key: cord-344316-mwnnmwnw authors: Herst, C.V.; Burkholz, S.; Sidney, J.; Sette, A.; Harris, P.E.; Massey, S.; Brasel, T.; Cunha-Neto, E.; Rosa, D.S.; Chao, W.C.H.; Carback, R.; Hodge, T.; Wang, L.; Ciotlos, S.; Lloyd, P.; Rubsamen, R. title: An Effective CTL Peptide Vaccine for Ebola Zaire Based on Survivors’ CD8+ Targeting of a Particular Nucleocapsid Protein Epitope with Potential Implications for COVID-19 Vaccine Design date: 2020-04-28 journal: Vaccine DOI: 10.1016/j.vaccine.2020.04.034 sha: doc_id: 344316 cord_uid: mwnnmwnw The 2013-2016 West Africa EBOV epidemic was the biggest EBOV outbreak to date. An analysis of virus-specific CD8+ T-cell immunity in 30 survivors showed that 26 of those individuals had a CD8+ response to at least one EBOV protein. The dominant response (25/26 subjects) was specific to the EBOV nucleocapsid protein (NP). It has been suggested that epitopes on the EBOV NP could form an important part of an effective T-cell vaccine for Ebola Zaire. We show that a 9-amino-acid peptide NP44-52 (YQVNNLEEI) located in a conserved region of EBOV NP provides protection against morbidity and mortality after mouse adapted EBOV challenge. A single vaccination in a C57BL/6 mouse using an adjuvanted microsphere peptide vaccine formulation containing NP44-52 is enough to confer immunity in mice. Our work suggests that a peptide vaccine based on CD8+ T-cell immunity in EBOV survivors is conceptually sound and feasible. Nucleocapsid proteins within SARS-CoV-2 contain multiple class I epitopes with predicted HLA restrictions consistent with broad population coverage. A similar approach to a CTL vaccine design may be possible for that virus. mouse using an adjuvanted microsphere peptide vaccine formulation containing NP44-52 is enough to confer immunity in mice. Our work suggests that a peptide vaccine based on CD8+ T-cell immunity in EBOV survivors is conceptually sound and feasible. Nucleocapsid proteins within SARS-CoV-2 contain multiple class I epitopes with predicted HLA restrictions consistent with broad population coverage. A similar approach to a CTL vaccine design may be possible for that virus. Keywords: Ebola Zaire vaccine, CTL Vaccine, controller, YQVNNLEEI, COVID-19, SARS-CoV-2, Flow Focusing Development of safe and effective vaccines for some viruses such as HIV and EBOV has been challenging [19] . Although vaccine development has been almost exclusively focused on eliciting a humoral immune response in the host 5 through inoculation with whole protein antigen [51] [69] [59] [29], CTL peptide vaccines producing a T-cell response may offer an important alternative approach [23] . For HIV and EBOV and influenza in particular, the potential of CTL vaccines has been discussed [21] [7] [56] . Although computational prediction alone has been used for T-cell vaccine design [2] [14], we saw a unique 10 opportunity to see if a preventative EBOV T-cell vaccine could be successfully designed based on the specific epitopes targeted by survivors of documented EBOV infection. The notion of HLA restricted HIV control has been described [58] . Pereyra- Heckerman conducted an analysis of virus-specific CD8+ T-cell immunity in 15 individuals living with HIV [43] . They reported that HIV controllers, individuals living with HIV not undergoing treatment who do not progress to AIDS, have CD8+ cells targeting different HLA restricted class I epitopes on HIV compared with progressors, individuals with HIV who progress to AIDS in the absence of therapy. Pereyra-Heckerman suggested that this observation could guide the 20 in-silico development of a CTL vaccine for HIV and other diseases. Acquired immunity has been documented after EBOV infection [4] . Antibody as well as T-cell responses have been described [44] . Sakebe et al. have shown that of 30 subjects surviving the 2013-2016 EBOV outbreak in West Africa, CD8+ T-cells from 26 of those survivors responded to at least one 25 EBOV antigen, with 25 of the 26 responders targeting epitopes on EBOV NP [50] . One of the most commonly targeted EBOV eptitopes on EBOV NP in the survivor group (targeted by CD8+ cells from four survivors) was NP41-60 (IPVYQVNNLEEICQLIIQAF). They also suggested that a CTL vaccine could be designed using epitopes targeted by CD8+ T-cells identified in these EBOV 30 