Abstract
The M protein of group A streptococci (Strep A) is a major virulence determinant and protective antigen. The N-terminal sequence of the protein defines the more than 200 M types of Strep A and also contains epitopes that elicit opsonic antibodies, some of which cross-react with heterologous M types. Current efforts to develop broadly protective M protein-based vaccines are directed at identifying potential cross-protective epitopes located in the N-terminal regions of cluster-related M proteins for use as vaccine antigens. In this study, we have used a comprehensive approach using the recurrent neural network ABCpred and IEDB epitope conservancy analysis tools to predict 16 residue linear B-cell epitopes from 117 clinically relevant M types of Strep A (~88% of global Strep A infections). To examine the immunogenicity of these epitope-based vaccines, nine peptides that together shared ≥60% sequence identity with 37 heterologous M proteins were incorporated into two recombinant hybrid protein vaccines, in which the epitopes were repeated 2 or 3 times, respectively. The combined immune responses of immunized rabbits showed that the vaccines elicited significant levels of antibodies against all nine vaccine epitopes present in homologous N-terminal 1–50 amino acid synthetic M peptides, as well as cross-reactive antibodies against 16 of 37 heterologous M peptides predicted to contain similar epitopes. The epitope-specificity of the cross-reactive antibodies was confirmed by ELISA inhibition assays and functional opsonic activity was assayed in HL-60-based bactericidal assays. The results provide important information for the future design of broadly protective M protein-based Strep A vaccines.
Keywords: Streptococcus pyogenes (S. pyogenes), M protein, Vaccine development, Neural networks, Linear epitopes, Bioinformatics
1. Introduction
Streptococcus pyogenes (group A Streptococcus, Strep A) is a major human pathogen that causes a wide spectrum of diseases, ranging from streptococcal pharyngitis and skin infections to more severe illnesses including necrotizing fasciitis, toxic shock, acute glomerulonephritis, acute rheumatic fever, and rheumatic heart disease (RHD) [1–3]. The greatest global burden of serious disease is RHD, which is disproportionately borne in low- and middle- income countries [4]. Despite previous efforts, there is not a commercially available vaccine against Strep A. Although multiple Strep A antigens have been proposed as potential vaccine candidates [5], only M protein-based vaccines have progressed to clinical studies [6–9]. Multivalent recombinant vaccines composed of tandemly linked M peptides from the N-terminal hypervariable region (HVR) of clinically-relevant M types have been shown to be safe and immunogenic in humans [8,9].
One of the obstacles to the development of multivalent M protein-based vaccines is the multiplicity of M types, which now number more than 200. Based on the classical view of “type-specific” immunity, a multivalent vaccine designed to prevent the majority of infections in diverse geographic regions would of necessity be highly complex. However, recent studies have shown that the M proteins may be grouped into sequence-related clusters [10] and that immunity may be a combination of M cluster-specificity and type-specificity [11,12]. The shared sequences within the N-terminal 50 amino acids (aa) of the M proteins likely result from structural constraints imposed by the requirement to bind human serum proteins, such as factor H and C4BP, which are involved in complement regulation and virulence of Strep A [13,14].
We have previously shown that the sequence and structural similarities within M clusters can be exploited to formulate vaccines containing a minimum number of N-terminal M peptides predicted to cross-react with heterologous M types in the same cluster [12,15]. In these studies, the predictive algorithms included sequence identity, three-dimensional conformation, and chemin formatics of the 1–50 aa or 16–50 aa N-terminal M peptides. In the current study, we have used a different approach to identify linear 16 aa peptides that contain shared epitopes within the N-terminal regions of M proteins from 117 epidemiologically relevant M types of Strep A. The goal of focusing on relatively short epitopes rather than the entire N-terminal region of the M proteins is to reduce the total amount of protein necessary to elicit broadly protective immunity against a significant number of prevalent M types of Strep A. The recurrent neural network ABCpred and IEDB epitope conservancy analysis tools were used to predict linear B-cell epitopes within the 50 aa N-terminal sequences and identify epitopes that are conserved in multiple M types of group A streptococci. Tandem repeats of linear epitopes have been shown to exhibit increased immunogenicity [16–18] compared to nonrepeats of the same sequences. To investigate this effect in the present design effort, two prototype recombinant multivalent vaccines were constructed which shared nine different 16 aa peptides repeated in tandem either two or three times. Rabbits immunized with one of the two different prototype vaccines developed antibodies that cross-reacted with 16/37 heterologous M peptides predicted to contain cross-reactive epitopes. The implications of the results for the future design of a broadly protective vaccine are discussed.
