Abstract
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 COVID-19 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.
1. Introduction
Development of safe and effective vaccines for some viruses such as HIV and EBOV has been challenging [15]. Although vaccine development has been almost exclusively focused on eliciting a humoral immune response in the host through inoculation with whole protein antigen [40][54][46][24], CTL peptide vaccines producing a T cell response may offer an important alternative approach [19]. For HIV and EBOV and influenza in particular, the potential of CTL vaccines has been discussed [17][6][44]. Although computational prediction alone has been used for T-cell vaccine design [2][11], we saw a unique 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 [45]. PereyraHeckerman conducted an analysis of virus-specific CD8+ T-cell immunity in individuals living with HIV [33]. 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 in-silico development of a CTL vaccine for HIV and other diseases.
Acquired immunity has been documented after EBOV infection [3]. Antibody as well as T-cell responses have been described [34]. 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 EBOV antigen, with 25 of the 26 responders targeting epitopes on EBOV NP [39]. 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 for could be designed using epitopes targeted by CD8+ T-cells identified in these EBOV 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 [53], and human immunodeficiency virus (HIV) RGPGRAFVTI [4]. The existence of such epitopes makes possible a range of pre-clinical vaccine experiments 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 Zaire: NP43-53 (VYQVNNLEEIC)[57]. Wilson et al came to this conclusion based on studying splenocytes harvested from mice vaccinated with Ebola Zaire NP using a Venezuelan equine encephalitis (VEE) vector. Their experiments showed that splenocytes from the vaccinated mice re-stimulated with NP43-53 had high levels of cytotoxic activity against target cells loaded with the EBOV NP peptide. Remarkably, NP43-53 also happens to be an 11 amino acid subsequence of the epitope identified by Sakebe et al, as most commonly favored for T-cell attack by survivors of the 2013-2016 EBOV outbreak in West Africa.
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), and to subsequently conduct an in-vivo EBOV challenge study to see if this pep-tide 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 antigen presenting cell (APC) at the same time [29]. 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 (Supplementary Material).
2. Results
We used a previously described biodegradable dry powder, PLGA microsphere, synthetic vaccine platform adjuvanted with TLR-4 and TLR-9 ligands [37] to immunize C57BL/6 mice with NP43-53 11-mer, the CTL+ class I peptide antigen from the Ebola Ziare NP protein identified by Wilson et al [57]. 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 [37]. Splenocytes from eight mice receiving the active vaccine, and eight mice receiving microspheres and adjuvants only, were harvested on day 14 and subjected to ELISPOT analysis evaluating IFN−γ release in response to stimulation with the peptide used in the vaccination. As illustrated in Figure 1, there was no statistically significant difference between the ELISPOT data for the vaccinated mice versus the response seen in the negative control group.
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-Db 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 2. This is consistent with the predicted H2-Db binding affinity of YQVNNLEEI as shown in Table 6.
We then loaded one population of adjuvanted microspheres with NP44-52 and a second population of adjuvanted microspheres loaded with VG19 from EBOV Zaire NP 273-291 (VKNEVNSFKAALSSLAKHG), a Class II epitope predicted to be relevant to NP43-53 based on the TEPITOPE algorithm using a technique described by Cunha-Neto at al [11]. This peptide has a predicted favorable H2-Ib binding affinity as shown in Table 8.
We vaccinated 16 mice with those two populations of microspheres via intradermal (tail) administration and an 12 additional mice with two populations of microspheres, one population containing NP43-53 and the other VG19. IFN−γ release as quantified by ELISPOT after the spleens were harvested on day 14 after immunization showed that the immune response to the 9mer NP44-52 was higher than the immune response after vaccination with NP43-53 and that this difference was statistically significant (P < 0.0001) Figure 3.
We conducted a pilot study demonstrating that intraperitoneal injection produced a statistically superior immune response by ELISPOT compared with intradermal tail injection in C57BL/6 mice (Supplementary Material). Based on the data from that study, we chose to proceed with intraperitoneal administration for the challenge portion of this study.