controllers. Human pathogen-derived peptide antigens that are also recognized by C57BL/6 T-cells have been previously described. These include peptides from vesicular stomatitis virus (VSV) RGYVYQGL [68] , and human immunodeficiency virus (HIV) RGPGRAFVTI [5] . The existence of such epitopes makes a range of pre- 35 clinical vaccine experiments possible without having to rely on non-human primates and expensive and complex-to-manage humanized mouse models. Wilson et al. showed that the EBOV nucleoprotein (NP) is an immunogen that provides protective, CTL-mediated immunity against EBOV in a C57BL/6 mouse model and that this protection was conferred by a peptide sequence within Ebola 40 Zaire: NP43-53 (VYQVNNLEEIC) [73] . Wilson We set out to see if we could drive CTL expansion directed against NP43-53 to occur after vaccinating C57BL/6 mice with Ebola Zaire NP43-53 (VYQVNNLEEIC), 50 and to subsequently conduct an in-vivo EBOV challenge study to see if this peptide was protective. We fabricated adjuvanted microspheres for this study as a room temperature stable dry powder using the Flow Focusing process to be 11µM in diameter so as to prevent more than one microsphere from being phagocytosed by any given 55 antigen presenting cell (APC) at the same time [37] . By loading only one peptide sequence per microsphere, we maximized the peptide payload and mitigated the possibility of multiple, different peptide sequences being delivered to the APC simultaneously, which could possibly result in competitive inhibition at the motif which could interfere with antigen presentation and subsequent T-cell expansion 60 (Supplementary Material Section 1). We also set out to see if a similar approach to a CTL vaccine design for SARS-CoV-2 would be feasible based on an analysis of the HLA binding characteristics of peptide sequences on SARS-CoV-2 nucleocapsid. 65 We used a previously described biodegradable dry powder, PLGA microsphere, synthetic vaccine platform adjuvanted with TLR-4 and TLR-9 agonists for this study [48] . In that article, we showed that the TLR-4 and TLR-9 agonists given together with a peptide in a mouse model did not produce T-cell expansion by ELISPOT and that microencapsulation of the peptide and the 70 TLR-9 ligand, with the TLR-4 ligand in the injectate solution, was required to elicit an immune response to the delivered peptide antigen as determined by ELISPOT. That study also demonstrated that the microencapsulated peptides alone were insufficient to induce an adequate immune response without the presence of the TLR-4 and TLR-9 agonists administered as described. The TLR 75 agonists used for this vaccine formulation are used in FDA approved vaccines and can be sourced as non-GMP or GMP material for pre-clinical and clinical studies. We show here that the H2-D b restricted epitopes VSV (RGYVYQGL) and OVA (SIINFEKL), when administered to C57BL/6 mice, each produce a CD8+ We used this adjuvanted microsphere peptide vaccine platform to immunize C57BL/6 mice with NP43-53, the CTL+ class I peptide antigen from the Ebola Ziare NP protein identified as protective by Wilson et al. [73] . Microspheres containing NP43-53 and CpG were prepared as a dry powder formulation and suspended before use in a PBS injectate solution containing MPLA, and administered intradermally via injection at the base of the tail into mice as described in a previous publication [48] . As illustrated in Figure 1c , there was no statistically significant difference between the ELISPOT data for the vaccinated mice versus the response seen in the negative ELISPOT controls. Wilson reported that protection seen in her experiment was due to a peptide sequence within NP-43-53. We hypothesized that the NP43-53 epitope was inefficiently processed into MHC binding sub-sequences during antigen presentation. In order to explore possible H2-D b matches for peptide sequences contained within Ebola Zaire NP43-53 (VYQVNNLEEIC), we prepared three peptide vaccine formulations, each containing one of the three possible 9mer sub-sequences within NP43-53. These sequences are shown in Table 1 . We then vaccinated, via intradermal (tail) injection, three groups of mice with microspheres containing one of the three 9mer sub-sequences of NP43-53 (6 per group). ELISPOT analysis was