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Article

An Algorithm for the Development of a Recombinant Antiherpetic Subunit Vaccine Combining the Crystal Structure Analysis, AlphaFold2-Based Modeling, and Immunoinformatics

by
Tatiana V. Rakitina
1,2,*,
Evgeniya V. Smirnova
2,
David D. Podshivalov
1,3,4,
Vladimir I. Timofeev
1,3,
Aleksandr S. Komolov
1,
Anna V. Vlaskina
1,
Tatiana N. Gaeva
1,
Raif G. Vasilov
1,
Yulia A. Dyakova
1 and
Mikhail V. Kovalchuk
1
1
National Research Center, Kurchatov Institute, 123182 Moscow, Russia
2
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 Moscow, Russia
3
Shubnikov Institute of Crystallography, Federal Scientific Research Centre “Crystallography and Photonics”, Russian Academy of Sciences, 119333 Moscow, Russia
4
Research Computing Center, Lomonosov Moscow State University, 127473 Moscow, Russia
*
Author to whom correspondence should be addressed.
Crystals 2023, 13(10), 1416; https://doi.org/10.3390/cryst13101416
Submission received: 23 August 2023 / Revised: 19 September 2023 / Accepted: 20 September 2023 / Published: 24 September 2023
(This article belongs to the Section Biomolecular Crystals)
Browse Figures
Versions Notes

Abstract

:
Using the envelope glycoprotein B (gB) crystal structure and digital prediction algorithm, the B- and T-cell antigenic determinants (epitopes) of human herpesvirus 1 (HHV-1), also known as herpes simplex virus 1 (HSV-1), were generated, and the method for their production in the form of recombinant proteins was proposed. First, the structure of the surface topological domain (ectodomain or spike) of gB with mapped epitopes was analyzed, and the most stable and immunogenic (due to their enrichment with B-and T-cell epitopes) subdomains were selected for the modeling of subunit vaccine prototypes using the AlphaFold2 (Google DeepMind, London, UK) artificial intelligence system. The proposed candidate vaccines included both small (about 100 amino acids) monomeric polypeptides, which were ideal for recombinant expression as fusion proteins, and a more complex polypeptide, which, due to its trimeric fold, looks like a miniature analog of the gB ectodomain. In this miniature analog, the ectodomain regions with the potential to interfere efficacious expression of soluble recombinant protein in Escherichia coli have been removed. The structural stability of the modeled proteins, confirmed by molecular dynamics simulation and host immune responses, predicted in silico, indicates the suitability of the two suggested polypeptides for generating subunit vaccines using recombinant DNA technology.

