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. Increased serum and plasma levels of MCP-1 have been observed in EBOV infected NHPs [22], [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 saw increased levels of IFN-γ in survivors. Other vaccine studies have associated IFN-γ with protection [70], [38].

We achieved protection against maEBOV challenge with a single injection of an adjuvanted microsphere peptide vaccine loaded with a Class I peptide in a region on EBOV nucleocapsid favored for CD8+ attack by survivors of the 2013–2016 West Africa EBOV outbreak. There is evidence that a CTL response could be beneficial in the context of a coronavirus infection. [13], [41], [64], [11], [28], [36] Peng et al. have found survivors of the SARS-CoV-1 outbreak who had circulating 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 9 amino acids in length. 9mers were chosen because they typically represent the optimal length for binding to the vast majority of HLA [1], [18], [3]. The resulting peptides were compared to the MSA to ensure than these sequences are conserved within all of the sequencing samples available and not affected by an amino acid variant that could complicate subsequent analysis, specifically the calculation of population coverage. A selection of HLA were selected to encompass the vast majority of the worlds population at over 97% coverage.

Peptides were run through artificial intelligence algorithms, netMHC and netMHCpan which were developed using training data from in vitro binding studies. The pan variant of netMHC is able to integrate in vitro data from a variety of HLA to allow for predictions to be made if limited in vitro data is available for the specified target HLA [30], [1]. This in silico analysis utilizes the neural networks ability to learn from the in vitro data and report back predicted values based on the imputed SARS-CoV-2 nucleocapsid phosphoprotein peptides. Peptides with a predicted HLA IC50 binding affinity of 500 nm or less in either of the algorithms, were included in the candidate list of targets for the vaccine [30], [1], [40].

A subset of these SARS-CoV-2 peptide sequences are present on SARS-CoV-1 nucleocapsid phosphoprotein and as a result had in vitro binding data in Immunology Epitope Database and Analysis Resource (IEDB) collected after a previous outbreak [65]. Predicted values of these peptides were cross referenced with actual in vitro binding measurements from identical 9mer peptides when that data was available.

3. Summary and discussion

Most preventative vaccines are designed to elicit a humoral immune response, typically via the administration of whole protein from a pathogen. Antibody vaccines typically do not produce a robust T-cell response. [72] A T-cell vaccine is meant to elicit a cellular immune response directing CD8+ cells to expand and attack cells presenting the HLA Class I restricted pathogen-derived peptide antigen. [47] Difficulty in obtaining a reliable immune response from peptide 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 peptide vaccines for protection against infectious disease for which development of an effective traditional whole protein vaccine has proved to be difficult. The adjuvanted microsphere peptide vaccine platform described here incorporates unmodified peptides making possible rapid manufacture and deployment to respond to a new viral threat.

CTL targeting of the EBOV NP protein has been described [42], [64], [28], [49], [24]. Nucleocapisid proteins are essential for EBOV replication [61]. Recent advances in T-cell based vaccines have focused on avoiding all variable viral epitopes and incorporating only conserved regions [7], [25]. EBOV NP may be more conserved than nucleocapsid proteins VP35 and VP24 making it more suitable as a CTL vaccine target [9], [73]. The nucleocapsid proteins in SARS-CoV-1 are also essential for that virus to function normally [10]. This suggests that a CTL vaccine targeting coronavirus nucleocapsid could be effective against SARS-CoV-1 or SARS-CoV-2.

We have shown that an H2-D^b restricted Class I peptide exists within the NP41-60 epitope identified by Sakebe et al. as the most commonly favored NP epitope for CD8+ attack by survivors of the 2013–1016 EBOV outbreak in West Africa. We have demonstrated, when delivered in conjunction with a predicted-matched Class II epitope using an adjuvanted microsphere peptide vaccine platform, NP44-52 protection against mortality and morbidity for the maEBOV challenge doses tested in a C57BL/6 mouse model. We accomplished this with an adjuvanted, microsphere-based, synthetic CTL peptide vaccine platform producing a protective immune response 14 days after a single administration.

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.

Sakebe et al. identified A∗30:01:01 as the only HLA type common to all four survivors in their study with CD8+ targeting of NP41-60. The A∗30 supertype is relatively common in West Africa: 13.3% for Mali, 15.4% for Kenya, 16.3% for Uganda, and 23.9% for Mozambique [32]. Although peptide vaccines are by their nature HLA restricted, it may be possible to create a CTL vaccine directed against EBOV for use alone or in conjunction with a whole protein vaccine to produce an antibody response in tandem, by incorporating additional Class I peptides from epitopes targeted by controllers to broaden the HLA coverage of the vaccine. MHC binding algorithms hosted by the IEDB predict that YQVNNLEEI will bind strongly to the MHC of HLA-A∗02:06, HLA-A∗02:03 and HLA-A∗02:01 individuals (Supplementary Material Table 2) [65]. HLA-DR binding database analysis also suggests that VKNEVNSFKAALSSLAKHG demonstrates sufficiently promiscuous binding characteristics cover that same population (Supplementary Material Table 4) [65]. Taken together, a peptide vaccine based on YQVNNLEEI and VKNEVNSFKAALSSLAKHG could produce a cellular immune response in about 50% of the population of the Sudan and about 30% of the population of North America.

The internal proteins located within influenza virus, in contrast to the glycoproteins 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].

SARS-CoV-1 infection survivors have been found to have a persistent CTL 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 conserved regions in nucleocapsid which is located within the virus (see multiple sequence alignment in Supplementary Material Fig. 7 and Supplementary Material Fig. 8). Antigenic escape allows a virus to retain fitness despite an immune response to vaccination [20]. Picking conserved regions for vaccine targeting is an important part of mitigating this problem. Coronavirus spike protein, for example, may be particularly susceptible to mutation meaning that antigenic escape would be likely if the spike protein was targeted by a coronavirus vaccine, making it difficult to achieve durable protection. [74] A recent paper conducted a population genetic analysis of 103 SARS-CoV-2 genomes showing that the virus has evolved into two major types: L and S, with changes in their relative frequency after the outbreak possibly due to human intervention resulting in selection pressure [62].

We took all possible 424 9mer peptide sequences from the SARS-CoV-2 nucleocapsid protein sequences available and evaluated each peptide for HLA restriction using NetMHC 4.0 and NetMHCpan 4.0 [65], [30], [1]. We analyzed 9mer peptide sequences because these are often associated with superior MHC binding properties than Class I peptides of other lengths [63], [18]. We found 53 unique peptides with predicted binding below 500nM from NetMHC 4.0 and/or NetMHCpan 4.0. These results are shown in Supplementary Material Table 6, Supplementary Material Table 7, Supplementary Material Table 8 and Supplementary Material Table 9.