We dosed three groups of mice, ten mice per group, with the adjuvanted microsphere vaccine formulation containing NP44-52 and VG-19, with each peptide in a distinct microsphere population, and challenged these mice 14 days after vaccine administration with escalating IP administered doses of mouse adapted EBOV (maEBOV) (Group 3 - 100 PFU, Group 5 - 1000 PFU and Group 7 - 10,000 PFU). The composition of the vaccine used for the exposure study is described in Supplementary Material. A second set of three control groups of mice (groups 2, 4 and 6), ten mice per group (mock groups), received PBS buffer solution alone and served as control animals for the study and were similarly challenged with maEBOV. Group 1 animals served as study controls and received no PBS buffer, vaccine or maEBOV injections. All mice were sourced from Jackson Labs and were 6-8 weeks of age and 15-25 grams at the time of vaccination. The dosing regimen is outlined in 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 Figure 5. Clinical observation data shown in Figure 6 and Figure 7 and daily weight data shown in Figure 8 and Figure 9 show protection from morbidity in all active vaccinated mice exposed to 1,000 PFU maEBOV.
PBS buffer mock-vaccinated mice showed mortality increasing from the 100 PFU to 1,000 PFU as shown in Figure 10a and Figure 5. We saw a paradoxical effect in control animals with survival increasing between 1,000PFU (Figure 5) and 10,000 PFU (Figure 11a). We believe this was caused by innate immunity triggered by the very large maEBOV challenge. All mice in all vaccinated groups across both experiments survived and showed no morbidity by clinical observation scores and weight data.
For each of the three challenge levels, the difference between the number of survivors in the vaccinated group versus the PBS control group was statistically significant by chi square (100 PFU P = 0.001; 1000 PFU P = 0.0003; 10,000 PFU P = 0.003)
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 4.
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 [23][14]. EBOV infected humans have also shown elevated IL-6 levels and these elevated levels have been associated with increased morality [56].
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 [18][23][14] and elevated levels of MCP-1 were associated with fatalities in EBOV infected human subjects [56]. 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 [56].
We saw increased levels of IFN−γ in survivors. Other vaccine studies have associated IFN − γ with protection [55][30].
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. In contrast, 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 pathogenderived peptide antigen. 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 [59]. However, observations derived from individuals able to control HIV infection [33] and EBOV infection [39] 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.
NP44-52 is located within one of the EBOV nucleocapsid proteins considered essential for virus replication. This epitope resides in a sequence conserved across multiple EBOV strains as shown in Figure 12. A 7.3Å structure for NP and VP24 is shown for context in Figure 13a [52]. A 1.8Å resolution structure rendering for EBOV NP shown in Figure 13b illustrates that NP44-52 is a buried structural loop, which is likely to be important to the structural integrity of the EBOV NP protein [13]. This structural role of NP44-52 likely explains its conservation across EBOV strains.
CTL targeting of the EBOV NP protein has been described [38][20]. Nucleocapisid proteins are essential for EBOV replication [48]. Recent advances in T-cell based vaccines have focused on avoiding all variable viral epitopes and incorporating only conserved regions [6][21]. EBOV NP may more conserved than nucleocapsid proteins VP35 and VP24 making it more suitable as a CTL vaccine target [7][57]. The nucleocapsid proteins in SARS-CoV are also essential for that virus to function normally [8]. This suggests that a CTL vaccine targeting coronavirus nucleocapsid could be effective against SARS-CoV or COVID-19.
We have shown that an H2-Db 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 predictedmatched 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.
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 [28]. Because the adjuvanted microsphere vaccine used in this experiment incorporates a TLR-4 agonist, we dosed 10 mice with adjuvanted microspheres without peptides and found the level of protection after exposure to 100 PFU EBOV to be statistically no different from that seen in PBS buffer controls. We conclude that level of protection conferred by the adjuvanted vaccine described in this study is dependant on delivering peptides with the microspheres.
EBOV can cause severe pulmonary problems in exposed subjects [31]. These problems can be especially severe when the virus is delivered by aerosol [12] [26]. 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 [27]. 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 (Table 5)[51]. HLA-DR binding database analysis also suggests that VKNEVNSFKAALSSLAKHG demonstrates sufficiently promiscuous binding characteristics cover that same population (Table 7)[51]. Taken together, a peptide vaccine based on YQVNNLEEI and VKNEVNS-FKAALSSLAKHG could produce a cellular immune response in about 50% of the population of the Sudan and about 30% of the population of North America. This same approach could be applied to COVID-19 which also has conserved regions in nucleocapsid proteins as shown in Figure 14 and Figure 15. Antigenic escape allows a virus to retain fitness despite an immune response to vaccination [16]. 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[58].
We took all possible 424 9mer peptide sequences from the COVID-19 nucleocapsid protein sequences available and evaluated each peptide for HLA restriction using NetMHC 4.0 and NetMHCpan 4.0 [51][25][1]. We analyzed 9mer peptide sequences because these are often associated with superior MHC binding properties than class I peptides of other lengths [49]. We found 53 unique peptides with predicted binding below 500nM from NetMHC 4.0 and/or NetMHCpan 4.0. These results are shown in Table 9, Table 10, Table 11 and Table 12.