1. Introduction

The discovery of vaccines has radically changed the treatment and prevention of infectious diseases. In the field of public health, vaccination represents one of the world’s most successful initiatives, as vaccination has completely eradicated smallpox and significantly reduced the incidence of socially threatening diseases, such as polio and measles. It has been established that vaccination currently prevents 4 to 5 million deaths annually. (WHO, https://www.who.int/news-room/facts-in-pictures/detail/immunization (accessed on 5 December 2019)). Despite such noteworthy advances, developing vaccines for many diseases remains elusive. Therefore, the new strategies for the development, manufacture, and delivery of specific and immunogenic vaccines with a high potential to induce immune response, either therapeutic or prophylactic, remains an urgent task [1,2].
Conventionally, most vaccines have been produced as live attenuated microorganisms or inactivated microorganisms with various delivery routes, typically by intramuscular injection (https://www.cdc.gov/vaccines/hcp/admin/administer-vaccines.html (accessed on 1 August 2019)). However, in vitro cultivation of many infectious agents is complicated; therefore, producing live attenuated or inactivated vaccines against these pathogens is a complex challenge [3]. In addition, modified characteristics of attenuated microorganisms may lead to an adverse response of the host’s immune system. Furthermore, vaccines of that type may comprise components that may induce undesirable immune responses [4,5].
Therefore, a beneficial strategy is to utilize an approach of subunit vaccines, wherein the only components of the infectious agent that are administered as a vaccine are those necessary to generate an appropriate immune response and which are nongenetic per se [6]. The subunit vaccines, comprising essentially proteins or peptides, are widely investigated and used in the clinic [6]. These subunit vaccines offer significantly improved safety, can be robustly produced (with high lot-to-lot consistency), and can elicit immune responses toward specific viral/microbial antigenic determinants (epitopes). Additional advantages of these types of vaccines are their inability to incorporate unwanted impurities and a significant reduction in the costs of production and storage [7]. Thus, as compared to traditional vaccines, which utilize attenuated or inactivated microorganisms, subunit vaccines provide a number of benefits regarding safety and ease of manufacture. At the same time, subunit vaccines have their disadvantages as well, such as low immunogenicity and failure to induce potent T-cell responses and generate memory cells [8,9,10].
Currently, there is a growing interest in the use of peptides in vaccine development approaches due to recent advances in the enhancement of their delivery and stability [11]. Moreover, the knowledge of how the immunogenic protein epitopes are processed in specific tissues and the discovery of extraordinarily long cytotoxic T-cell epitopes give clues for understanding how to enhance the immunogenicity of peptides, which consequently attracts interest in the use of peptides in vaccine development and broaden the range of their targets [12].
Human herpesvirus 1 (HHV-1), which is also called human herpes simplex virus 1 (HSV-1) from the Herpesviridae family, is a common cause of sexually transmitted infections, a leading cause of corneal blindness in Western countries and the most prevalent cause of fatal sporadic encephalitis in the United States [13]. It also makes a significant contribution to neonatal mortality [14]. By 2016, about 70% of people in the world aged 15–49 were infected with HSV-1 [15].
In the Herpesviridae family, all members share common features, i.e., comparatively large, double-stranded linear genomic DNA encoding 100–200 genes enclosed in a capsid. The viral capsid consists of a lipid bilayer and about 12 glycoproteins: gB, gC, gD, gE, gG, gH, gI, gJ, gK, gL, gM, and gN [16,17]. Such surface-exposed viral glycoproteins are known to be traditional targets in vaccine development approaches because they are required for the fusion of viruses with cell membranes and play a role in immune system evasion [18,19,20]. Due to that, their structures and functions have been extensively studied in attempts to create antiherpetic vaccines, which are currently at various stages of development, from research and preclinical trials to phase II and III clinical trials. For example, vaccine development against HSV-1 (and cognate HSV-2) around the globe includes live attenuated [21,22,23,24,25] and replication-defective viral vaccines [26,27,28], as well as DNA-based vaccines [29,30,31,32,33]. However, the most profound results have been achieved with subunit vaccines [34,35,36,37,38,39], particularly with adjuvanted subunit vaccines based on viral gD (phase III, [38]). However, despite years of research, there are still no vaccines approved to prevent the onset of or treat existing HSV-1 infection. At the same time, a therapeutic Shingrix vaccine against shingles, an adjuvanted subunit vaccine consisting of a single recombinant gE [19], and a prophylactic Varilrix vaccine against chickenpox have already been developed [40]. The causative agent for both diseases is cognate Varicella Zoster virus (VZV).
VZV and HSV-1 are both members of the Herpesviridae family; however, the immune responses elicited by VZV and HSV-1 and their immune evasion mechanisms are different. One of the reasons that complicate the vaccine development is the latency of the HSV-1 infection, which allows viruses to remain in the host body lifelong [41], and the HSV’s immediate-early proteins that provide virus evasion of the host immune system response [42]. Due to the host immune system evasion, a promising strategy for vaccine development against HSV-1 involves a multi-subunit approach in which multiple glycoproteins are used as antigens, e.g., gD in combination with gE and gC [34]. Another approach is based on expanding the spectrum of glycoproteins used as antigens; for example, both separated domains and trimeric form of gB were successfully implemented for the vaccine development against cytomegalovirus (CMV), which is also a member of the Herpesviridae family [43].
At present, the accumulation of a large amount of genetic and structural information about pathogens in specialized databases makes it possible to use the crystal structures of viral proteins to identify potential immunogenic epitopes and predict subunit vaccines based on them using immunoinformatics approaches [44,45,46,47,48]. In addition, the development of bioinformatic tools for predicting the host’s immune response allows in silico evaluation of the effectiveness of these vaccines. Considering the ensuing production of the subunit vaccines based on recombinant DNA (rDNA) technology, the time required to produce such vaccines has been significantly reduced [46,47].
In this work, an algorithm for the development of a subunit vaccine against HSV-1 is described. This algorithm is a completely in silico technique. It is based on using the crystal structure of the HSV-1 gB and artificial intelligence for epitope prediction, as well as the modeling of subunit candidate vaccines that can be easily produced using rDNA technology. Thus, the main advantage of this algorithm is an in-depth analysis of the structure of the target antigen, which allows one to take into account the spatial relationships within the protein structure and reproduce them in the modeled subunit vaccine candidates. This feature distinguishes our algorithm from those previously described in [46,47,48]. As a result, two types of subunit vaccine candidates have been proposed: a small (about 100 amino acids) monomeric polypeptide, ideally suited for recombinant expression as a fusion protein, and a more complex polypeptide that, due to its trimeric fold, is a miniature analog of the gB ectodomain.

2. Materials and Methods

2.1. Amino Acid Sequence Analysis

The protein sequence of the HSV-1 (strain 17) gB (Uniprot P10211, GB_HHV11) was obtained from the Uniprot knowledgebase (accessed on 1 June 2023) using the BLAST algorithm [49]. To confirm its conservancy, the amino acid sequence of GB_HHV11 was compared to those of gBs from other representatives of the Herpesviridae family. Multisequence alignment (MSA) was performed using the Clustal Omega program, version 1.4.2 (https://www.ebi.ac.uk/Tools/msa/clustalo/).

2.2. Analysis of the Target Antigen Structure

The gB crystal structure (PDB ID 5V2S) obtained with 3.6 Å resolution for postfusion conformation of the glycoprotein [50], as well as the gB molecular model built with AlphaFold-2 [51], were used. The topology of the protein relative to the cell membrane was confirmed using the TMHMM program [52]. Further structural analysis was performed using PyMol2.4 (https://pymol.org/2/).

2.3. Prediction and Analysis of T- and B-Cell Epitopes

T cell epitopes were predicted using NetCTL software [53] only for HLA class I [54]. The following parameters were used: weight of tap—0.05; weight of MHC—1; weight of Cleavage—0.15; the epitope identification threshold was 0.75.
Both discontinuous (conformational) and linear B cell epitopes were predicted using the—prediction tool (http://tools.iedb.org/ellipro/) located at the IEDB server [55]. The lowest score and the maximum distance in Ångström were calibrated using the default values of minimum score—0.5 and Maximum distance (Å)—6, accordingly. A simulated 3D protein structure was used to predict both conformational (discontinuous) and linear B-cell epitopes.
The AllerTOP program was used to analyze the allergenicity of the found epitopes [56]. To analyze toxicity, the ToxinPred program was used [57]. The immunogenicity of the found epitopes was assessed using VaxiJen [58]. The conservancy of the identified epitopes was analyzed using Epitope Conservancy Analysis [59].
The parameters and thresholds used for prediction tools were selected as described in [46,47,48].

