses to pooled S peptides in the majority of vaccinated animals by IFN-γ ELISPOT assays at week 5 (). 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 (). S-specific IL-4+ CD4+ and CD8+ T cell responses were marginal (), 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 × 108 VP (1.1 × 104 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 () 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) log10 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 log10 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 (). High levels of sgmRNA were observed in the sham controls () 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) log10 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 (), including &gt;3.1 and &gt;3.7 log10 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) (). 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 (). 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 log10 sgmRNA in both BAL (P &lt; 0.0001, R = −0.6877, two-sided Spearman rank-correlation test) and NS (P = 0.0199, R = −0.4162) (). Similarly, the log10 live virus NAb titer at week 5 inversely correlated with peak log10 sgmRNA levels in both BAL (P &lt; 0.0001, R = −0.7702) and NS (P = 0.1006, R = −0.3360) (). 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 log10 sgmRNA levels in BAL (P = 0.0041, R = −0.4733) (fig. S6). Vaccine-elicited ELISPOT responses (fig. S7), CD4+ ICS responses (fig. S8), and CD8+ ICS responses (fig. S9) did not correlate with protection.We next explored the potential contribution of other antibody effector functions to immune correlates of protection. In addition to NAb titers, S- and RBD-specific ADCD responses inversely correlated with peak log10 sgmRNA levels in BAL (, 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 (, bottom left panel). Moreover, NAb titers correlated with most antibody effector functions, except for antibody-mediated NK cell activation (, 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, two-sided Mann-Whitney test), RBD-specific ADCD responses (P = 0.0001), S-specific RBD responses (P = 0.0010), and RBD-specific ADCP responses (P = 0.0005) compared with partially protected animals ().All animals exhibited anamnestic humoral and cellular immune responses following challenge, including increased ELISA titers (fig. S10), pseudovirus NAb titers (fig. S11), live virus NAb titers (fig. S12), and IFN-γ ELISPOT responses (fig. 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 (, ). 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 &gt;3.1 and &gt;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 (, –). Phase 1 clinical studies for SARS and MERS vaccine candidates have also been conducted (), 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 (). 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 (–). 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 demonstrate effective vaccine protection against SARS-CoV-2 in rhesus macaques and define NAb titers as an immune correlate of protection, which will accelerate the development of SARS-CoV-2 vaccines for humans.Materials and MethodsFigs. S1 to S13ReferencesThis is an open-access article distributed under the terms of the , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited., , , , , , , , , , , , , , , , , , , . Nature , – ()., , , , , , , , , , , , , , , , , , , , , , , , , , , , , . Nature , – ()., , , , , , , , , , , , , , , , , , , , , , , ; , . N. Engl. J. 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Funding: We acknowledge support from the Ragon Institute of MGH, MIT, and Harvard, Mark and Lisa Schwartz Foundation, Beth Israel Deaconess Medical Center, Massachusetts Consortium on Pathogen Readiness (MassCPR), Janssen Vaccines &amp; Prevention BV, and the National Institutes of Health (OD024917, AI129797, AI124377, AI128751, AI126603 to D.H.B.; AI007151 to D.R.M.; AI146779 to A.G.S.; AI121394, AI139538 to D.R.W.; 272201700036I-0-759301900131-1, AI100625, AI110700, AI132178, AI149644, AI108197 to R.S.B.). We also acknowledge a Burroughs Wellcome Fund Postdoctoral Enrichment Program Award to D.R.M. Author contributions: D.H.B. designed the study. J.Y., L.H.T., L.P., N.B.M., K.M., S.H.M., J.P.N., J.L., Z.L., A.C., E.A.B., G.D., M.S.G., X.H., C.J.D., M.K., N.K., Z.L., L.F.M., F.N., R.N., J.V., and H.W. performed the immunologic and virologic assays. D.R.M. and R.S.B. performed the live virus neutralization assays. C.L., C.A., S.F., J.S.B., M.D.S., and G.A. performed the systems serology. Y.C., A.Z., F.J.N.L., M.T., S.H., and D.R.W. provided the convalescent human specimens. L.P., A.V.R., K.B., R.B., A.C., B.F., A.D., E.T., J.D.V., H.A., and M.G.L. led the clinical care of the animals. R.Z. and F.W. participated in study design and interpretation of data. Y.C., B.C., and A.G.S. provided purified proteins. D.H.B., J.L., Z.L., and B.C. designed the immunogens. D.H.B. wrote the paper with all co-authors. Competing interests: The authors declare no competing financial interests. D.H.B. is a co-inventor on related vaccine patents. R.Z. and F.W. are employees of Janssen Vaccines &amp; Prevention BV. Data and materials availability: All data are available in the manuscript or the supplementary materials. Correspondence and requests for materials should be addressed to D.H.B. (dbarouch@bidmc.harvard.edu). This work is licensed under a Creative Commons Attribution 4.0 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 . 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.Vol 368, Issue 6495© 2020 . All rights reserved. AAAS is a partner of&nbsp;,&nbsp;, , ,&nbsp;,&nbsp;&nbsp;and&nbsp;.Science ISSN 1095-9203.

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...

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Science, also widely referred to as Science Magazine, is the peer-reviewed academic journal of the American Association for the Advancement of Science (AAAS) and one of the world's top academic journals. It was first published in 1880, is currently circulated weekly and has a subscriber base of around 130,000. Because institutional subscriptions and online access serve a larger audience, its estimated readership is 570,400 people.

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

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