performed, stimulating harvested splenocytes with the three possible 9mer sub-sequences. Splenocytes from mice receiving the NP44-52 sub-sequence had a statistically higher ELISPOT response than mice vaccinated with the other two possible sub-sequence 9mers (P < 0.0001) as shown in Figure 1a . This is consistent with the predicted H2-D b binding affinity of YQVNNLEEI as shown in Supplementary Material Table 3 . We then loaded one population of adjuvanted microspheres with NP44-52 . This peptide has a predicted favorable H2-I b binding affinity as shown in Supplementary Material Table 5 . We showed that vaccination of 6 mice with the adjuvanted microsphere vaccine loaded with VG19 and NP44-52 showed an ELISPOT response to NP44-52 whereas 6 mice vaccinated with adjuvanted microspheres not loaded with peptide did not ( Figure 1d ). We also showed that mice vaccinated with VG19 alone did not show an ELISPOT response to NP44-52 ( Figure 2a ) and, conversely, mice vaccinated with NP44-52 did not show a response to VG19 (Figure 2b ). We conducted a pilot study demonstrating that intraperitoneal injection of the adjuvanted microsphere vaccine produced a statistically superior immune response by ELISPOT compared with the same dose delivered by intradermal tail or intramuscular injection in C57BL/6 mice (Supplementary Material Section 2). Based on the data from that study, and the fact that the volume of the intraperitoneal space would allow larger amounts of microsphere suspension to be delivered, we chose to proceed with intraperitoneal administration for the challenge portion of this study delivering 20mg of microspheres per dose. We dosed three groups of mice, ten mice per group, with the adjuvanted mi- Table 2 . Peak mortality across all groups tested was seen in mice challenged with 1,000 PFU maEBOV versus PBS buffer control as shown in the survival curve in We saw what appears to be an innate immune response at the 10,000 PFU EBOV exposure level. It has been suggested that EBOV can mediate an innate immunity response through stimulation of TLR-4 [33] . Because the adjuvanted This provides some evidence that the protective effect of vaccination using this adjuvanted microsphere vaccine is reproducible. Serum samples from sacrificed animals exposed to EBOV who did not receive vaccine were quantitatively assayed for various cytokines using BioPlex plates. Animals having unwitnessed demise did not have serum samples collected. A Pearson Correlation Analysis was performed to assess relationships between specific cytokine levels and survival. The results are shown in Table 3 . We observed low levels of IL-6 in surviving mice. NHPs infected with EBOV have been determined by other researchers to have elevated levels of IL-6 in plasma and serum [27] [17] . EBOV infected humans have also shown elevated IL-6 levels and these elevated levels have been associated with increased mortality [71] . Similarly, we observed low levels of MCP-1, IL-9 and GM-CSF in survivors. [27] [17] and elevated levels of MCP-1 were associated with fatalities in EBOV infected human subjects [71] . Human survivors of EBOV have been found to have very low levels of circulating cytokines IL-9 and elevated levels of GM-CSF have been associated with fatality in humans exposed to EBOV [71] . We lating T-cells targeting SARS-CoV-1 nucleocapsid two years after initial infection. [42] We decided to investigate the feasibility of designing a SARS-CoV-2 peptide vaccine targeting SARS-CoV-2 nucleocapsid. All available SARS-CoV-2 protein sequences were obtained from the NCBI viral genomes resource within GenBank, an NIH genetic sequence database [8] . Retrieved sequences were processed using multiple sequence alignment (MSA) via Clustal for the nucleocapsid phosphoprotein [34] . The nucleocapsid phosphoprotein sequences were trimmed down to every possible peptide sequence Dosing [65] . Predicted values of these peptides were cross referenced with actual in-vitro binding measurements from identical 9mer peptides when that data was available. Most preventative vaccines are designed to elicit a humoral immune response, typically via the administration of whole protein from a pathogen. Antibody antigens and the HLA restricted nature of CTL vaccines have limited their utility to protect individuals from