2.4. Modeling of Candidate Subunit Vaccines

First, the gB spatial structure with immunogenic epitopes mapped to it was analyzed. Next, the most stable and immunogenic (due to their enrichment with B-and T-cell epitopes) subdomains were chosen from the gB ectodomain. Unwanted ectodomain regions and the regions with the potential to interfere with the efficacious expression of soluble recombinant protein in Escherichia coli were removed. Then, Cys residues were replaced by either Ser and His residues (in the case of removal of the second amino acid residue involved in the disulfide bond formation) or a pair of oppositely charged residues able to form a salt bridge instead of a disulfide bond. Finally, using the AlphaFold2 artificial intelligence system [51], the optimal way to link the separate subdomains with each other was chosen, which was based on the elucidation of the best model using the quality control of the batch of predicted structures using the AlphaFold LDDT-Ca metric.

2.5. Stability Assessment of the Predicted Polypeptides

Molecular dynamics simulations (MD) were used to test the structural stability of three proposed subunit vaccine candidates validated using the AlphaFold LDDT-Ca metric. The research was conducted using the “Lomonosov-2” supercomputer of the Lomonosov Moscow State University [60].
At first, for each system, MD simulation was carried out using the Amber-20 simulation package [61] with the ff99SB-ildn force field [62]. The minimum distance from the protein molecule to the cell boundary was 15 Å. The TIP3P water, as well as K+ and Cl− ions, were added to the cell to neutralize the overall charge of the protein and maintain a physiological salt concentration (0.15 M). After the minimization procedure, the system was heated sequentially from 0 to 310 K at constant volume using a Langevin thermostat with a collision frequency of 3.0 ps−1 [63,64]. For constraining the bonds involving hydrogen atoms, the SHAKE algorithm was used. The system was pressurized to 1 atmosphere with a Berendsen barostat for 500 ps [65]. NPT dynamics of 400 ns long were carried out using the PME algorithm and a cutoff radius of 10.
The changes in the protein folding along the MD trajectories were evaluated by calculating the root mean square deviations (RMSD) for the backbone atoms, gyration radii (Rg) for the three polypeptides as well as residual root mean square fluctuations (RMSF) along the polypeptide chains. The values for the above parameters were obtained from the cpptraj package of Amber20. The graphs were created in RStudio.
Since the stability of the most complex trimeric structure was questionable, the MD experiment for this system was repeated. In the second simulation, the ff19SB force field was used, which significantly improved the stability of the system.

2.6. Assessment of the Host Immune Responses

The in silico simulation of the host immune responses to the modeled vaccines was performed using the C-IMMSIM online server (http://kraken.iac.rm.cnr.it/C-IMMSIM [66]).

3. Results and Discussion

3.1. Experimental Design

As mentioned in the Introduction section, the accumulation of a large amount of genetic and structural information in specialized databases, as well as the extensive development of bioinformatic tools for predicting the host’s immune response, make it possible to develop subunit vaccines in silico. In this work, to design subunit vaccines against HSV-1, a cascade of computational procedures similar to that used in [48] was engaged. The crucial steps of the cascade are described in Table 1.

3.2. Herpesviridae Family

Herpesviridae is a large family of DNA-containing viruses that cause various diseases in humans and animals [67]. In humans, they can result in the development of life-threatening diseases, including tumors and leukemia. There are 8 herpesvirus species that affect humans, which are divided into three subfamilies: α, β, and γ (Table 2) [68].
The most important herpesviruses belong to the α-Herpesviridae subfamily of the Herpesviridae family. Mammals serve as natural hosts. This subfamily is distinguished primarily by faster reproduction than other subfamilies of the Herpesviridae family. Currently, there are 45 known species in this subfamily, divided into 5 genera, with one species not belonging to any genus. Symptoms, which are associated with diseases caused by this subfamily, include skin vesicles or mucosal ulcers; in rare cases, HSV-1 and HSV-2, as well as HSV-3, may cause encephalitis and meningitis. Viruses of the α-Herpesviridae subfamily can invade the nervous systems of their mammalian hosts [69].
Mammals serve as natural hosts for the β-Herpesviridae subfamily of the Herpesviridae family. There are 26 species assigned to this subfamily, divided into 5 genera. Diseases associated with this subfamily include congenital cytomegalovirus (CMV) infection (HHV-5), “sixth disease” also known as Roseola infantum or subitumenal exanthema (HHV-6), chronic fatigue syndrome (HHV-7). Members of this family are lymphotropic and infect T cells and monocytes [70].
The reproduction rate of γ-Herpesviridae subfamily viruses is more variable than that of the viruses from other Herpesviridae subfamilies. This subfamily contains 43 species, divided into 7 genera, with three species not belonging to any genus. Diseases associated with this subfamily include infectious mononucleosis (HHV-4) and Kaposi’s sarcoma (HHV-8) [71,72].
All herpesviruses share similar virion structures and penetrate host cells by merging viral envelopes with cell membranes [73]. In the case of HSV-1 and HSV-2, four viral glycoproteins (gB, gD, gH, and gL) and a host receptor for gD are essential for this fusion. For other herpesviruses, the three core glycoproteins, gB, gH, and gL, paired with different partners, are essential [73]. The key fusogen, which merges the viral and cell membranes, is gB. This type I membrane protein anchored to the viral envelope is conserved among herpesviruses from both structural and functional points of view [74]. Comparisons of amino acid sequences of gB from 8 subfamilies of the Herpesviridae family and from different strains of closely related HSV-1 and HSV-2 viruses, which are shown in Figure 1A,B, respectively, demonstrate high conservation of the glycoprotein sequences between HSV-1 and -2 viruses.
Crystal and electron cryotomography (CryoET) structures of gB from HHV-1, HHV-4, and HHV-5 are available [50,75,76,77,78,79].