2. Methods
2.1. Linear epitope prediction via machine learning
The ABCpred server [19], a recurrent neural network trained to identify linear B-cell epitopes, was used to predict 16 aa linear epitopes from the N-terminal (1–50 aa) M protein sequences from 117 M types. Using a default ABCpred score of 0.51, linear epitopes were scored using a range from 0 (non-epitope) to 1 (epitope). Predicted linear epitopes with scores closer to 1 are more likely to be linear epitopes based on the dataset of 700 experimentally defined linear epitopes used to train the neural network. To maximize the selection of cross-reactive epitopes, ABCpred linear epitopes were submitted to the IEDB epitope conservancy analysis tool, which searches for sequences similar to user-defined epitope sequences within a set of antigens [20]. A sequence identity threshold of 60% was chosen to identify potential cross-reactive epitopes conserved among heterologous M types, based on previous observations of cross-reactive immune responses among sequence-based clusters of N-terminal M peptides [12,15].
2.2. Vaccine construction, expression, and purification
Based on the calculations, two recombinant protein vaccines were constructed for this study (Fig. 1a). A 9-valent vaccine contained 16 aa peptides predicted to contain cross-reactive M epitopes repeated three times in tandem. The second 12-valent vaccine contained the same nine peptides in the same order but repeated twice. This construct was extended to contain three additional 16 aa peptides in order to assess the maximum number of immunogenic epitopes that could be included in a single protein and to maintain a molecular size that approximated that of the 9-valent vaccine (Fig. 1b). Peptides containing predicted epitopes were selected from a set of epitope sequences that were found in more than one M type (60% linear identity threshold), and did not contain the XENE motif, which is similar to GLKTENE that has been associated with T cell-mediated myocardial infiltrates [21]. Nucleotide pairs encoding the two vaccine proteins were synthesized (GenScript, Piscataway, NJ) to contain the 16 codon fragments in tandem. The synthetic genes, which also contained a T7 promoter and a 3′ His-tag, were ligated into pUC57, which was used to transform E. coli carrying the T7 RNA pol. The recombinant proteins were expressed and then purified by metal chelate chromatography, as previously reported [22].
Fig. 1.
Schematic of the 9-valent (a) and 12-valent (b) epitope-based recombinant vaccines.
2.3. Immunization of rabbits
Two groups of three female New Zealand white rabbits each were immunized I.M. with 100 μg of either the 9-valent trimeric or 12-valent dimeric recombinant protein mixed with AddaVax (wt/vol) with 10 μg monophosphorylated lipid A (MPLA) per dose (adjuvant and TLR4 agonist both from Invivogen, San Diego, CA) at time 0, 4, and 8 weeks. A booster injection was given at 12 weeks and serum was obtained 2 weeks after the final injection by standard methods. All animal procedures were approved by the University of Tennessee Health Science Center Institutional Animal Care and Use Committee.
2.4. Antibody assays
Rabbit antisera were assayed for M peptide antibodies by ELISA, as previously described [23]. Antibody titers were defined as the inverse of last serum dilution that resulted in an O.D. of >0.1. All assays were performed using N-terminal 1–50 aa synthetic M peptides (Peptide2go, Manassas, VA). Peptide-specificity of the antisera was determined using ELISA inhibition assays, as previously described [12]. Briefly, N-terminal M peptides (1–50 aa) containing the 16 aa epitopes in the vaccines were used as solid-phase ELISA antigens. A constant dilution of antiserum, predetermined to yield an O.D. of ~0.8–1.0, was pre-incubated with the homologous or heterologous soluble inhibiting peptide (10 μg/ml) and then added to the wells containing the solid-phase peptide. Control wells were reacted with the same dilution of serum without inhibiting peptide. All reactions were performed in duplicate. Percent inhibition of antibody binding was calculated using the O.D. of the uninhibited serum dilution compared to the O.D. of the reaction mixture containing the inhibitor peptide.
HL-60 opsonophagocytic killing (OPK) assays were performed with a subset of vaccine M types and cross-reactive M types of Strep A, as previously described [24]. The assay mixtures contained HL-60 cells, baby rabbit serum as a source of complement, and pre-immune or immune rabbit serum, diluted 1:4. All assays were performed in quadruplicate. Percent killing was calculated based on the growth of the test organism during a two-hour rotation in pre-immune serum compared to immune serum.
3. Results
3.1. ABCpred analysis of 117 N-terminal M peptides
Our initial assessment was comprehensive and designed to identify all potential cross-reactive epitopes present in N-terminal M sequences from 117 epidemiologically significant [15] Strep A M types. These 117 M types account for approximately 88% of all Strep A infections globally [25]. ABCpred predicted at least one 16 aa linear B-cell epitope in each of the 117 M types analyzed. A total of 472 linear epitopes were predicted, 141 of which were type-specific while the remaining 331 predicted epitope sequences shared at least 60% identity with at least one heterologous M type (Supplemental Excel Document). Using the criteria described above, nine 16 aa peptides were selected to construct a recombinant hybrid protein vaccine, in which the peptides were repeated three times in tandem (Fig. 1a). These nine epitopes were predicted to cross-react with similar epitopes in 37 heterologous M peptides, resulting in predicted immune responses against 46 M types of Strep A. In addition to the 9-valent vaccine, a 12-valent vaccine was constructed that contained the same 9 peptides plus three additional peptides (M94, M104, and M232), all repeated two times, in order to assess the maximum number of immunogenic epitopes that could be included in a single protein and to maintain a molecular size that approximated that of the 9-valent vaccine (Fig. 1b).
