key: cord-0918537-uvhzw6c9
authors: Dangi, Tanushree; Class, Jacob; Palacio, Nicole; Richner, Justin M.; Penaloza-MacMaster, Pablo
title: Combining spike- and nucleocapsid-based vaccines improves distal control of SARS-CoV-2
date: 2021-08-17
journal: Cell Rep
DOI: 10.1016/j.celrep.2021.109664
sha: 02d6230cb73650e5fe86e9d1aeaef5554a0d64e0
doc_id: 918537
cord_uid: uvhzw6c9

SARS-CoV-2 infection causes respiratory insufficiency and neurological manifestations, including loss of smell and psychiatric disorders, and can be fatal. Most vaccines are based on the spike antigen alone, and although they have shown efficacy at preventing severe disease and death, they do not always confer sterilizing immunity. Here, we interrogate whether SARS-CoV-2 vaccines could be improved by incorporating nucleocapsid as an antigen. We show that after 72 hr of challenge, a spike-based vaccine confers acute protection in lung, but not in brain. However, combining a spike-based vaccine with a nucleocapsid-based vaccine confers acute protection in both lung and brain. These findings suggest that nucleocapsid-specific immunity can improve the distal control of SARS-CoV-2, suggesting the inclusion of nucleocapsid in next-generation COVID-19 vaccines.

Most SARS-CoV-2 vaccines used in humans are based on the spike antigen. These vaccines have shown high efficacy against severe disease and death, but they do not always confer sterilizing immunity (Baden et al., 2021; Logunov et al., 2021; Polack et al., 2020; Sadoff et al., 2021; Voysey et al., 2021; Zhu et al., 2020) . In particular, breakthrough infections can be detected in nasopharyngeal swabs of vaccinated individuals (Keehner et al., 2021; Levine-Tiefenbrun et al., 2021; Tande et al., 2021) .

Nasopharyngeal swabs typically contain viruses derived from the proximal site of challenge (the respiratory system), which may not reflect ongoing virus replication at distal sites of the body. It is unknown whether SARS-CoV-2 vaccines can prevent acute viral dissemination to distal sites of the body, such as the central nervous system, which is considered an immune-privileged site that is relatively "impermeable" to circulating antibodies (Forrester et al., 2018) . The central nervous system has become increasingly important in the management of COVID-19 disease, especially because even mild or asymptomatic infections can trigger neurological manifestations, including "brain fog" and psychiatric conditions (Nalbandian et al., 2021) . SARS-CoV-2 is a respiratory virus, but prior reports suggest that it may also disseminate to the brain via the olfactory mucosa (Cantuti-Castelvetri et al., 2020; Meinhardt et al., 2021a; Song et al., 2021) . Post-mortem analyses of COVID-19 patients have shown the presence of SARS-CoV-2 in the brain (Gasmi et al., 2021; Kumari et al., 2021; Meinhardt et al., 2021b; Song et al., 2021) . Knowing whether J o u r n a l P r e -p r o o f vaccines can block viral dissemination to the central nervous system is important, because this would help elucidate if vaccinated people who get exposed to the virus could still develop neurological complications. Due to ethical reasons it is not feasible to sample the brain of vaccinated individuals to assess breakthrough infections in this distal site.

The SARS-CoV-2 spike protein is a critical antigen in coronavirus vaccines. This antigen alone is the basis for the Pfizer/BioNTek vaccine, Moderna vaccine, Johnson & Johnson's (J&J) Janssen vaccine, AstraZeneca vaccine, CanSino vaccine, Sputnik V vaccine, Novavax vaccine, among others. Vaccines that contain the spike antigen generate robust neutralizing antibodies that prevent the initial entry of SARS-CoV-2 into the respiratory system, but it is unclear whether other viral antigens could provide equally important immune protection. It is also unknown whether inclusion of multiple viral antigens could provide a synergistic improvement in vaccine-elicited protection. In this study, we compared the efficacy of spike-based versus nucleocapsid-based vaccines after an intranasal SARS-CoV-2 challenge in K18-hACE2 mice, which are highly susceptible to SARS-CoV-2 infection. Surprisingly, we show that a spike-based vaccine does not provide acute protection to the central nervous system; protection against distal viral dissemination to the nervous system was only observed when a spike-based vaccine was co-administered with a nucleocapsid-based vaccine. These findings demonstrate a synergy between spike-specific and nucleocapsid-specific immune responses, and provide a framework for the rational design of next-generation coronavirus vaccines.