3.3. Structural Analysis of the HSV-1 Envelope Glycoprotein B in the Postfusion Conformation

The HSV-1 gB is a homotrimer (Figure 2A). Each mature gB monomer consists of 904 amino acid residues and contains an ectodomain (residues 31–725 (Figure 2B)) and three other regions: an external membrane proximal region (MPR, residues 726–774), transmembrane domain (TMD, residues 775–795), and intraviral or cytoplasmic domain (CTD, residues 796–904). The ectodomain is a virion surface topological domain, which represents about 80% of gB. All other regions represent about 20% of gB. As shown in Figure 2A, MPR, TMD, and CTD form a pedestal for the ectodomain spike.
The N-terminal (residues 31–88) and central (residues 474–500) regions of the gB ectodomain are completely disordered and not visible in the electron density of the crystal structures. The rest of the ectodomain can be divided into the proximal (closest to the virion membrane) subdomain P (subP, residues 140–154 and 362–473), intermediate subdomain I (subI, residues 155–361), and apical (distant from the virion membrane) subdomain A (subA, residues 573–664), which is linked with subdomain I through the long helical region (subdomain H (subH)), residues 501–555). Because of the domain swap, the β3-strand is a part of subI, while consequent β-strands β4-β19 form subP, which contains two regions involved in fusion (residues 173–179 and 258–265). In addition to the β3-strand, subI includes the α3- and α4-helices as well as three β-strands (β20-β22). Helical subH consists of the α5- and α6-helices, which participate in the ectodomain trimerization. The β-turn formed by β23- and β24-strands follows subH and precedes subA, which contains several short β-strands (β25-β34) and is the most distal to the viral membrane. SubA from three gB monomers forms the apical end of the ectodomain, after which the polypeptide chains turn backward toward the viral membrane. This C-terminal part of the ectodomain is partially unstructured but contains several α-helical regions. The entire structure of the ectodomain is fastened by four intermolecular disulfide bonds. Three disulfide bonds are located in subdomains A, I, and P, while one bond connects subH and the N-terminal region preceding subP.
The described structural arrangement is characteristic of the gB trimer in the most stable and well-studied postfusion conformation, while in the prefusion conformation, a metastable state detected on the virion surface using cryoET, the aforementioned gB subdomains are significantly rearranged [76,79]. There are currently no high-resolution spatial structures for gB in the prefusion conformation.

3.4. Designing of Subunit Vaccines against HSV-1 Based on the Distribution of T- and B-Cell Epitopes in the gB Ectodomain

The search for antigenic determinants in the HSV-1 gB was carried out using the ElliPro program [55], which selects antigenic residues on the three-dimensional structure of the protein, as described in the Materials and Methods section.
To search for T-cell epitopes, the entire gB sequence was divided into 896 peptides of 9 amino acids long, of which 20 were identified as antigenic determinants. A complete list of the identified epitopes is presented in Table S1. Four peptides located outside the gB ectodomain were excluded from consideration. The locations of the remaining 16 peptides on the gB ectodomain are shown in Figure 3A.
B-cell epitopes can be divided into two types depending on the spatial structure of the epitopes: continuous (linear) and discontinuous (conformational) epitopes [81]. Entire lists of predicted epitopes of both types are provided in Tables S2 and S3. Peptides located outside the gB ectodomain were excluded from consideration.
Locations of 9 linear B-cell epitopes and 7 conformational B-cell epitopes on the gB ectodomain are shown in Figure 3B,C, respectively.
According to Figure 3, the majority of antigenic determinants belong to subdomains A, P, and I. However, the proximity of the subdomain P to the viral envelope and its partial association with the viral membrane could become an obstacle to its expression in E. coli in a soluble form. At the same time, bacterial expression of well-structured globular and strongly exposed to solvent subA and subI should not be a problem. In addition, small sizes allow using them in various combinations with each other and with all sorts of tags employed to improve the expression of recombinant proteins and to facilitate their purification by high-performance affinity chromatography.
As we already noted in the Introduction section, gB was not the focus of researchers for the development of vaccines against HHV-1, but it was actively used in the development of vaccines against HHV-5 [43]. It was shown that both the trimeric gB ectodomain expressed in eukaryotic cells and individual gB subdomains, some of which were expressed in E. coli, exhibited immunogenic potentials, with the trimeric variant being the most immunogenic. Since subH connects subA and subI in the native gB ectodomain and is known to be responsible for its oligomeric nature [79], we combined these three subdomains to obtain a trimeric variant of the subunit vaccine.
To generate such a trimeric variant in silico, we removed all unsuitable regions from the gB ectodomain and linked subdomains A, H, and I with each other. Figure 4 shows antigenic epitopes mapped on the gB ectodomain truncated to subA, subH, and subI. Immunogenicity, allergenicity, and toxicity of the epitopes located in the selected areas were evaluated as described in Table 1.

