Protective CD8⁺ T-cell response against Hantaan virus infection induced by immunization with designed linear multi-epitope peptides in HLA-A2.1/K^b transgenic mice

Rodent-borne orthohantaviruses of the family Hantaviridae are widespread zoonotic pathogens that cause two diseases in humans, hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS) [1, 2]. At present, over 28 hantaviruses have been identified. The Old World hantaviruses, such as the prototypic member Hantaan virus (HTNV), Puumala virus (PUUV) and Dobrava virus (DOBV), are etiological agents in Asia and Europe, and together with the worldwide distributed Seoul virus (SEOV), they cause the majority of cases of HFRS [3, 4]. While New World hantaviruses, including the Andes virus (ANDV) and Sin Nombre virus (SNV), were found predominantly in the Americas—causing HCPS with a fatality rate of up to 40% of cases [5]. Hantaviruses are maintained in rodent reservoirs and are usually transmitted to humans in aerosols of rodent excreta, causing syndromes ranging from acute renal failure to pulmonary oedema and severe hemorrhagic illness [3]. So far, around 1000 HCPS cases have been reported, whereas more than 100,000 HFRS cases are estimated to occur worldwide each year [3]. China has the largest number of HFRS infections, with a fatality rate of up to 15% of cases, which accounted for nearly 90% of the global HFRS cases [6, 7]. Regarding their impacts on public health, hantaviruses-associated diseases merit significantly more attention from researchers and officials, and are considered to be a high-priority public health concern in many countries [8].

Approved HFRS inactivated vaccines are a reality in China and Korea [9]. Approximately 2 million doses of inactivated rodent brain or cell-culture-derived HFRS vaccines are administered annually in China [10, 11]. Although vaccination, along with other preventative measures, has coincided with a reduction in HFRS cases in these regions, the inactivated vaccines fails to demonstrate a statistically significant reduction in the disease severity of HFRS [12]. Currently, there are no U.S. Food and Drug Administration (FDA)-approved vaccines or treatments for these hantavirus diseases [10]. The World Health Organization-approved HFRS vaccines also remain unavailable. One of the new strategies for innovative vaccine development is a multi-epitope-based peptide vaccine, which has been under study in many infectious diseases and cancers, and has even progressed to preclinical or clinical trials [13‐15]. A multi-epitope-based peptide vaccine offering the capability of eliciting both cell-mediated responses and antibody (Ab) responses has several advantages, such as being easily produced, chemically stable and free of bacterial/viral contaminating substances [16]. Thus, the identification of the epitopes, especially the immunoprotective epitopes, could provide a basis for the development of epitope-based peptide vaccines. However, there are hardly any studies on the design and evaluation of the multi-epitope-based peptide vaccine for HFRS infected with HTNV.

The antigenicity of hantaviruses largely depends upon two structural proteins, nucleocapsid protein (NP) and envelope glycoprotein (GP), encoded by the negative-sense RNA genome. Both NP and GP, heterodimers of mature glycoprotein Gn and Gc, have been confirmed to be responsible for vigorous cellular and humoral immune responses [12, 13]. Several studies have demonstrated that both vigorous cytotoxic T cell (CTL) responses and multifunctional T helper (Th) cell responses are important for protection against HTNV infection [17‐19]. In our previous study, eight CTL epitopes on HTNV-NP with different HLA restrictions and seven CTL epitopes on HTNV-GP-restricted by HLA-A*02 have been identified, meanwhile several CD4⁺ T-cell epitopes from HTNV-GP were also defined [20‐23]. Importantly, these epitopes have been confirmed for the induction of immunoprotective responses in HFRS patients or in mice, which exactly provided the basis to develop an epitope-based vaccine for HFRS [20‐23].

Notably, the specific CD4⁺ T-cells could exert the effects further through the stimulation of B-cells for neutralizing antibody production and promoting the CD8⁺ T-cell response during infections. Thus, CD4⁺ T-cell epitopes may be of great importance in the design of multi-epitope peptide vaccines. The pan HLA-DR binding epitope (PADRE), which can bind various MHC class II molecules with high affinity, is capable of generating antigen-specific CD4⁺ T-cells and has been widely used to construct the multi-epitope peptide vaccines [24]. Several epitope-based vaccines for diseases such as HIV, cytomegalovirus infection and hepatocellular carcinoma, represented T-cell incorporated alongside a universal PADRE to improve the potency of their immune-response stimulation [25‐28]. Therefore, we propose that PADRE peptide may provide a powerful immunogen in the development of multi-epitope peptide vaccines of HTNV.

In this study, HTNV linear multi-epitope peptide, composed of single HLA-A*02-restricted HTNV CTL epitope and PADRE were designed and constructed as candidate vaccine. Moreover, specific CD8⁺ T-cells responses and characteristics induced by the multi-epitope peptide were analyzed in HLA-A2.1/K^b transgenic (Tg) mice to evaluate the immunogenicity and protective effects of the peptide, which may provide some important information for the development of a novel peptide vaccine for HFRS infected by HTNV.

