Vaccines are one of the most effective interventions to prevent the spreading of infectious diseases.
They trigger the immune system to produce antibodies that protect the body against infection. However, we still lack efficacious vaccines for many important pathogens like the flu or dengue fever.
“When a vaccine doesn’t work well, we tend to think that it’s because the antibodies produced are not protective,” says Bruno Correia, a professor at the Laboratory of Protein Design & Immunoengineering (LPDI) in EPFL’s School of Engineering.
“It’s usually because our immune system is simply making the wrong type of antibodies.” Scientists in Correia’s lab have now developed a strategy to design artificial proteins that very precisely instruct the body’s immune system which antibodies to produce. The study has been published in the journal Science.
Building proteins like Legos
The EPFL team created artificial proteins designed using computational methods. “They don’t exist in nature,” says Che Yang, a Ph.D. student and co-leading author in the study.
“We developed a protein design algorithm called TopoBuilder. It lets you construct proteins virtually as if you were putting Lego bricks together. Assembling artificial proteins that have novel functions is absolutely fascinating.” says Fabian Sesterhenn, a Ph.D. student and co-leading author.
Correia’s team focused on the design of de novo proteins that can result in a vaccine for the respiratory syncytial virus (RSV).
RSV causes serious lung infections and is a leading cause of hospitalization in infants and the elderly. “Despite several decades of research, up to today, there is still no vaccine or cure for respiratory syncytial virus,” says Correia.
The artificial proteins were created in the laboratory and then tested in animal models, and triggered the immune system to produce specific antibodies against weak spots in RSV.
“Our findings are encouraging because they indicate that one day we will be able to design vaccines that target specific viruses more effectively, by prompting the immune system to generate those particular antibodies,” says Correia.
“We still have a lot of work ahead to make the vaccine we developed more effective—this study is a first step in that direction.”
Methods for creating de novo proteins have applications well beyond immunology – they can also be used in various branches of biotechnology to expand the structural and functional range of natural proteins.
“We can now use the protein design tools to create proteins for other biomedical applications such as protein-based drugs or functionalized biomaterials,” concludes Sesterhenn.
Although Human Immunodeficiency Virus (HIV-1) is one of the best-characterized viruses, there is no efficient vaccine against this pathogen so far. Giving credit for notable progress in approaches to antiretroviral therapy that considerably prolongs the lifespan of HIV-infected patients, it should be noted that these are only palliative means to control the virus which cannot stop the HIV-1 pandemic [1,2].
For the most effective control of HIV-1 spread, a prophylactic vaccine should be used widely [3,4]. However, vaccine development is associated with particular well-known issues. First of all, HIV-1 genetic and consequent antigenic drift allows for evasion of the protective effects of the immune system. Therefore, traditional vaccine strategies have failed to protect against the virus [5,6,7].
Development of artificial polyepitope HIV-1 immunogens using a broad range of protective B- and T-cell epitopes from the viral antigens that can induce broadly neutralizing antibodies and responses of cytotoxic (CD8+ CTL) and helper (CD4+ Th) T-lymphocytes is one of the promising strategies for antiviral vaccine design [6,8,9,10,11,12,13].
There are a number of efforts developing artificial polyepitope T-cell immunogens [10,14,15,16,17,18,19,20,21]. Some of them have proven successful in inducing CD4+ T-cell and CD8+ T-cell responses of much greater breadth and magnitude in non-human primates compared to the vaccines containing full-length HIV protein genes [6,10]. Several polyepitope T-cell vaccine candidates have undergone phase I clinical trials [22,23,24].
The development of artificial B-cell HIV-immunogens, including those constructed using epitopes of broadly neutralizing HIV-1 antibodies (bNAbs), is the most complicated problem, since the majority of them recognize conformational epitopes and, significantly more rarely, linear epitopes.
Furthermore, conformational B-cell epitopes on HIV surface glycoproteins are formed by lipids and glycans and their combinations [25,26], which further complicates the design of immunogens capable of inducing the required B-cell response. This task is believed to be solved using peptide mimics of conformational epitopes that can be obtained using combinatorial biology (the phage display technique) .