infectious disease [77] . However, observations derived from individuals able to control HIV infection [43] and EBOV infection [50] demonstrating that control may be associated with specific CTL targeting behavior, suggest that there may be an important role for HLA-restricted pep- EBOV can cause severe pulmonary problems in exposed subjects [39] . These problems can be especially severe when the virus is delivered by aerosol [15] [31]. Interaction of EBOV specific antibody, NHP lung tissue and EBOV delivered to NHPs via aerosol can produce a more lethal effect than in NHPs without circulating anti-EBOV antibody exposed to aerosolized EBOV (unpublished conference presentation). This suggests that a CTL vaccine may be more effective for prophylaxis against filovirus protection than an antibody vaccine if the anticipated route of EBOV exposure is via aerosol. coproteins present on the surface, show a high degree of conservation. Epitopes within these internal proteins often stimulate T-cell-mediated immune responses [57] . As a result, vaccines stimulating influenza specific T-cell immunity have been considered as candidates for a universal influenza vaccine [66] . response to SARS-CoV-1 nucleocapsid two years after infection. [42] This suggests that the same approach could be applied to SARS-CoV-2 which has con- Table 6 and Supplementary Material Table 7 . The remaining 46 SARS-CoV-2 peptides listed in could also be further qualified as potential vaccine candidates by confirming MHC binding predictions by in-vitro binding affinity and/or binding stability studies [54] [52] [26] . Another approach to evaluating the 53 SARS-CoV-2 candidate vaccine peptides though in-vitro testing is also possible. As we have shown in this paper, a peptide targeted by EBOV controllers could form the basis of a preventative vaccine for EBOV. ELISPOT analysis of PBMCs taken from the peripheral blood of COVID-19 controllers and progressors to assess the presence of a differential response to the 53 peptides could lead to a broadly applicable protective CTL vaccine against SARS-CoV-2 by incorporating peptides into the vaccine that are more commonly targeted for CD8+ attack by the controllers versus the progressors. A peptide vaccine for SARS-CoV-2, unlike a typical antibody vaccine, is not limited to virus surface antigen targets. This provides opportunities to attack other targets on SARS-CoV-2 besides spike which may be prone to mutation [74] . In addition, a peptide vaccine mitigates the risk of Antibody Disease Enhancement (ADE) seen in the context of a non-neutralizing antibody response to a whole protein vaccine [75] [55] . Also, neutralizing antibodies directed against spike protein in SARS-CoV-1 patients have been associated with an increased risk of Acute Lung Injury (ALI) [35] . Specifically, patients succumbing to SARS-CoV-1 were found to develop a neutralizing antibody (NAb) response to spike protein faster than survivors after the onset of symptoms and the NAb titers were higher in the patients who died compared with those who recovered [76] . To the extent to which antibody vaccines producing an antibody response against the spike protein in SARS-CoV-2 could increase the risk of ALI, this risk could also be mitigated by a using peptide vaccine as an alternative approach. The extent of the COVID-19 outbreak should allow many more controllers to be identified than the thirty individuals studied by Sakabe and the seven individuals identified in the Peng study [42] [50] . Furthermore, Sakebe and Peng did not report progressor data perhaps because of the difficulty in obtaining blood samples from those patients. If researchers act now during the COVID-19 outbreak, perhaps controller and progressor blood samples could be collected and prospectively analyzed, quickly creating a database of optimal candidate class I peptides for inclusion into a CTL vaccine with potentially broad HLA coverage for subsequent rapid manufacture and deployment. It would be interesting to see the extent to which the peptides favored by controllers appear on SARS-CoV-2 nucleocapsid, making SARS-CoV-2 a second example, across two different viruses, of controllers exhibiting CTL attack preferentially on the nucleocapsid protein. 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