3.5. Modeling and Validation of the Antiherpetic Subunit Vaccine Candidates

Since the volume of the UniProtKB/TrEMBL database, which contains over 200 million protein sequences, exceeds by three orders of magnitude the volume of the PDB database, which contains only about 200 thousand experimentally determined protein structures, the modeling of protein tertiary structure becomes a crucial step for the in silico vaccine development. One of the most novel, progressive, and accurate modeling applications is AlphaFold2, developed by DeepMind (London, UK) and based on both deep learning and a conventional neural network [51]. AlphaFold2 is an excellent tool that can solve various problems in modeling the three-dimensional structures of proteins and protein-containing complexes and is compatible with different experimental and computational techniques (see, e.g., [82] as one of numerous examples). The advantages of using AlphaFold2 predictions in viral research are reviewed in [83].
As mentioned above, in our case, the structure of the target antigen was available; however, AlphaFold2 was employed at the step of modeling subunit vaccine candidates from selected subdomains of the gB ectodomain. For example, after subP was deleted, the amino acid linker between two portions of subI was optimized. Similarly, the selection of the optimal linker was carried out after removing the unstructured region between subI and helical subH. These optimizations included AlphaFold2-based modeling of spatial structures of several vaccine candidates having different linkers between fused regions of the protein and selection of the best linker using AlphaFold LDDT-Ca metric, which estimates a per-residue confidence score (pLDDT) of the models on a scale from 0 to 100. pLDDT score >90 indicates the highest confidence, 70–90 indicates good confidence, 50–70 indicates low confidence, while pLDDT score <50 indicates a lack of structure.
Figure 5 shows that the best structural models of subI, subA, and subIHA—combinations of subI, subH, and subA, a trimeric miniature analog of the gB ectodomain—predominantly have high to good confidence levels. Only subIHA has a small problem area at the junction of subI with subH.
A comparison of the models with the original structure shows that the subA and subI folds copy their original structures (Figure 5). In trimeric subIHA, all three subdomains and junctions between subA and subH are similar to those in the original structure, while subI is replaced from its original position regarding subH. The reason for this replacement is that to increase the confidence of the modeled structure of subIHA, the unstructured region between subI and subH was deleted along with the part of the α4-helix. It should be noted that due to the absence of subP in the trimeric subIHA, the exposure of the surface (and epitopes) of subI to the solvent (and immunoglobulins or other immune molecules) remained the same in both the crystal structure of the native trimeric gB ectodomain in the postfusion conformation [50] and the CryoET-based model of the gB prefusion conformation [79].
Stable 3D-folding is one of the requirements for the successful expression of recombinant proteins in E. coli since unstructured polypeptides are prone to proteolytic degradation. Due to this, the structural stability of the suggested vaccine candidates, subA, subI, and subIHA, was confirmed by MD simulation in addition to the AlphaFold LDDT-Ca metric. Several standard indicators were calculated to verify the simulation quality and the structural stabilities of the three models (subI, subA, and subIHA) (Figure 6 and Figure 7).
According to the backbone root mean square deviation (RMSD) and residual root mean square fluctuation (RMSF) levels, the subI and subA polypeptides had reasonable stabilities throughout the MD trajectory (Figure 6); moreover, the radius of gyration (Rg) values suggested that the proteins remained in their compact forms throughout 400 ns simulation at 310 K (Figure 6).
At the same time, in the case of the trimeric vaccine model (SubIHA), an increase in RMSD level along the MD timeline was observed together with a decrease of Rg (an indication of system compaction) and the enhanced level of the residual RMSF of the one monomer (Figure 7). Time evolution of the secondary structure throughout the MD trajectory in the subIHA trimer shows that a significant disruption in the long α-helix of subH had occurred in one of the monomers (Supplementary Figure S1). Visual analysis of the behavior of the subIHA structure showed that this disruption was associated with bending at the level of subH, consequent rearrangement of the subI and subA positions relative to each other, and compaction of the entire system. It should be noted that, despite the structural rearrangement, the trimer itself did not collapse, and the main parts of the individual subdomain surfaces remained accessible to the solvent. The observed phenomenon could potentially reflect conformational dynamics of the native gB ectodomain, whereas according to the cryoET results [76,79], the bend at the level of subH and more condensed state are distinctive features of the trimeric gB ectodomain in the prefusion conformation. However, by repeating the experiment using another force field, it was not possible to reproduce this phenomenon. The system showed a fairly high level of stability, as evidenced by the absence of a significant increase in the RMSD level along the MD trajectory and the absence of significant bursts of residual RMSF levels (Figure 7). In addition, a constant Rg level was observed along the MD trajectory. These results indicate that in the case of such a complex system as trimeric subIHA, the choice of force field significantly affects the simulation results. Thus, the results of the MD experiment allowed us to conclude that the structural stability of the modeled subunit vaccine candidates is suitable for expression in E. coli.
The final validation step of the subunit vaccine development algorithm was an in silico simulation of an immune response elicited by injection of each of all three modeled polypeptides (Figure 8). According to Figure 8A, in the case of subI and subIHA, high immunoglobulin titers were generated after the second and third antigen injections. The increase of immunoglobulin M levels was characteristic of the primary responses in both cases. The high immunoglobulin titers were observed during both the stimulation period and even after it (when the antigen levels were undetectable). At the same time, there were significant increases in the interferon-gamma (INF-γ) levels and moderate increases in the levels of various interleukins, interferon-β, and tumor necrosis factor (TNF) after all three consequent injections (Figure 8B). Low (close to zero) levels of Simpson indexes (D), also known as danger factors, indicate the absence of abnormal cytokine activations after injections of subI and subIHA (Figure 8B).
However, in the case of in silico modulation of the immune response after injection with subA antigen, a completely opposite pattern was observed, where zero increase in immunoglobulin titers was observed, accompanied by an abnormally high increase in cytokine levels (cytokine storm) (Figure 8A,B). Thus, in silico simulation of the immune response showed that out of three modeled polypeptides, only two variants (subI and subIHA) can be considered prototypes of the antiherpetic subunit vaccine. In connection with these results, we would like to note once again that, according to cryoET [76,79], subI is a part of the gB ectodomain, which is strongly exposed to the solvent in both postfusion and prefusion conformations, while subIHA imitates the trimeric form of the gB ectodomain that has shown to be the most promising variant in the development of a subunit vaccine against HHV-5 [43].