3.2. Cross-reactive immunogenicity of the ABCpred vaccines
The 9-valent and 12-valent vaccines were immunogenic in rabbits and elicited antibodies against both the M peptides contained in the vaccines and non-vaccine M peptides predicted to contain cross-reactive epitopes (Fig. 2). The 16 aa peptides from M94, M104, and M232 that were added to the 12-valent vaccine to extend its length were immunogenic and the highest titer observed against each homologous 50 aa M peptide was 3200, 3200, and 800, respectively (data not shown). Because one objective was to compare the immunogenicity of vaccines containing either two or three tandem 16 aa peptides, our analysis of the results was confined to the nine epitopes present in both vaccines. Using a threshold titer of 1600 (16-fold increase in antibody level over pre-immune titers <200), one or both of the vaccines evoked significant levels of antibodies in at least one rabbit against each of the nine N-terminal 50 aa M peptides containing the vaccine epitopes. Although there was considerable variation in antibody responses among the three rabbits immunized with each vaccine (Supplemental Table S1), cross-reactive antibody titers of at least 1600 were observed against 16/37 M peptides predicted to contain epitopes similar to those contained in the vaccines (Fig. 2 and Table 1). A comparison of the 9-valent (trimeric repeating peptides) and 12-valent (dimeric) vaccines revealed that of the 25 reactive peptides (vaccine-specific and cross-reactive), there were 6 peptides (M121, M33, M79, M15, M175, and M77) that had significant antibody levels (≥1600) elicited exclusively by the trimeric vaccine. Conversely, there were 3 peptides (M1, M103, and M238) which had significant antibody levels (≥1600) elicited exclusively by the dimeric epitope configuration (Fig. 2).
Fig. 2.
Vaccine-specific and cross-reactive antibodies evoked by the 9-valent (3× peptide repeats) and 12-valent (2× peptide repeats) ABCpred vaccines. Bars represent the highest titer from one of three rabbits immunized with each of the indicated vaccines. All pre-immune sera had titers of <200. Only the 16 non-vaccine M peptides that resulted in cross-reactive antibody titers of ≥1600 are shown. All six rabbit antisera had titers of <1600 against the remaining 21 peptides (see Table 1).
Table 1.
Cross-reactive immunogenicity of ABCpred M peptide vaccines versus percent sequence identity within the 16 aa predicted epitopes.
| Vaccine M Type (Underlined) and Predicted Cross-reactive M type | Vaccine Sequence and Predicted cross-reactive M type similar AA sequence | % sequence identity | Highest antibody titer1 |
|---|---|---|---|
| M2 | LFEKLDKVEEEHKKVE | 25,600 | |
| M73 | LFDKLEKVEEENKKVK | 75 | 3200 |
| M77 | LLNKLDKVEEEHKKDH | 75 | 1600 |
| M102 | LLNDLDKVKEEHKKDH | 63 | 800 |
| M1102 | LKEKLDKDQEEREKIE | 63 | 800 |
| M175 | LLNKLDKVEEEHKKDH | 75 | 6400 |
| M124 | LFEKLDKVEEEHKKVE | 100 | 6400 |
| M11 | NSLWDENKTLREKQEE | 6400 | |
| M152 | NKLLDENEKLKEKNEE | 63 | 12,800 |
| M42 | NSLWEENKTLREEQEE | 88 | 1600 |
| M44 | DKLSDENDILREKQDE | 63 | 1600 |
| M85 | NSLWDENKTLREKQGE | 94 | 6400 |
| M158 | GKLWDENETLREKQEE | 81 | 6400 |
| M13 | DKHGELLSEYDALKEK | 6400 | |
| M27 | TKHEELLGENDALKEK | 75 | 400 |
| M76 | TKHEELLAEHDALKEK | 75 | 400 |
| M92 | AKHGELLSEYDALKEK | 93 | 12,800 |
| M106 | TKHEELLGENDALKEK | 75 | 400 |
| M110 | ELHEELWKEYDILKEK | 63 | 800 |
| M186 | GSLWQRQREEIDKLKS | 6400 | |
| M43 | GILWLRQKEENDKLKL | 69 | 400 |
| M80 | AHLWYYQKEENDKLKS | 63 | 800 |
| M91 | GNVWYRQHQEIGKLKS | 63 | 800 |
| M123 | ASAWYYQKEENDKLKS | 63 | 800 |
| M178 | GTVWYRQKEEIDKLKS | 75 | 400 |
| M1 | LRHENKDLKARLENAM | 25,600 | |
| M227 | IRHENKDLKARLENAM | 93 | 25,600 |
| M238 | LKAKNEDLKARLENAM | 75 | 12,800 |
| M239 | LRSENQKLKESLENAR | 63 | 400 |
