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Combining spike- and nucleocapsid-based vaccines improves distal control of SARS-CoV-2

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 h of challenge, a spike-based vaccine confers acute protection in the lung, but not in the brain. However, combining a spike-based vaccine with a nucleocapsid-based vaccine confers acute protection in both the lung and brain. These findings suggest that nucleocapsid-specific immunity can improve the distal control of SARS-CoV-2, warranting the inclusion of nucleocapsid in next-generation COVID-19 vaccines.


In brief
Most SARS-CoV-2 vaccines are based on the spike antigen alone, and it is unknown whether including other viral antigens improves protection. Dangi et al. show that combining a spike vaccine with a nucleocapsid vaccine improves the control of a SARS-CoV-2 infection, warranting the inclusion of nucleocapsid in next-generation SARS-CoV-2 vaccines.

INTRODUCTION
Most of the severe acute respiratory syndrome-coronavirus-2 (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 coronavirus disease 2019 , 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., 2021;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., 2021;Song et al., 2021).
Knowing whether vaccines can block viral dissemination to the central nervous system is important, because this would help elucidate whether vaccinated people who become exposed to the virus could still develop neurological complications. For 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/Oxford vaccine, CanSino vaccine, Sputnik V vaccine, Novavax vaccine, and 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 the 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 observed only 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.
After 3 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 ( Figures 1B-1F). We also confirmed the immunogenicity of the Ad5-S and Ad5-N vaccines by intracellular cytokine staining (ICS) assays, which showed interferon-g (IFN-g)-expressing CD8 T cells ( Figures  2A-2C), and IFN-g-expressing CD4 T cells (Figures 2D and 2E). Similar to prior studies with other Ad5-vectored vaccines (Larocca et al., 2016;Penaloza-MacMaster et al., 2013;Provine et al., 2016), we observed modest IFN-g expression on virusspecific CD8 and CD4 T cells following Ad5 vaccination. The Ad5-S and Ad5-N vaccines also elicited their respective antibody responses by enzyme-linked immunosorbent assay (ELISA) (Figure 3). These results show that the spike-based 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 5 3 10 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. 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 the 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 the 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 lung at day 3 post-challenge, relative to spike-based vaccine alone . These data show that a spike-based vaccine alone is sufficient to protect the site of initial viral entry, the respiratory system.
Effect of nucleocapsid-specific immunity in the distal control of a SARS-CoV-2 infection At first glance, the data above suggested that nucleocapsidspecific 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 the 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., 2021). 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 the brain (Figure 4B). However, combining a spike-based vaccine with a nucleocapsid-based vaccine resulted in significantly improved protection in the brain ( Figure 4B). 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 nucleocapsidspecific immunity.
Insidious neurological manifestations are common after SARS-CoV-2 infection. It is unknown whether vaccines can prevent long-term neurological manifestations of SARS CoV-2. An unanticipated result from our studies is that a spike-based vaccine does not fully protect the central nervous system early following infection. This observation could help explain symptomatic infections that are reported in a fraction of vaccinated individuals, which can be associated with the loss of smell and other neurological manifestations.
Spike-specific antibodies are critical to prevent initial infection because they sterically block the binding of the spike protein to the host angiotensin-converting enzyme 2 (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 protein of SARS-CoV-2 has been suggested as an important target for T cell responses (Joag et al., 2021;Line-burg et al., 2021;Nguyen et al., 2021;Rydyznski Moderbacher et al., 2020). First, this protein contains conserved crossreactive 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). Second,  (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 the 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. 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.
A limitation of our study is that we only measured viral loads at a very early time point. We did not compare viral loads at later time points, because unvaccinated animals succumb (or must 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 1 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 nucleocapsid-specific antibodies versus T cells, and whether vaccines that encode other viral antigens (e.g., envelope, 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 the 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 nucleocapsid antigens in COVID-19 vaccines. These Larocca, R.A., Provine, N.M., Aid, M., Iampietro, M.J., Borducchi, E.N., Badamchi-Zadeh, A., Abbink, P., Ng'ang'a, D., Bricault, C.A., Blass, E., et al. (2016). Adenovirus serotype 5 vaccine vectors trigger IL-27-dependent inhibitory CD4 + T cell responses that impair CD8 + T cell function. Sci. Immunol. 1, eaaf7643.

Lead contacts
Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact Pablo Penaloza-MacMaster (ppm@northwestern.edu) and/or Justin Richner (richner@uic.edu).

Data and code availability
This article does not report original code. The article includes all the datasets 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.

EXPERIMENTAL MODELS AND SUBJECT DETAILS
Animals and ethics statement Mice were purchased from Jackson laboratories and were housed at Northwestern University or University of Illinois at Chicago (UIC) animal facility. All procedures were performed with the approval of the center for comparative medicine at Northwestern University and the UIC IACUC. Adult mice, approximately half females and half males were used for the immunogenicity experiments included in this study. For the challenge studies, female mice were used.

METHOD DETAILS
Cell lines 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.
Mice and vaccinations 6-8-week-old K18-hACE2 mice were used. These mice express the human ACE2 protein behind the keratin 18 promoter, directing expression in epithelial cells. Mice were purchased from Jackson laboratories (Stock No: 034860). Mice were immunized intramuscularly (50 mL per quadriceps) with an Ad5 vector expressing SARS-CoV-2 spike protein (Ad5-S), or nucleocapsid protein (Ad5-N), or both vectors combined; diluted in sterile PBS, at 10 9 PFU per mouse. Ad5-N was a kind gift of the Masopust/Vezys laboratory (Joag et al., 2021). These are non-replicating Ad5 vectors (E1/E3 deleted). The vectors contain a CMV (Cytomegalovirus) promoter driving the expression of the respective proteins. The Ad5 vectors were propagated on trans-complementing HEK293 cells (ATCC), purified by cesium chloride density gradient centrifugation, titrated, and then frozen at À80 C.

SARS-CoV-2 virus and infections
SARS-Related Coronavirus 2, Isolate USA-WA1/2020, NR-52281 was deposited by the Centers for Disease Control and Prevention and obtained through BEI Resources, NIAID, NIH. Virus was propagated and tittered on Vero-E6 cells (ATCC). In brief, Vero cells were passaged in DMEM with 10% Fetal bovine serum (FBS) and Glutamax. Cells less than 20 passages were used for all studies. Virus