4. Conclusions

Vaccination is one of the most successful public health initiatives ever achieved, but despite such noteworthy advances, the development of vaccines for many diseases remains elusive. For example, despite years of research, there are still no approved vaccines to prevent oral and genital herpes infections caused by human herpesvirus 1 (HHV-1), also called human herpes simplex virus 1 (HSV-1) from the Herpesviridae family, which is the prevalent cause of sexually transmitted infection and infected more than 60% of the human population aged 15–49.
Here, we described an in silico algorithm for the development of a recombinant antiherpetic subunit vaccine, which is based on the combination of the detailed structural analysis of the HSV-1 surface glycoprotein B crystal structure and recent advances in the artificial intelligence-based immunoinformatics prediction techniques and structural modeling using AlphaFold2.
This algorithm is based on the following basic steps: Search for suitable candidate proteins (target-protein) and detailed analysis of their spatial structure, including oligomeric state, topology, and domain (subdomain) organization. Search for B-cell and T-cell epitopes and assessment of their localization. Analyze immunogenicity, allergenicity, toxicity, and conservancy of the selected epitopes. Drafting subunit vaccines based on stable and conserved structural elements (sub-domains) enriched with antigenic determinants. Modeling of the spatial structures of the vaccine candidates. Validation of the stabilities of the modeled structures of subunit vaccines using MD simulation. Validation of the effectivities of the subunit vaccine candidates using an in silico host’s immune response prediction. The main advantages of the algorithm presented are an in-depth analysis of the structure of the target antigen, which allows taking into account spatial relationships within the protein structure, as well as its simplicity, providing an opportunity for further development and adaptation to solve a wide range of problems.
As a result, two types of subunit vaccine candidates, which can be easily produced using rDNA technology, have been proposed: a small (about 100 amino acids) monomeric polypeptide, ideally suited for recombinant expression as a fusion protein, and a more complex polypeptide that, due to its trimeric fold, is a miniature analog of the gB ectodomain. Both proteins had good physical and chemical properties and high structural stability, which ensured their successful production in the bacterial expression system and also demonstrated good antigenic potential for the activation of both branches (cellular and humoral) of the immune response.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/cryst13101416/s1, Table S1: T-cell epitopes; Table S2: Linear B-cell epitopes; Table S3: Conformational B-cell epitopes; Figure S1: Time-evolution of the secondary structure throughout the MD trajectory in all monomers of the subIHA trimer.

Author Contributions

Conceptualization, T.V.R. and V.I.T.; methodology, T.V.R., D.D.P. and V.I.T.; validation, E.V.S. and T.N.G.; formal analysis, T.N.G.; investigation, T.V.R., D.D.P., V.I.T., E.V.S., A.S.K. and A.V.V.; resources, D.D.P., V.I.T., E.V.S., R.G.V., Y.A.D. and M.V.K.; data curation, R.G.V.; writing—original draft preparation, T.V.R., E.V.S. and V.I.T.; writing—review and editing, T.N.G. and R.G.V.; visualization, T.V.R., D.D.P. and V.I.T.; supervision, R.G.V., Y.A.D. and M.V.K.; project administration, R.G.V.; funding acquisition, R.G.V., Y.A.D. and M.V.K. All authors have read and agreed to the published version of the manuscript.

Funding

The work was supported by the NRC “Kurchatov Institute” (in part of structural analysis, epitopes prediction and immunosimulation) and within the State Assignment of FSRC “Crystallography and Photonics” RAS (in part of the molecular dynamic simulation analysis).

Data Availability Statement

All data are included in the manuscript or the Supplementary Materials.

Acknowledgments

This work was carried out using computing resources of the shared research facilities of HPC computing resources at Lomonosov Moscow State University and the federal collective usage center Complex for Simulation and Data Processing for Mega-science Facilities at NRC “Kurchatov Institute”.

Conflicts of Interest

The authors declare no conflict of interest.