| M121 | PNHPGYTEANNAVLNG | 1600 | |
| M52 | VDHHRYTEANNAVLQG | 69 | 200 |
| M56 | ENHPGYTAAQNGVLSE | 63 | 3200 |
| M64 | RLHPGYTAANNAARNE | 63 | 800 |
| M119 | PNHPRYTDANNAVRNG | 81 | 400 |
| M192 | VYHDRYTEANNAVTQG | 63 | 800 |
| M49 | TDKNGEYLERIGELEE | 12,800 | |
| M9 | QEKSAEYLERIGELEE | 75 | 6400 |
| M15 | KEKNEEYLEKIGEQEE | 69 | 12,800 |
| M118 | TDKNGEYLERIEELEE | 94 | 800 |
| M183 | QEQNGEYLKKIGELEE | 69 | 3200 |
| M33 | FGPLLASTMRDNHNLK | 6400 | |
| M70 | FAPLLANAIRDNNNLT | 63 | 800 |
| M108 | FAPLLAETMRDNNNLR | 75 | 400 |
| M225 | FGPLLESKIRENDNLK | 69 | 400 |
| M79 | TMWKQKAEEAKAKASN | 3200 | |
| M87 | SVWKKKVEEAKEKASK | 63 | 800 |
| M103 | SMWKKKAEEAEAKASK | 75 | 1600 |
| M209 | QMWKQQAEEAKAKVSK | 75 | 400 |
Highest antibody titer from either 9-valent trimeric or 12-valent dimeric vaccine antisera.
M110 also shares sequence with M13; M15 also shares sequence identity with M49.
3.3. Antibody cross-reactivity predictions and sequence identity thresholds
The ABCpred epitopes included in the recombinant 9- and 12- valent vaccines were selected based on a threshold of 60% sequence identity across the entire 16 aa peptide with at least one heterologous N-terminal M protein sequence in the 117 peptides analyzed. In total, 37 M peptides were predicted to cross-react with at least one of the antisera against either the 9- or 12- valent vaccines (Table 1). A comparison of maximum observed antibody titer with percent sequence identity resulted in a correlation coefficient of 0.39 (Pearson, p = 0.01). Based on these results, the actual threshold of sequence identity that resulted in a significant consistent level of cross-reactive antibody binding (titer ≥ 1600) is estimated to be ~65–70%.
3.4. Epitope-specificity of M peptide cross-reactive antibodies
ELISA inhibition experiments were performed to confirm the epitope-specificity of the cross-reactive M antibodies elicited by the recombinant hybrid 9-valent or 12-valent vaccines (Table 2). In these experiments, the solid-phase ELISA antigens were the N-terminal 50 aa synthetic M peptides that contained the 16 aa vaccine peptide sequences. The rabbit antiserum with the highest titer against the solid-phase ELISA antigen was chosen as the test serum. To confirm that each of the nine 16 aa vaccine peptides contained epitopes that were unique and not present in any of the remaining eight 16 aa vaccine peptides and/or the 50 aa test peptides, peptide inhibition experiments were first performed using antiserum against either the 9- or 12-valent vaccine reacted with each of the 9 vaccine peptides as solid-phase antigens and all 9 peptides as inhibitors. In each case, only the M peptide containing the homologous 16 aa predicted epitope inhibited the antibody binding (inhibition ranging from 54 to 88%) while all of the heterologous peptides resulted in <10% inhibition, confirming the specificity of the nine epitopes included in the vaccine (data not shown). Next, to determine if the cross-reactive vaccine antibodies were specific for the predicted cross-reactive sequences in the heterologous M peptides, inhibition experiments were performed using the vaccine peptides as solid-phase ELISA antigens and the cross-reactive peptides as inhibitors (Table 2). In each case, only the peptides predicted to contain epitopes that cross-reacted with the vaccine peptides resulted in significant inhibition of antibody binding. Taken together, these results indicate that the ABCpred vaccine epitopes were immunogenic in rabbits and elicited cross-reactive antibodies against similar epitopes that displayed sufficient sequence identity. Additionally, none of the vaccine antibodies cross-reacted with epitopes in heterologous M peptides that were not predicted based on linear sequence similarities.
Table 2.