Concerning studies related to the development of artificial B-cell immunogens, a protein scaffold approach should be mentioned. Such scaffolds can expose one or several epitopes of broadly neutralizing antibodies to provide the most efficient exposure of the desired epitopes to the immune system [28,29,30,31,32].
Epitope scaffolds developed by rational design were able to elicit 4E10 and 2F5-like antibodies in laboratory animals [28,29]. Zhu et al. proposed computationally designed epitopes that mimic carbohydrate-occluded neutralization epitopes (CONEs) of Env through ‘epitope transplantation’, in which the target region is presented on a carrier protein scaffold.
Although a tested anti-CONE serum demonstrated a modest magnitude of inhibitory activity on HIV-1 infectivity, the consistency of the effect against multiple isolates of HIV-1 Env pseudoviruses allows us to suggest that this approach could provide a broad neutralizing antibody response .
Another HIV vaccine strategy is based on the use of soluble stabilized Env trimer spikes for inducing broadly neutralizing antibodies. These trimeric antigens are comprised of cleavage products of gp120 and gp41 subunits forming a native-like Env conformation exposing vulnerable sites recognized by bNAbs [3,34,35,36].
However, as well as scaffolds, trimers could contain undesirable epitopes, diverting the protective humoral immune response [36,37]. To date, several approaches are used to decrease the immunogenicity of such epitopes [38,39,40,41].
Considering all of the above, it seems reasonable to create an immunogen which contains only the HIV-specific epitopes crucial for inducing a protective immune response. This approach focuses the immune response specifically on protective antigenic determinants and excludes the undesirable vaccine epitopes that could induce autoreactive antibodies or antibodies intensifying viral infectivity.
The paper represents the results of a study on constructing and investigating immunogenic properties of an artificial nTBI molecule comprising epitopes recognized by bNAbs 2F5, 10E8 [42,43], and a linear peptide mimic of a conformational epitope recognized by VRC01  (Figure 1).
Eroshkin et al. have previously designed an artificial polyepitope TBI protein (T- and B-cell epitopes containing immunogen) composed of conservative epitopes from Env and Gag HIV-1 and based on a well-known protein structural motif, i.e., a four-helix bundle . TBI included four Th-cell epitopes (amphipathic α-helix) and five B-cell epitopes (regions with flexible hydrophilic loops) [46,47].
The rationale for the TBI design was that combining T- and B-cell epitopes in one construct would stimulate both proper B-cell and T-cell responses and the necessary interplay between B- and T-cells.
The TBI protein had a CD spectra similar to α-helical proteins and showed crystal-yielding capacity, which was demonstrated for the first time in an artificial protein with a predicted tertiary structure .
It was shown that TBI induced both cellular and humoral responses to HIV-1 in immunized mice and rhesus macaques, and TBI-induced antibodies showed virus-neutralizing activity to HIV-1 . Lately, TBI was included into the composition of the CombiHIVvac candidate vaccine that had undergone phase I clinical trials [24,58].
Based on its ability to crystallize, we assumed that the TBI protein structure was similar to that of the natural protein. We decided to design a modified protein based on TBI with an enhanced ability to induce HIV-neutralizing antibodies.
First, in order to increase the yield of recombinant protein, we changed the expression vector to pET21a, which led to an increase of gene expression level and allowed the use of the 6xHis/Ni-NTA system for protein purification. Additionally, we added an E. coli infB gene fragment encoding an N-terminal expressivity tag to its structure .
This protein was referred to as TBI-tag and used for further modifications. At the next step, we replaced three B-cell epitopes from TBI-tag with epitopes recognized by bNAbs. We substituted the Env (255–266), Gag (99–109), and Gag (351–361) epitopes (Figure 2).
The first two epitopes were replaced with peptides from the MPER region, i.e., (NEQELLELDKWASLWNK) and (NWFNITNWLWYIK), recognized by bNAbs 2F5 and 10E8, respectively. Instead of Gag (351–361), we added a phage display-selected linear peptide mimic of the epitope recognized by the VRC01 antibody, into the protein structure .
According to the PSSpred tool modeling data, the epitopes for 10E8 and 2F5 have retained their peculiar α-helical structure, which was shown to be required for their antigenic and immunogenic properties (Figure 2).