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Crystals 13 01416 g001
Figure 1. Percent identity matrices, which show % of identities among amino acid sequences of gB from 8 subfamilies of the Herpesviridae family (A) and from different strains of HSV-1 and HSV-2 viruses (B). The percent identity matrices were generated using the Clustal Omega MSA program.
Figure 1. Percent identity matrices, which show % of identities among amino acid sequences of gB from 8 subfamilies of the Herpesviridae family (A) and from different strains of HSV-1 and HSV-2 viruses (B). The percent identity matrices were generated using the Clustal Omega MSA program.
Crystals 13 01416 g001
Crystals 13 01416 g002
Figure 2. The crystal structure of HSV-1 envelope glycoprotein B (PDB 5V2S) and distribution of the secondary structure elements along the amino acid sequence of the gB ectodomain. (A) One monomer of the gB trimer is colored as a rainbow: N-terminus (N) is blue, and C-terminus (C) is red, while disulfide bonds (red balls) and N-glycosylation are shown. MPR, TMD, and CTD regions, as well as major subdomains of the ectodomain described in the text as subA, subP, subI, and subH, are indicated. (B) The scheme of the secondary structure distribution along the amino acid sequence is prepared using ESPript (http://espript.ibcp.fr, [80]).
Figure 2. The crystal structure of HSV-1 envelope glycoprotein B (PDB 5V2S) and distribution of the secondary structure elements along the amino acid sequence of the gB ectodomain. (A) One monomer of the gB trimer is colored as a rainbow: N-terminus (N) is blue, and C-terminus (C) is red, while disulfide bonds (red balls) and N-glycosylation are shown. MPR, TMD, and CTD regions, as well as major subdomains of the ectodomain described in the text as subA, subP, subI, and subH, are indicated. (B) The scheme of the secondary structure distribution along the amino acid sequence is prepared using ESPript (http://espript.ibcp.fr, [80]).
Crystals 13 01416 g002
Crystals 13 01416 g003
Figure 3. Mapping of antigenic determinants on the monomer of the gB ectodomain. (A) T-cell epitopes. (B) B-cell linear epitopes. (C) B-cell conformational epitopes. Monomers are rainbow-colored from blue (N-terminus) to red (C-terminus). Antigenic determinants are highlighted in gray. Major subdomains of the ectodomain, described in the text as subA, subP, subI, and subH, are indicated in panel C.
Figure 3. Mapping of antigenic determinants on the monomer of the gB ectodomain. (A) T-cell epitopes. (B) B-cell linear epitopes. (C) B-cell conformational epitopes. Monomers are rainbow-colored from blue (N-terminus) to red (C-terminus). Antigenic determinants are highlighted in gray. Major subdomains of the ectodomain, described in the text as subA, subP, subI, and subH, are indicated in panel C.
Crystals 13 01416 g003
Crystals 13 01416 g004
Figure 4. Mapping of antigenic determinants on the monomer of the gB ectodomain reduced to subdomains A, H, and I. (A) T-cell epitopes. (B) B-cell linear epitopes. (C) B-cell conformational epitopes. Monomers are rainbow-colored from blue (N-terminus) to red (C-terminus). Antigenic determinants are highlighted in gray. Major subdomains of the ectodomain, described in the text as subA, subI, and subH, are indicated in panel (A).
Figure 4. Mapping of antigenic determinants on the monomer of the gB ectodomain reduced to subdomains A, H, and I. (A) T-cell epitopes. (B) B-cell linear epitopes. (C) B-cell conformational epitopes. Monomers are rainbow-colored from blue (N-terminus) to red (C-terminus). Antigenic determinants are highlighted in gray. Major subdomains of the ectodomain, described in the text as subA, subI, and subH, are indicated in panel (A).
Crystals 13 01416 g004
Crystals 13 01416 g005
Figure 5. AlphaFold2-built 3D models of subunit vaccine candidates, subI, subA, and subIHA, shown together with the gB ectodomain truncated to subdomains A, H, and I from (Figure 4A). The models are colored according to the model confidence level: pLDDT > 90 (red), pLDDT > 80 (orange), pLDDT > 70 (yellow), and pLDDT > 60 (green), pLDDT > 50 (emerald), pLDDT > 40 (blue), pLDDT > 30 (dark blue).
Figure 5. AlphaFold2-built 3D models of subunit vaccine candidates, subI, subA, and subIHA, shown together with the gB ectodomain truncated to subdomains A, H, and I from (Figure 4A). The models are colored according to the model confidence level: pLDDT > 90 (red), pLDDT > 80 (orange), pLDDT > 70 (yellow), and pLDDT > 60 (green), pLDDT > 50 (emerald), pLDDT > 40 (blue), pLDDT > 30 (dark blue).
Crystals 13 01416 g005
Crystals 13 01416 g006
Figure 6. The behavior of the modeled structures of subunit vaccine candidates subI and subA over the 400 ns MD simulation. Time development of the RMSD and Rg throughout the MD trajectory relative to the AlphaFold2-built models of subI and subA and residual RMSF levels are shown in the top, medium, and bottom panels.
Figure 6. The behavior of the modeled structures of subunit vaccine candidates subI and subA over the 400 ns MD simulation. Time development of the RMSD and Rg throughout the MD trajectory relative to the AlphaFold2-built models of subI and subA and residual RMSF levels are shown in the top, medium, and bottom panels.