K18-hACE2 mice constitute a robust pre-clinical model that recapitulates salient features of SARS-CoV-2 infection in humans, including respiratory insufficiency, a dysregulated inflammatory response, neurological complications and death. Therefore, this mouse model has been useful for investigating antiviral therapies, vaccines, and COVID-19 pathogenesis (Bao et al., 2020; Oladunni et al., 2020; Rosenfeld et al., 2021; Winkler et al., 2020; Zheng et al., 2021) . We first vaccinated K18-hACE2 mice intramuscularly with an adenovirus vector expressing: SARS-CoV-2 spike (Ad5-S) or nucleocapsid (Ad5-N), or both (Ad5-S + Ad5-N), at a dose of 10 9 PFU per vector per mouse ( Figure 1A ).

After three weeks, we evaluated SARS-CoV-2-specific CD8 T cell responses in peripheral blood mononuclear cells (PBMCs) and tissues by tetramer binding assays (K b VL8 to detect spike-specific CD8 T cells; D b N219 to detect nucleocapsid-specific CD8 T cells). Ad5-S and Ad5-N vaccination elicited detectable CD8 T cell responses in PBMCs and tissues ( Figure 1B-1F) . We also confirmed the immunogenicity of the Ad5-S and Ad5-N vaccines by intracellular cytokine staining (ICS) assays, which showed IFN-expressing CD8 T cells (Figure 2A-2C) , and IFN-expressing CD4 T cells ( Figure   2D -2E). Similar to prior studies with other Ad5-vectored vaccines Penaloza-MacMaster et al., 2013; Provine et al., 2016) , we observed modest IFN expression on virus-specific CD8 and CD4 T cells following Ad5 vaccination. The Ad5-S J o u r n a l P r e -p r o o f and Ad5-N vaccines also elicited their respective antibody responses by enzyme-linked immunosorbent assay (ELISA) (Figure 3) . Altogether, these results show that the spikebased and nucleocapsid-based vaccines elicit CD8 T cells, CD4 T cells, and antibody responses.

A spike-based vaccine confers proximal protection in the lung, but not distal protection in the brain.

We performed intranasal challenges with 5x10 4 PFU of SARS-CoV-2 (isolate USA-WA1/2020), followed by euthanasia 3 days post-challenge to evaluate acute viral loads in tissues. We also challenged unvaccinated K18-hACE2 mice as controls. First, we compared viral loads in the lung, which represents a proximal site of challenge.

Intranasal challenge of unvaccinated K18-hACE2 mice with SARS-CoV-2 normally results in high viral replication in lung, followed by distal dissemination to the brain by day 2 post-challenge (Winkler et al., 2020) . Consistent with prior studies (Oladunni et al., 2020; Rosenfeld et al., 2021; Winkler et al., 2020; Zheng et al., 2021) , the unvaccinated mice showed high viral loads in lung ( Figure 4A ). In contrast, mice vaccinated with the spike-based vaccine alone (but not the nucleocapsid-based vaccine alone) exhibited significant antiviral protection in the lung at day 3 post-challenge ( Figure 4A ). Importantly, co-immunization of mice with the spike-based vaccine and the nucleocapsid-based vaccine did not confer any synergistic protective advantage in the J o u r n a l P r e -p r o o f lung at day 3 post-challenge, relative to spike-based vaccine alone ( Figure 4A ). These data show that a spike-based vaccine alone is sufficient to protect the site of initial viral entry, the respiratory system.

At first glance, the data above suggested that nucleocapsid-specific immunity plays a dispensable role during a SARS-CoV-2 infection. Nucleocapsid-specific immunity has recently garnered attention as a main target for T cell responses (Lineburg et al., 2021; Nguyen et al., 2021; Rydyznski Moderbacher et al., 2020) . Antibody responses can block the initial entry of virus at proximal sites of challenge, but if the virus escapes the antibody response, T cell responses are critical for controlling second-round infections and subsequent viral dissemination to distal sites. SARS-CoV-2 is thought to disseminate to the nervous system after second-round infections that progress from the olfactory mucosa to the brain (Meinhardt et al., 2021a) . Although current spike-based SARS-CoV-2 vaccines show high efficacy in protecting the respiratory system via their induction of neutralizing antibodies, it is not clear whether they can also provide acute protection at distal sites, outside the respiratory system.