Peptide-specificity of cross-reactive antibodies elicited by epitope-based multivalent M vaccines.
| Cross-reactive peptide | % Inhibition of antibody binding to cross-reactive M peptides by vaccine peptides (% sequence identity between vaccine peptide and cross-reactive peptide in a 16 aa window) | ||||||||
| M2* | M11* | M13* | M186* | M1* | M121* | M49* | M33* | M79* | |
| M731 | 87.2 (75) | <10 (25) | <10 (25) | <10 (31) | <10 (25) | <10 (19) | <10 (38) | <10 (19) | <10 (31) |
| M77 | 77.8 (75) | <10 (31) | <10 (25) | <10 (12) | <10 (25) | <10 (19) | <10 (31) | <10 (12) | <10 (25) |
| M175 | 80.4 (75) | <10 (31) | <10 (31) | <10 (25) | <10 (19) | <10 (19) | <10 (38) | <10 (12) | <10 (31) |
| M124 | 25.6 (62) | <10 (38) | <10 (25) | <10 (19) | <10 (25) | <10 (31) | <10 (25) | <10 (12) | <10 (31) |
| M42 | <102 (25) | 71.2 (88) | <10 (31) | <10 (19) | <10 (38) | <10 (13) | <10 (31) | <10 (25) | <10 (31) |
| M85 | <10 (25) | 71.1 (94) | <10 (38) | <10 (31) | <10 (31) | <10 (19) | <10 (38) | <10 (25) | <10 (25) |
| M158 | <10 (19) | 80.2 (81) | <10 (38) | <10 (33) | <10 (31) | <10 (13) | <10 (38) | <10 (25) | <10 (19) |
| M44 | <10 (25) | 43.2 (38) | <10 (38) | <10 (25) | <10 (25) | <10 (13) | <10 (38) | <10 (12) | <10 (19) |
| M92 | <10 (44) | <10 (38) | 80.4 (94) | <10 (25) | <10 (31) | <10 (25) | <10 (38) | <10 (19) | <10 (13) |
| M227 | <10 (19) | <10 (38) | <10 (25) | <10 (25) | 84.3 (94) | <10 (6) | <10 (31) | <10 (19) | <10 (38) |
| M238 | <10 (13) | <10 (44) | <10 (31) | <10 (31) | 85.3 (75) | <10 (19) | <10 (19) | <10 (25) | <10 (38) |
| M56 | <10 (19) | <10 (25) | <10 (44) | <10 (19) | <10 (44) | 33.3 (63) | <10 (44) | <10 (19) | <10 (13) |
| M9 | <10 (31) | <10 (38) | <10 (38) | <10 (25) | <10 (19) | <10 (25) | 84.2 (75) | <10 (6) | <10 (38) |
| M183 | <10 (31) | <10 (31) | <10 (31) | <10 (38) | <10 (31) | <10 (19) | 38.3 (69) | <10 (13) | <10 (25) |
| M15 | <10 (25) | <10 (63) | <10 (44) | <10 (38) | <10 (38) | <10 (19) | 85.5 (69) | <10 (19) | <10 (25) |
| M103 | <10 (31) | <10 (13) | <10 (19) | <10 (25) | <10 (19) | <10 (13) | <10 (19) | <10 (13) | 80.6 (75) |
ELISA peptides and inhibiting peptides were N-terminal 1–50 aa synthetic peptides containing the 16 aa vaccine sequences.
Percent inhibition ranged from 0 to <10%.
M type included in 9-valent trimeric vaccine and 12-valent dimeric vaccine.
3.5. Functional bactericidal activity of antibodies against epitope-based vaccines
To determine the level of functional activity of the antisera against the epitope-based vaccines, opsonic activity was assessed in in vitro bactericidal assays with the vaccine M types and a subset of the cross-reactive M types of Strep A (Table 3). In these assays, we selected the immune sera with the highest peptide antibody levels to illustrate functional antibody activity. HL-60 cells served as the effector phagocytes and baby rabbit serum was the source of complement. The average percent killing of the M types of Strep A represented in the vaccine averaged 41% (range 0–92). The average killing of the cross-reactive M types tested was 21% (range 0–41).
Table 3.
Maximum observed opsonophagocytic killing of a subset of vaccine and non-vaccine M types of Strep A promoted by rabbit antisera against either 9-valent or 12-valent epitope-based vaccines.
| Vaccine M Types | % Killing (±S.D.) | Non-vaccine M Types | % Killing (±S.D.) |
|---|---|---|---|
| M2 | 0 | M15 | 16 ± 9 |
| M11 | 48 ± 8 | M9 | 14 ± 5 |
| M13 | 0 | M85 | 15 ± 34 |
| M186 | 31 ± 3 | M92 | 0 |
| M1 | 481 ± 11 | M73 | 41 ± 17 |
| M121 | 71 ± 5 | M42 | 23 ± 21 |
| M49 | 19 ± 5 | M238 | 25 ± 13 |
| M33 | 93 ± 1 | M56 | 35 ± 4 |
| M79 | 601 ± 9 |
Assays performed with 12-valent vaccine antiserum. All other assays performed with one antiserum against the 9-valent vaccine.