T-helper epitopes and spacer sequences between epitopes remained unchanged, since they form amphipathic α-helices which seem to stabilize protein structure (Figure 2). Significance of the helical amphipathicity of epitopes for the recognition of T-cells is evident, since 70% of T-helper epitopes have an α-helical conformation.
Previously, it was shown that TBI elicited HIV-specific T-cell response in mice, which supported the idea that TBI processing as well as presentation of T cell epitopes were adequate .
Since antigen-presenting cells present T-cell epitopes as linear peptides after antigen processing, we suppose that the functional activity of T-helper epitopes in the context of nTBI should have remained at about the same level as in TBI.
Modeling the secondary structure enabled us to assume that accomplished modifications didn’t significantly affect the recombinant protein organization, since the predicted secondary structures of TBI_tag and nTBI were found to be similar (Figure 1 and Figure 2).
To evaluate the secondary structure of the TBI_tag and nTBI proteins, we used a circular dichroism spectroscopic method. CD spectra analysis of the proteins in a 20% trifluoroethanol solution revealed that TBI_tag contains 54% α-helices and 4% β-sheets, which is consistent with theoretical data predicted with PSSpred; 52% and 2%, respectively.
The corresponding predicted values for nTBI were the following: 59% of α-helices and 3% of β-sheets. However, we failed to determine its secondary structure using CD. The probable reason is that the inclusion of the peptide mimic of the VRC01 conformational epitope into an internal area of the molecule TBI_tag resulted in a destabilizing of the protein structure and changing of its physico-chemical properties, since it became prone to aggregation.
Western blot analysis revealed that the mAb VRC01 poorly interacted with the peptide mimic as part of nTBI as compared to the same peptide as part of protein p3 of bacteriophage M13 . Two other peptides, 2F5 and 10E8, included in the nTBI molecule are effectively recognized by their corresponding MPER bNAbs (2F5 and 10E8) (Figure 3A).
Antibody titers of rabbits’ antisera derived after three-fold immunization with both TBI-tag and nTBI were more than 1:3,125,000, which indicates the high immunogenicity of both proteins. New Lav Blot 1 analysis showed that induced antibodies in rabbits by immunization with TBI_tag and nTBI were capable of binding to HIV-1 proteins (Figure 5). Consequently, both immunogens were able to elicit HIV-specific antibodies.
The analysis of virus neutralizing activity of sera from animals immunized with both TBI_tag and nTBI revealed that both proteins elicit antibodies capable of neutralizing tier 1 SF162.LS. Additionally, anti-nTBI IgG neutralizing activity proved to be higher than that of the antibodies induced with TBI_tag.
The IC50 of anti-nTBI antibodies was six times lower than the IC50 for anti-TBI_tag antibodies (Figure 6). To test whether the neutralizing activity of anti-nTBI IgGs from rabbits’ immune sera is mediated by the antibodies which were elicited against substituted epitopes, we performed a peptide competition neutralization assay.
The 10E8 peptide was the most immunogenic, since its inhibition capacity of anti-nTBI IgG significantly differed from control (Figure 7A). Thus, enhancement of virus neutralizing activity of antibodies induced by nTBI seems to be connected to the inclusion of peptide 10E8 in the compound of the TBI protein.
VRC01 mimotope VSWPELYKWTWS contributes poorly to the induction of neutralizing antibodies. When immunized with the nTBI protein, the neutralizing activity of the antibodies induced against this mimotope decreased after the addition of the VSWPELYKWTWS peptide; although the difference between the control group was not significant (Figure 7C).
Probably, this may be explained by a shielding of the peptide mimic by flanking amino acid sequences in the nTBI compound. Thereby, placing the peptide mimetic of the VRC01 epitope into the internal region of nTBI could alter the immunogenicity of this peptide. Importantly, the peptide mimic of the VRC01 epitope efficiently inhibited the binding of bNAbs VRC01 to the SF162 pseudovirus, which is consistent with the literature .
Concerning 2F5 peptide, we failed to demonstrate inhibition of SF162 neutralization by IgGs from rabbits immunized with TBI_tag and nTBI.