We proceeded to determine the predicted HLA population coverage of a vaccine incorporating all 53 peptides using median values of the ANN, SMM, NetMHC 4.0 and NetMHCpan 4.0 algorithms hosted by IEDB [51]. These 53 peptides, taken together, had predicted HLA coverage of greater than 97% of the world’s population as shown in Table 14. We also calculated HLA coverage based on alleles specific to populations in China and found that coverage across those individuals could be expected to be within 3% percent of the world wide coverage estimate as shown in Table 15. This same population coverage could be achieved with 16 of the 53 unique peptides as shown in Table 13.
Seven of the 53 peptides with a predicted HLA match have been tested invitro for HLA binding affinity by various researchers [51]. These binding affinity assays were originally performed with the SARS virus during a previous out-break. Specific literature references for these in-vitro assays for each peptide sequence are as follows: ASAFFGMSR, LSPRWYFYY, QQQGQTVTK: [42], FPRGQGVPI: [42][22][36][47], GMSRIGMEV: [22][50][10][32][9], KTFPPTEPK: [42][22][35][47][5] and LLLDRLNQL: [32][10][9][50][60]. These seven peptides are shown in red in Table 9, Table 10 and Table 13.
The remaining 46 COVID-19 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 [43][41][22]. Another approach to evaluating the 53 COVID-19 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 COVID-19 by incorporating peptides into the vaccine that are more commonly targeted for CD8+ attack by the controllers versus the progressors. The extent of the COVID-19 outbreak should allow many more controllers to be identified then the 30 individuals studied by Sakabe. Furthermore, Sakebe 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 COVID-19 nucleocapsid, making COVID-19 a second example, across two different viruses, of controllers exhibiting CTL attack preferentially on the nucleocapsid protein.
5. Declaration of Interest Statement
CV Herst, Scott Burkholz, Lu Wang, Peter Lloyd and Reid Rubsamen are employees of Flow Pharma, Inc. all receiving cash and stock compensation. Alessandro Sette, Paul Harris, William Chao and Tom Hodge are members of Flow Pharmas Scientific Advisory Board. Alessandro Sette and Paul Harris have received cash and stock compensation as SAB members. Richard Carback and Serban Ciotlos are consultants to Flow Pharma, both receiving cash and stock compensation. John Sidney works with Alessandro Sette at the La Jolla Institute of Allergy and Immunology. Flow Pharma, Inc. has previously contracted with the La Jolla Institute of Allergy and Immunology to support other research not related to this study funded under STTR contract CBD18-A002-0016. Reid Rubsamen, CV Herst, Scott Burkholz, Lu Wang, Peter Lloyd, Richard Carback, Serban Ciotlos and Tom Hodge are named inventors on various issued and pending patents relating to Flow Pharma’s technology. All of the rights to all of these patents have been assigned by each of the inventors to Flow Pharma. Shane Massey, Trevor Brasel, Edecio Cunha-Neto and Daniela Rosa have nothing to declare.
4. Acknowlegements
All animal handling was done in accordance with NIH and institutional animal care and use guidelines by Aragen Bio-sciences in Morgan Hill California and the University of Texas, Medical Branch, Galveston Texas working in conjunction with the Galveston National Laboratory. The research was funded by Flow Pharma, Inc.
Footnotes
Corrected misspelled author name.
References
- [1].↵
- [2].↵
- [3].↵
- [4].↵
- [5].↵
- [6].↵
- [7].↵
- [8].↵
- [9].↵
- [10].↵
- [11].↵
- [12].↵
- [13].↵
- [14].↵
- [15].↵
- [16].↵
- [17].↵
- [18].↵
- [19].↵
- [20].↵
- [21].↵
- [22].↵
- [23].↵
- [24].↵
- [25].↵
- [26].↵
- [27].↵
- [28].↵
- [29].↵
- [30].↵
- [31].↵
- [32].↵
- [33].↵
- [34].↵
- [35].↵
- [36].↵
- [37].↵
- [38].↵
- [39].↵
- [40].↵
- [41].↵
- [42].↵
- [43].↵
- [44].↵
- [45].↵
- [46].↵
- [47].↵
- [48].↵
- [49].↵
- [50].↵
- [51].↵
- [52].↵
- [53].↵
- [54].↵
- [55].↵
- [56].↵
- [57].↵
- [58].↵
- [59].↵
- [60].↵