4. Discussion
Our approach to the development of Strep A vaccines has focused on the N-terminal region of the surface M proteins. Previous studies have shown that peptides as short as 10 aa coupled to a carrier are immunogenic and elicit opsonic antibodies against the homologous M type of Strep A [26,27]. Multivalent M protein-based vaccines composed of N-terminal M peptides ranging in size from 35 to 50 aa have been shown to elicit opsonic antibodies in animals [22,28,29] and humans [7–9] and to protect animals from lethal intranasal, soft tissue, and systemic challenge infections [30]. A recent analysis indicated that the coverage of the 26- and 30-valent M protein-based vaccines may be sufficient in high-income countries; however, coverage in low- and middle-income countries, which have the greatest burden of serious infections, complications, and morbidity, is predicted to be low [31]. Therefore, there is a need to develop more broadly protective vaccines that will have an impact on the global incidence of Strep A infections and their complications.
Our previous work used structure-based approaches to the development of broadly protective M protein-based vaccines, including analysis of calculated three-dimensional structures of sequence-related M peptides to identify potential cross-reactive vaccine antigens [12] and the sequence identity of polar amino acids within the heptad repeats in coiled-coil regions that are found to aid in the prediction of the binding of cross-reactive antibodies [15]. In the current work, we took a direct approach to identify the M epitopes that are shared among multiple M proteins and expressed the 16 aa peptides containing the shared epitopes in hybrid proteins. The neural network ABCPred identified linear B-cell epitopes within the N-termini of the M proteins and, in conjunction with the IEDB epitope conservancy analysis tool, enabled the calculation of which epitopes are conserved and likely to elicit cross-reactive antibodies. Because our main goal was to identify potential cross-reactive epitopes, we focused our analysis on positive antibody responses to gain a better understanding of the linear sequences of heterologous cross-reactive peptides. The combined immunogenicity results from the 9- and 12-valent vaccines showed that cross-reactive antibodies (titer ≥ 1600) were elicited against 16/37 peptides predicted to contain similar epitopes based on a sequence identity threshold of 60% within a 16 aa peptide. Further analysis of the observed antibody levels indicated that sequence identity of 65–70% may be a more reliable predictor of cross-reactive immunogenicity. Within these parameters, we also observed exceptions to this prediction. For example, the M2 vaccine peptide and the cross-reactive M124 peptide share 100% sequence identity yet the ELISA titers were discordant (25,600 vs.6400, respectively, Table 1). Additionally, the M2 cross-reactive peptides M77 and M175 have the same sequence but the vaccine antibody titers against each were 1600 and 6400, respectively. The predicted cross-reactive epitopes were present in different locations within the N-terminal 1–50 aa ELISA peptides, which may have altered the conformations of the cross-reactive epitopes or their accessibility for antibody binding.
Previous studies have shown that increasing the epitope density of fusion proteins is associated with greater immunogenicity and higher antibody affinity [16–18]. In the current study, there was not an obvious effect on overall immunogenicity or cross-reactive immunogenicity comparing the results from vaccines that contained 2 or 3 repeated peptide sequences, although the highly variable immune responses among the three rabbits in each group precluded a formal comparison.
The results of this study provide important information to guide the design and formulation of future broadly protective M protein-based vaccines. Although low levels of opsonic antibodies were detected against several homologous and cross-reactive M types of Strep A, there was also some discordance between peptide antibody levels and functional antibody activity. In addition, the observed levels of opsonophagocytic killing were lower than previously obtained with rabbit antisera against multivalent vaccines containing N-terminal 1–50 M peptides [24]. In the previous study, the mean level of killing using 21 different M types of Strep A was 66% (range 23–98%). In the present study, the mean level of killing observed with vaccine M types was 41% (range 0–92%). Opsonization of bacteria requires a threshold level of antibody binding to the surface M protein [32] necessary for activation of the classical complement pathway and deposition of C3b. The short peptide sequences contained in the vaccines may have elicited a limited repertoire of antibodies insufficient to overcome the counteracting effects of complement regulatory proteins, such as C4BP and factor H [33]. Additionally, the linear vaccine epitopes stimulated production of antibodies that reacted with the 50 aa peptides but many of the functional M epitopes on the surface of the bacteria may be conformational and defined by the alpha-helical structure of the native proteins. This may at least partially explain why the M2 peptide antibody titer was 25,600 but there was no detectable killing of the M2 bacteria (Tables 1 and 3). Similarly, the antibody titer against the M13 peptide was 6400 but the antibodies did not promote killing of M13 bacteria. These observations indicate that future vaccine design might benefit from the following considerations:
the inclusion of multiple cross-reactive epitopes within the N-terminal regions of the M proteins to ensure a critical threshold of antibody binding,
identification and selection of cross-reactive epitopes located in regions of the N-terminus of M proteins predicted to have high alpha-helical potential and designing the hybrid vaccines to retain the heptad periodicity to generate antibodies more likely to recognize epitopes in their native conformations,
the identification and/or addition of native or universal T-helper epitopes to facilitate consistent antibody responses and higher affinity cross-reactive antibodies, and
an assessment of different adjuvants to optimize the immunogenicity of multivalent vaccines containing limited peptide sequences from multiple M proteins.