Crystals 13 01416 g006
Crystals 13 01416 g007
Figure 7. The behavior of the modeled structure of the trimeric subunit vaccine candidate subIHA over the 400 ns MD simulation. Time development of the RMSD and Rg throughout the MD trajectory relative to the AlphaFold2-built model of subIHA and residual RMSF levels are shown for two independent experiments performed using the Amber-18 simulation package with two different force fields: the ff99SB-ildn (red lines) and ff19SB (blue lines). The shades of colors on the RMSF graphs show different monomers.
Figure 7. The behavior of the modeled structure of the trimeric subunit vaccine candidate subIHA over the 400 ns MD simulation. Time development of the RMSD and Rg throughout the MD trajectory relative to the AlphaFold2-built model of subIHA and residual RMSF levels are shown for two independent experiments performed using the Amber-18 simulation package with two different force fields: the ff99SB-ildn (red lines) and ff19SB (blue lines). The shades of colors on the RMSF graphs show different monomers.
Crystals 13 01416 g007
Crystals 13 01416 g008
Figure 8. Computational in silico Immune Simulation (C-ImmSim) using the suggested subunit vaccine candidates as antigens. (A) Immunoglobulin/antibodies production in response to antigen injections. (B) Production of cytokines and interleukins expressed as ng/mL in response to antigens injections.
Figure 8. Computational in silico Immune Simulation (C-ImmSim) using the suggested subunit vaccine candidates as antigens. (A) Immunoglobulin/antibodies production in response to antigen injections. (B) Production of cytokines and interleukins expressed as ng/mL in response to antigens injections.
Crystals 13 01416 g008
Table 1. Flowchart of the in silico design of the subunit vaccines.
Table 1. Flowchart of the in silico design of the subunit vaccines.
Computational ProceduresPrograms or DatabasesInternet Addresses and/or [References]
1Search for target antigen and its validationUniProtKB, NCBI
Clustal Omega1.4.2		https://www.ebi.ac.uk/uniprot/TrE, https://blast.ncbi.nlm.nih.gov/Blast.cgi, https://pubmed.ncbi.nlm.nih.gov
https://www.ebi.ac.uk/Tools/msa/clustalo/
2Search for crystal structure or construction of a 3D model of the selected proteinProtein Data Bank (PDB), AlphaFold-2https://www.rcsb.org/,
[51]
3Analysis of the oligomeric state, topology, and domain (subdomain) organizationPyMol2.4,
TMHMM		https://pymol.org/,
[52]
4Search for B-cell and T-cell epitopes and assessment of their localizationNetCTL,
ElliPro		[53],
http://tools.iedb.org/ellipro/, [55]
5Analysis of immunogenicity, allergenicity, toxicity, and conservancy of the selected epitopesVaxiJen, AllerTOP, ToxinPred, Epitope Conservancy Analysis[59]
6Modeling of the subunit vaccine candidates AlphaFold2[51]
7Validation of the stability of the modeled polypeptides using MD simulationAmber20[61]
https://ambermd.org/
8Prediction of the host immune responsesC-IMMSIMhttp://kraken.iac.rm.cnr.it/C-IMMSIM, [66]
Table 2. List of Human Herpesvirus species with associated diseases.
Table 2. List of Human Herpesvirus species with associated diseases.
Herpesvirus SpeciesVirus SubfamiliesAssociated Diseases
Human Herpes Virus 1 (HHV-1)/Herpes Simplex virus 1 (HSV-1)αOral and genital herpes
Human Herpes Virus 2/Herpes Simplex virus 2 (HHV-2/HSV-2)αOral and genital herpes
Human Herpes Virus 3 (HHV-3)/Varicella Zoster virus (VZV)αChickenpox, shingles
Human Herpes Virus 4 (HHV-4)/Epstein-Barr virus γInfectious mononucleosis, lymphomas, carcinomas
Human Herpes Virus 5 (HHV-5)/cytomegalovirus (CMV)βAcute respiratory infection, congenital infection, abdominal infections, hepatitis, mucoepidermoid carcinoma
Human Herpes Virus 6 (HHV-6)βNeuroinflammatory diseases (HHV-6A), sixth disease (HHV-6B)
Human Herpes Virus 7 (HHV-7)βChronic fatigue syndrome
Human Herpes Virus 8 (HHV-8)γKaposi’s sarcoma
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Rakitina, T.V.; Smirnova, E.V.; Podshivalov, D.D.; Timofeev, V.I.; Komolov, A.S.; Vlaskina, A.V.; Gaeva, T.N.; Vasilov, R.G.; Dyakova, Y.A.; Kovalchuk, M.V. An Algorithm for the Development of a Recombinant Antiherpetic Subunit Vaccine Combining the Crystal Structure Analysis, AlphaFold2-Based Modeling, and Immunoinformatics. Crystals 2023, 13, 1416. https://doi.org/10.3390/cryst13101416

AMA Style

Rakitina TV, Smirnova EV, Podshivalov DD, Timofeev VI, Komolov AS, Vlaskina AV, Gaeva TN, Vasilov RG, Dyakova YA, Kovalchuk MV. An Algorithm for the Development of a Recombinant Antiherpetic Subunit Vaccine Combining the Crystal Structure Analysis, AlphaFold2-Based Modeling, and Immunoinformatics. Crystals. 2023; 13(10):1416. https://doi.org/10.3390/cryst13101416

Chicago/Turabian Style

Rakitina, Tatiana V., Evgeniya V. Smirnova, David D. Podshivalov, Vladimir I. Timofeev, Aleksandr S. Komolov, Anna V. Vlaskina, Tatiana N. Gaeva, Raif G. Vasilov, Yulia A. Dyakova, and Mikhail V. Kovalchuk. 2023. "An Algorithm for the Development of a Recombinant Antiherpetic Subunit Vaccine Combining the Crystal Structure Analysis, AlphaFold2-Based Modeling, and Immunoinformatics" Crystals 13, no. 10: 1416. https://doi.org/10.3390/cryst13101416

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