In the current study, we designed the HTNV linear multi-epitope peptide consisting of HLA-A*02-restricted HTNV CTL epitope and PADRE, and evaluated the multi-epitope peptide as candidate vaccine in HLA-A2.1/K^b Tg mice as a virus infection model of immunizing the Tg mice following HTNV challenge. Compared with HTNV single VV9 epitope, linear multi-epitope peptide PADRE-VV9 could induce higher level of HTNV-specific CD8⁺ T-cell responses in Tg mice. Moreover, the HTNV-specific CTL responses induced by PADRE-VV9 was more effective than that of single VV9 epitope to reduce the HTNV RNA loads in Tg mice.These results may be important foundation for the development of novel peptide vaccines for HTNV.

It has been well established that severe or critical HFRS after HTNV infection is associated with decreased responses of CD4⁺ T-cells and CD8⁺ T-cells specific to multiple epitopes of HTNV [21, 22]. HTNV CTL epitope-specific T-cell responses have been proven to play a critical role in the control of virus infection through cytotoxic effects to clear the HTNV-infected cells directly. Given that T-cells are crucial for the control and clearance of HTNV infection, peptide vaccines inducing specific T-cell responses may offer promising approaches for the prevention of HTNV infection. The incorporation of CTL epitopes into the peptide vaccine could induce specific CTL responses, which may be more effective in the prevention of HTNV infection. As confirmed by our previous data, several HLA-A*02-restricted HTNV CTL epitope could induce protective CD8⁺ T-cell responses among HLA A*02⁺ HFRS individuals [21, 23]. However, low immunogenicity of the single epitope is a big obstacle in developing such peptide vaccines. A simple T-cell epitope is inadequate to act as an effective immunogen, leading to the requirement of much more complex antigens. Therefore, different approaches have been applied to make the epitope-based vaccines more potent and efficient [34, 35], one of which is to design linear multi-epitope peptides as candidate vaccines. However, it should be noted that T-cell epitopes in the vaccines would be restricted by the highly polymorphic HLA-I and II molecules.

In fact, HLA-A*02 allele is most frequently represented in the Chinese Han population [36]. Therefore, the multi-epitope peptides designed in this study, containing HLA-A*02-restricted HTNV-GP CTL epitope VV9, which has been proven to induce a high level of protective CD8⁺ T-cell responses in HFRS patients [23], will cover 30–40% of the at-risk population in China. The second component required for the design of the multi-epitope peptide vaccine was Th cell epitope, which could provide strong support to promote CTL response and a potent humoral response. A universal synthetic PADRE, which was originally developed by optimization of binding to HLA-DR, with high affinity to 15 out of 16 different HLA-DR molecules tested so far, was chosen for our multi-epitope peptide design [24, 37, 38]. PADRE can also bind with high to intermediate affinity to mouse I-A^b/d and I-E^b/d MHC haplotypes [38]. Therefore, PADRE peptide, used as a Th-epitope, was able to generate effective Th-cell responses, which could overcome the problems posed by the extreme polymorphism of HLA-DR molecules in the human population [24, 38]. Thus, our design of the HTNV linear multi-epitope peptide would allow for extensive coverage of the human population.

Additionally, tandem fusion of CTL epitopes and PADRE without proper linkers may result in the generation of junctional epitopes or a new protein. Therefore, short amino acid motifs (linkers) must be used between different epitopes connection. In our design, the epitopes were linked by an A-A-A linker contained in PADRE, which could play a principal role in retaining the structural features of each epitope. Moreover, the linkers are usually appropriate for proteasome-mediated cleavage or binding to a transporter associated with an antigen processing protein [39‐41]. Therefore, the A-A-A linker may be helpful to achieve optimal processing for our multi-epitope peptides in vivo. The K-S-S at the amino-terminus of peptides was employed as an adaptor sequence, in which the lysine residue not only has a protective effect but also has the characteristic of forming connections easily with lipid components on the surface of the cell membrane, promoting the uptake of peptides by the antigen-presenting cells, then involved in T-cell induction following peptide immunization. The nature of the standard spacer S-S, positioned between the lysine and the epitope, was proven to be of low solubility and highly particulate, which is crucial for immunogenicity of the peptides [27, 37, 42].