The epitope recognized by the bNAb 2F5 was designed at the protein C-terminus, and was followed by an additional six histidine residues. Inclusion of this hexahistidine tag helped streamline protein purification, yet it could also affect the structure and accessibility of the adjacent epitope.
This could be one of the possible reasons why the 2F5 epitope retained antigenicity in the context of the nTBI, but failed to induce 2F5-like Abs (as shown by the competition analysis using the synthetic 2F5 peptide).
It seems that 2F5 epitope heavily relies on its natural environment to expose its immunogenic features . The obtained findings will be considered in the further enhancement of TBI immunogen. It is possible that removing the histidines from the C-terminus of nTBI will result in efficient presentation of the 2F5 peptide.
Another possible explanation of low neutralizing activity of the immune sera could be the animal model that we used for the experiment. Although rabbits can be used for characterization of HIV-1 immunogenicity, they are not a valid model for the analyses of novel immunogens driving an anti-HIV response.
One reason is that they are naturally resistant to HIV infection, and more importantly, sequences of their immunoglobulin genes are too diverged from those of primates. In our future experiments testing the elicitation of neutralizing antibodies, common marmosets will be used as the model animals.
Approach to the development of artificial proteins constructed using conservative T- and B-cell epitopes and their mimics is believed to be promising for the development of vaccines against variable viruses, including HIV.
In such constructs, all of the component helper T cell epitopes may be expected to provide T cell help for antibody production against all of the component B cell epitopes, thus overcoming the limitation of genetic restriction .
In theory, this approach makes it possible to evade the antigenic variability of the virus, focus the immune responses on protective determinants, and exclude from the vaccine compound undesired determinants capable of inducing autoantibodies or antibodies increasing virus infectivity.
In this study, we used a polyepitope-based HIV immunogen design strategy to develop an artificial nTBI protein exposing epitopes recognized by bNAbs 2F5, 10E8, and a phage display-selected peptide mimic of the VRC01 discontinuous epitope.
Antigenic properties of the incorporated peptides were saved in the compound of nTBI, except for the peptide mimic VRC01. nTBI became less soluble as compared to the initial TBI.
This is probably because substitution of the initial TBI B-cell epitopes by new ones negatively affected its conformation, despite the prediction results. However, compared to the initial TBI, nTBI demonstrated a better capacity to induce virus neutralizing antibodies in comparison with the initial TBI, at least for the SF162 pseudovirus the neutralizing activity of nTBI-induced IgGs outperformed that of the TBI_tag-induced IgGs (the IC50 of anti-nTBI antibodies was six times lower than the IC50 of anti-TBI_tag antibodies (Figure 6)).
Competition assay revealed that immunization of rabbits with nTBI induced mainly 10E8-like antibodies, whereas no 2F5 epitope and VRC01 mimotope-induced antibodies were detected. We considered several strategies for boosting the antigenicity and immunogenicity of nTBI.
The peptide mimetic targeted by VRC01 can be placed at the C- or N- terminus of TBI, which may translate into the induction of neutralizing antibodies. Our earlier results  indicate that the peptide mimetic selected using phage display was able to induce neutralizing antibodies, either in the context of the M13 phage or as individual synthetic peptide.
Furthermore, following affinity selection, we obtained many more VRC01 mimetics, and these may be considered viable alternatives for future modifications of TBI.
As for the 2F5 epitope, testing whether the C-terminal hexahistidine tag affects its immunogenicity remains an attractive experiment to do. Also, one may think of increasing the spacers between the epitopes, as this may translate into greater accessibility of the epitopes to B-cell receptors.
Establishing a broader range of TBI derivatives may therefore be highly productive, as this may help identify the variants showing greater activity. Finally, the lead immunogen should be thoroughly tested in various prime-boost immunization schemes.
The ultimate goal of this approach is to obtain an anti-HIV vaccine component that would provide the desired neutralizing immune response. It is possible that such an immunogen will comprise a cocktail of several lead TBI versions.
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More information: New Approach to Design Functional Antibodies for Precision Vaccines, Science (2020). science.sciencemag.org/cgi/doi … 1126/science.aay5051