We show that mice immunized with a spike-based vaccine (or a nucleocapsid-based vaccine) did not exhibit a statistically significant improvement in antiviral protection in brain ( Figure 4B ). However, combining a spike-based vaccine with a nucleocapsid-J o u r n a l P r e -p r o o f based vaccine resulted in significantly improved protection in brain ( Figure 4B ). Taken together, these findings show that spike-specific immunity confers acute protection at the proximal site of viral entry (the respiratory system), but acute protection at distal sites may critically depend on also having nucleocapsid-specific immunity. Novavax vaccine (Baden et al., 2021; Logunov et al., 2021; Polack et al., 2020; Sadoff et al., 2021; Voysey et al., 2021; Zhu et al., 2020) Spike-specific antibodies are critical to prevent initial infection, because they sterically block the binding of the spike protein to the host ACE2 receptor. However, in the context of a breakthrough infection, infected cells are eliminated by cytotoxic T cells, which do not need to be spike-specific to recognize infected cells. The nucleocapsid J o u r n a l P r e -p r o o f protein of SARS-CoV-2 has been suggested to be an important target for T cell responses (Joag et al., 2021; Lineburg et al., 2021; Nguyen et al., 2021; Rydyznski Moderbacher et al., 2020) . Firstly, this protein contains conserved cross-reactive T cell epitopes that are present among different coronaviruses, suggesting that it could be an ideal target for universal coronavirus vaccines (Dutta et al., 2020) . Secondly, the nucleocapsid protein is among the most abundant structural proteins in the coronavirus lifecycle (Chang et al., 2014; de Breyne et al., 2020; Weiss and Leibowitz, 2011) , which may facilitate early antigen presentation and recognition by T cells.

Why does the nucleocapsid-based vaccine fail to confer acute protection at the proximal site of challenge (the lung)? It is possible that acute viral loads in lung represent primarily viruses derived from the primary foci of infection. During the hyperacute phase, nucleocapsid-specific immune responses may not prevent breakthrough infection in the respiratory system, because they target an internal protein that is not involved in viral entry. However, during subsequent second-round infections that progress from proximal sites of challenge to distal sites, nucleocapsid-specific T cells may provide a synergistic antiviral effect by killing virally infected cells, curtailing further the dissemination of the virus to distal sites. Altogether, co-immunization with a spike-based vaccine and a nucleocapsid-based vaccine accelerates the acute control of SARS-CoV-2, suggesting an advantage of including other viral antigens besides the spike protein in future COVID-19 vaccines.

J o u r n a l P r e -p r o o f A limitation of our study is that we only measured viral loads at a very early timepoints.

We did not compare viral loads at later time points, because unvaccinated animals succumb (or have to be euthanized) after day 5 post-challenge (Oladunni et al., 2020) , and because it is already known that spike-based vaccines elicit immune responses that ultimately clear SARS-CoV-2 within a week of challenge (Feng et al., 2020; Wu et al., 2020) . Our study was focused on evaluating hyperacute viral control to assess breakthrough infection. Future studies will assess the contribution of nucleocapsidspecific antibodies versus T cells, and whether vaccines that encode other viral antigens (such as envelope or membrane) can improve further distal protection after a SARS-CoV-2 challenge. Although the K18-hACE2 model is considered useful to evaluate vaccines and antivirals, it is possible that the extensive viral dissemination in brain may be a peculiarity of this mouse model. Nevertheless, it is still reasonable to conclude that nucleocapsid-specific immunity improves the acute distal control of SARS-CoV-2. In conclusion, we show a substantial benefit of including both spike and 

This article does not report original code. The article includes all the data sets and analyses generated for this study. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Mice were purchased from Jackson laboratories and were housed at Northwestern University 

Adenoviral vectors were propagated using HEK293 cells purchased from ATCC (cat # CRL-1573).

Vero E6 cells were used to propagate SARS CoV-2 isolate USA-WA1/2020 (BEI resources, NR-52281). Cells were not authenticated as they were purchased from a reputable vendor and certificate of analysis was obtained.

6-8-week-old K18-hACE2 mice were used. These mice express the human ACE2 protein behind 

Tissues were isolated from infected mice and homogenized in sterile PBS. RNA was isolated with the Zymo 96-well RNA isolation kit (Catalog #: R1052) following the manufacturer's protocol. SARS-CoV-2 viral burden was measured by RT-qPCR using Taqman primer and probe sets from IDT with the following sequences: 

Binding antibody titers were quantified using ELISA as described previously Palacio et al., 2020) 

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