Based on the results of this initial comprehensive assessment of shared M protein epitopes and the immunogenicity of multivalent epitope-based vaccines, future development efforts could result in a broadly efficacious vaccine that could have a significant impact on the health of millions of people around the world.
Supplementary Material
Acknowledgments
Funding
Supported by research funds from the National Institutes of Health AI-R01AI132117.
Footnotes
Declaration of Competing Interest
The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: J.B.D. is the inventor of certain technologies related to the development of Strep A vaccines. The technology has been licensed from the University of Tennessee Research Foundation to Vaxent, LLC, of which J.B.D. is a member and Chief Scientific Officer. All other authors declare that they have no conflicts of interest with the contents of this article.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.org/10.1016/j.vaccine.2021.01.075.
References
- [1].Carapetis JR, Steer AC, Mulholland EK, Weber M. The global burden of group A streptococcal diseases. Lancet Infect Dis 2005. 10.1016/S1473-3099(05)70267-X. [DOI] [PubMed] [Google Scholar]
- [2].Donaldson PMW, Naylor B, Lowe JW, Gouldesbrough DR. Rapidly fatal necrotising fasciitis caused by Streptococcus pyogenes. J Clin Pathol 1993. 10.1136/jcp.46.7.617. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [3].Stevens DL. Streptococcal toxic-shock syndrome: spectrum of disease, pathogenesis, and new concepts in treatment. Emerg Infect Dis 1995. 10.3201/eid0103.950301. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [4].Watkins DA, Johnson CO, Colquhoun SM, Karthikeyan G, Beaton A, Bukhman G, et al. Global, regional, & national burden of rheumatic heart disease, 1990–2015. N Engl J Med 2017. 10.1056/NEJMoa1603693. [DOI] [PubMed] [Google Scholar]
- [5].JDale JB, Batzloff MR, Cleary PP, Courtney HS, Good MF, Grandi G, et al. Current approaches to group A streptococcal vaccine development; 2016. [PubMed] [Google Scholar]
- [6].Sekuloski S, Batzloff MR, Griffin P, Parsonage W, Elliott S, Hartas J, et al. Evaluation of safety and immunogenicity of a group a streptococcus vaccine candidate (mj8vax) in a randomized clinical trial. PLoS ONE 2018. 10.1371/journal.pone.0198658. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [7].Kotloff KL, Corretti M, Palmer K, Campbell JD, Reddish MA, Hu MC, et al. Safety and immunogenicity of a recombinant multivalent group A streptococcal vaccine in healthy adults: Phase 1 trial. J Am Med Assoc 2004. 10.1001/jama.292.6.709. [DOI] [PubMed] [Google Scholar]
- [8].McNeil S a, Halperin S a, Langley JM, Smith B, Warren A, Sharratt GP, et al. Safety and immunogenicity of 26-valent group a streptococcus vaccine in healthy adult volunteers. Clin Infect Dis 2005. 10.1086/444458. [DOI] [PubMed] [Google Scholar]
- [9].Pastural É, McNeil SA, MacKinnon-Cameron D, Ye L, Langley JM, Stewart R, et al. Safety and immunogenicity of a 30-valent M protein-based group a streptococcal vaccine in healthy adult volunteers: a randomized, controlled phase I study. Vaccine 2020. 10.1016/j.vaccine.2019.12.005. [DOI] [PubMed] [Google Scholar]
- [10].Sanderson-Smith M, De Oliveira DMP, Guglielmini J, McMillan DJ, Vu T, Holien JK, et al. A systematic and functional classification of Streptococcus pyogenes that serves as a new tool for molecular typing and vaccine development. J Infect Dis 2014. 10.1093/infdis/jiu260. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [11].Frost HR, Laho D, Sanderson-Smith ML, Licciardi P, Donath S, Curtis N, et al. Immune cross-opsonization within emm clusters following group A streptococcus skin infection: broadening the scope of type-specific immunity. Clin Infect Dis 2017. 10.1093/cid/cix599. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [12].Dale JB, Smeesters PR, Courtney HS, Penfound TA, Hohn CM, Smith JC, et al. Structure-based design of broadly protective group a streptococcal M protein-based vaccines. Vaccine 2017. 10.1016/j.vaccine.2016.11.065. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [13].Buffalo CZ, Bahn-Suh AJ, Hirakis SP, Biswas T, Amaro RE, Nizet V, et al. Conserved patterns hidden within group A Streptococcus M protein hypervariability recognize human C4b-binding protein. Nat Microbiol 2016. 10.1038/nmicrobiol.2016.155. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [14].Gustafsson MCU, Lannergård J, Nilsson OR, Kristensen BM, Olsen JE, Harris CL, et al. Factor H binds to the hypervariable region of many streptococcus pyogenes M proteins but does not promote phagocytosis resistance or acute virulence. PLoS Pathog 2013. 10.1371/journal.ppat.1003323. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [15].Aranha MP, Penfound TA, Spencer JA, Agarwal R, Baudry J, Dale JB, et al. Structure-based group A streptococcal vaccine design: helical wheel homology predicts antibody cross-reactivity among streptococcal M protein-derived peptides. J Biol Chem 2020. 10.1074/jbc.RA119.011258. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [16].Zheng B, Graham FL, Johnson DC, Hanke T, McDermott MR, Prevec L. Immunogenicity in mice of tandem repeats of an epitope from herpes simplex gD protein when expressed by recombinant adenovirus vectors. Vaccine 1993. 10.1016/0264-410X(93)90042-V. [DOI] [PubMed] [Google Scholar]