Our previous studies indicated that protective immunity against HTNV involved strong epitope-specific CD8⁺ T-cell responses, promoting the clearance of the virus both in ex vivo HFRS patients and in vivo animal models [20, 21, 23]. In this study, the optimal immunization effects of the designed HTNV multi-epitope peptide in Tg mice were proven by the capacity of multi-epitope peptide to activate CD8⁺ T-cells responses. Evidence was initially shown from the finding that a much higher frequency of epitope VV9-specific CTLs was observed in the splenocytes of Tg mice stimulated with multi-epitope peptide than single VV9, as detected by IFN-γ ELISPOT assay. In line with the findings, multi-epitope peptide was also demonstrated to be capable of generating specific CTLs secreting granzyme B, demonstrating the potential cytotoxic effects of the specific CTLs induced by multi-epitope peptide. Meanwhile, expansion of VV9-specific CD8⁺ T-cells was observed from multi-epitope peptide-immunized Tg mice. The overall evidence suggested that multi-epitope peptide had good specificity and immunogenicity to induce specific-CD8⁺ T-cell response against HTNV antigens in vivo. Interestingly, there was a certain percentage of granzyme B⁺ CD8⁺ T-cells in unrelated VY9 peptide-immunized Tg mice and also in PBS-injection Tg mice, as shown in Fig. 3. Since there was peptide stimulation of the splenocytes from each Tg mice group, including PBS-immunized mice during the cell culture before granzyme B detection, we speculated that the peptide stimulation might be a driving factor for the CD8⁺ T-cell to produce granzyme B to some extent. Moreover, Arens R et al. showed that about 4% granzyme B⁺ CD8⁺ T-cells could be detected in wild-type C57BL/6 mice, which was similar to our findings and may prove that CD8⁺ T-cells in mice without any activation could also produce granzyme B [43]. However, the detection of CD107a, an important marker for the degranulation function of T cells, and the cytotoxic assay of the VV9-specific CD8⁺ T-cells in the immunized mice would be carried out in the future study, which may be more beneficial to prove the efficiency of CD8⁺ T-cell responses induced by HTNV linear multi-epitope peptide. Moreover, since the fluorescence intensity of CFSE will be weakened gradually with cell passages during the cell proliferation, the CFSE is widely used for cell proliferation assays. The wider peak with two small peaks shown in the PADRE-VV9-immunized mice after peptide stimulation indicated that the multi-epitope peptide had a good ability to stimulate specific CD8⁺ T-cell proliferation.

Notably, most of the susceptible animals to HTNV are rodents, however, HTNV-infected mice always carry HTNV in organs but without obvious symptoms after infection. The lack of an HFRS disease model that could recapitulate the features of human HFRS disease hampers the development of vaccines and the evaluation of post-exposure prophylactics, which may be a major shortcoming of the field. In the absence of a relevant disease animal model for HTNV infection, viral load reduction is considered as a more reliable indicator for HTNV vaccine efficacy in Tg mice [20, 23]. In this study, the effects of multi-epitope peptide-induced immune responses were evaluated in HLA-A2.1/K^b Tg mice with HTNV challenge as an infection model. The immunization of HTNV-inactivated vaccine in Tg mice was used as a positive control in our study, which has been proven to boost humoral responses to inhibit virus replication. Furthermore, the immunological mechanisms for viral clearance are also closely related with T-cell-mediated immune responses. The reduction in the levels of HTNV RNA loads in the liver, spleen and kidneys of Tg mice immunized with HTNV multi-epitope peptide could provide direct evidence to support that an effective CTL immunity could contribute to the clean of HTNV infection. The specific CTL responses induced by multi-epitope peptide immunization may also be considered as an important mechanism of immunoprotection in Tg mice. In this study, we selected peptide VV9 on HTNV-GP as CTL epitope to construct the multi-epitope vaccine. Although epitope VV9 has been proven to bind with HLA-A*02 with highest affinity among the seven HLA-A*02-restricted HTNV-GP CTL epitope, the HLA-A*02-restricted epitope LL9 (aa358-aa366, LIWTGMIDL) on HTNV-GP may be another appropriate candidate epitope for multi-epitope vaccine construction, given that epitope LL9 could induce higher frequencies of specific CTL responses and reduce the level of HTNV RNA loads in HTNV infected mice more effectively compared with that of epitope VV9. Therefore, we will try to research on epitope LL9 and explore the value and possibility of LL9 as a better candidate epitope for multi-epitope peptide vaccine construction in the future study.

Generally, there was a post-vaccination increase in VV9-specific CD8⁺ T-cell levels from Tg mice immunized with the multi-epitope peptide containing PADRE. Thus, it seems that the Th-cell epitope PADRE promoted the production of specific CTL responses. In fact, an important factor in a strong anti-viral approach is effective activation of CD4⁺ T-cell response, which is necessary for activation of CD8⁺ T-cell and generation of memory T-cells [44]. The success of linear multi-epitope peptide to elicit a potent CTL response against HTNV infection may be attributed to the existence of both CTL epitope and Th epitope, indicating that PADRE was critical to improving multi-epitope peptide vaccine-induced immune responses. However, a future study of CD4⁺ T-cell responses is necessary and essential to better evaluate the efficacy of the HTNV multi-epitope peptide vaccines.

The present study demonstrated that the designed HTNV linear multi-epitope peptide vaccine consisting of HLA-A*02-restricted HTNV CTL epitope and CD4⁺ T-cell activator PADRE, could induce protective CD8⁺ T-cell responses to reduce HTNV RNA loads in HLA-A2.1/K^b Tg mice. The presence of PADRE may enhance the immunogenicity and efficiency of the multi-epitope peptide vaccine, which may provide some important information for the development of novel HTNV peptide vaccines. Additional studies are expected to confirm the effectiveness of the multi-epitope peptide vaccine in humans in the future.