- [17].Sun Z, Zhu Y, Wang Q, Ye L, Dai Y, Su S, et al. An immunogen containing four tandem 10E8 epitope repeats with exposed key residues induces antibodies that neutralize HIV-1 and activates an ADCC reporter gene. Emerg Microbes Infect 2016. 10.1038/emi.2016.86. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [18].Liu W, Peng Z, Liu Z, Lu Y, Ding J, Chen YH. High epitope density in a single recombinant protein molecule of the extracellular domain of influenza A virus M2 protein significantly enhances protective immunity. Vaccine 2004. 10.1016/j.vaccine.2004.05.028. [DOI] [PubMed] [Google Scholar]
- [19].Saha S, Raghava GPS. Prediction of continuous B-cell epitopes in an antigen using recurrent neural network. Proteins Struct Funct Genet 2006. 10.1002/prot.21078. [DOI] [PubMed] [Google Scholar]
- [20].Bui HH, Sidney J, Li W, Fusseder N, Sette A. Development of an epitope conservancy analysis tool to facilitate the design of epitope-based diagnostics and vaccines. BMC Bioinf 2007. 10.1186/1471-2105-8-361. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [21].Cunningham MW, Antone SM, Smart M, Liu R, Kosanke S. Molecular analysis of human cardiac myosin-cross-reactive B- and T-cell epitopes of the group A streptococcal M5 protein. Infect Immun 1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [22].Dale JB. Multivalent group a streptococcal vaccine designed to optimize the immunogenicity of six tandem M protein fragments. Vaccine 1999. 10.1016/S0264-410X(98)00150-9. [DOI] [PubMed] [Google Scholar]
- [23].Hall MA, Stroop SD, Hu MC, Walls MA, Reddish MA, Burt DS, et al. Intranasal immunization with multivalent group A streptococcal vaccines protects mice against intranasal challenge infections. Infect Immun 2004. 10.1128/IAI.72.5.2507-2512.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [24].Salehi S, Hohn CM, Penfound TA, Dale JB. Development of an opsonophagocytic killing assay using HL-60 cells for detection of functional antibodies against streptococcus pyogenes. MSphere 2018. 10.1128/msphere.00617-18. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [25].Tariq Salie, Engel M. Rapid Review of Global Emm Types 2020. (Accessed October 27, 2020) 10.25375/uct.13056074.v1. [DOI]
- [26].Dale JB, Beachey EH. Localization of protective epitopes of the amino terminus of type 5 streptococcal M protein. J Exp Med 1986. 10.1084/jem.163.5.1191. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [27].Beachey EH, Seyer JM, Dale JB. Protective immunogenicity and T lymphocyte specificity of a trivalent hybird peptide containing NH2-terminal sequences of types 5, 6, and 24 M proteins synthesized in tandem. J Exp Med 1987. 10.1084/jem.166.3.647. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [28].Dale JB, Penfound TA, Chiang EY, Walton WJ. New 30-valent M protein-based vaccine evokes cross-opsonic antibodies against non-vaccine serotypes of group A streptococci. Vaccine 2011. 10.1016/j.vaccine.2011.09.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [29].Hu MC, Walls MA, Stroop SD, Reddish MA, Beall B, Dale JB. Immunogenicity of a 26-valent group A streptococcal vaccine. Infect Immun 2002. 10.1128/IAI.70.4.2171-2177.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [30].Penfound TA, Chiang EY, Ahmed EA, Dale JB. Protective efficacy of group A streptococcal vaccines containing type-specific and conserved M protein epitopes. Vaccine 2010. 10.1016/j.vaccine.2010.05.018. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [31].Steer AC, Law I, Matatolu L, Beall BW, Carapetis JR. Global emm type distribution of group A streptococci: systematic review and implications for vaccine development. Lancet Infect Dis 2009. 10.1016/S1473-3099(09)70178-1. [DOI] [PubMed] [Google Scholar]
- [32].Beachey EH, Stollerman GH, Johnson RH, Ofek I, Bisno AL. Human immune response to immunization with a structurally defined polypeptide fragment of streptococcal m protein. J Exp Med 1979. 10.1084/jem.150.4.862. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [33].Walker MJ, Barnett TC, McArthur JD, Cole JN, Gillen CM, Henningham A, et al. Disease manifestations and pathogenic mechanisms of group A Streptococcus. Clin Microbiol Rev 2014. 10.1128/CMR.00101-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
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