Immunogenicity and Safety of a Chemically Synthesized Divalent Group A Streptococcal Vaccine

Background Group A streptococcus (GAS) infections and poststreptococcal sequelae remain a health problem worldwide, which necessitates searching for an effective vaccine, while no licensed GAS vaccine is available. We have developed a divalent peptide vaccine composed of 84 amino acids to cover the main GAS serotypes (M1 and M12 streptococci) in China, and herein, we aimed to evaluate immunogenicity and safety of this vaccine. Methods Mice were immunized with the vaccine. ELISA, indirect bactericidal test, and immunofluorescent assay were used to study immunogenicity. GAS challenge assay was used to test the protective effect. Safety was tested by histopathological analysis. Results Immunized group mice (n=16) developed higher titer antibody after immunization than nonimmunized group mice (n=16) did. This antibody can deposit on the surface of GAS and promote killing of GAS, resulting in 93.1% decrease of M1 GAS and 89.5% of M12 GAS. When challenged with M1 and M12 streptococci, immunized group mice had a higher survival rate (87.5% and 75%) than nonimmunized group mice (37.5% and 25%). No autoimmune reactions were detected on organs of mice. Conclusion The results suggest that this vaccine shows fair immunogenicity and safety, which will lead our research on GAS vaccine into clinical trial.


Introduction
Group A streptococcus (GAS) is an exclusive human pathogen causing a wide spectrum of diseases varying from uncomplicated pharyngitis and pyoderma to lethal invasive diseases, such as bacteremia, streptococcal toxic shock syndrome, and necrotizing fasciitis. GAS infection also results in several autoimmune sequelae including rheumatic fever, rheumatic heart disease, and poststreptococcal glomerulonephritis [1]. ese diseases a ect many indigenous communities of developed countries and many developing countries where health care and antibiotic treatment are poor. However, there is a reemergence of GAS infection in industrialized countries where the incidence of GAS disease has declined in the past decades [2,3]. GAS diseases are important causes of morbidity and mortality in the world [4], which highlight the need for a safe and e ective vaccine.
E orts to develop a GAS vaccine have lasted for decades. Most strategies focus on the M protein, a surface protein with a high changeable amino terminal and a conserved carboxyl terminal [5]. M protein is an important virulence determinant and protective antigen of GAS. Previous studies have revealed that epitopes from the amino terminal of M protein are able to elicit type-speci c bactericidal antibody which has been the basis for the design of complex multivalent amino terminal vaccines including the 26-valent [6] vaccine and the 30-valent [7] vaccine, and that epitopes from carboxyl terminal are capable of evoking broad serotype antibody which provided the rationale for creating conserved carboxyl terminal M protein vaccines including J8-DT [8] and StreptInCor [9].
However, e ort to develop a GAS vaccine has been hindered by the diversity of streptococcal serotypes. More than 200 emm types have been identi ed; the distribution of GAS emm types varies from region to region [10]. Infection with one strain does not provide immunity against another strain. e 26-valent vaccine covered 90% of the serotypes in the USA but only 39% in Africa and 24% in the Paci c region [10].
Although the 30-valent vaccine evoked cross-opsonic antibodies against 24 of 40 nonvaccine serotypes [7], with more than 200 emm types, it is far away from being e ective worldwide. A vaccine covering all the serotypes is unlikely to be developed, while people in GAS endemic areas are in badly need of an e ective vaccine; thus geographical regionspeci c vaccines that encompass the multiple N-terminal epitopes of dominant serotype GAS in a given area may be feasible.
In China, emm1 and emm12 GAS are most prevalent according to our [11] and others' [12][13][14] investigations. In 2011, an emm12 GAS strain caused large outbreaks of scarlet fever in Shanghai [15] and Hong Kong [15,16], resulting in two children's death in Hong Kong.
us, we developed a divalent 84 amino acid peptide vaccine composed of three subunits: a 35 amino acid sequence from N terminal of M1 protein (M1 peptide), a 35 amino acid sequence from N terminal of M12 protein (M12 peptide), and the J14 peptide shared by conserved C terminal of these two M proteins [17].
is vaccine was demonstrated to be able to elicit high titer opsonic antibody in mice in our previous work, but two questions remain unanswered. First, is this antibody protective against GAS challenge? Second, does this vaccine induce deleterious reaction? is paper aims to answer these questions.

Chemically Synthesized Peptide Vaccine, GAS Strains, and
Mice.
e amino acid sequence of the divalent peptide vaccine is GFANQTEVKANGDGNPREVIEDLAANNPAIQNIRL-DHSDLVAEKQRLEDLGQKFERLKQRSELYLQQYYDASR-EAKKQVEKALE. e vaccine ( Figure 1) and its three subunit peptides were synthesized using a 9-a-uorenylmethoxycarbonyl (Fmoc) solid-phase strategy and puri ed by reverse-phase high-pressure liquid chromatography (GL Biochem, China). Speci c pathogen-free 5-to 6-week-old female BALB/c mice were purchased from and maintained in a SPF animal room of Laboratory Animal Center, Sun Yat-sen University, China. e mice were housed in cages with free access to food and water. All procedures were reviewed and approved by the Animal Ethical and Welfare Committee of Sun Yat-sen University (project no. IACUC-DB-16-0407). e emm1 and emm12 GAS were isolated from human tonsil or throat samples, identi ed by emm gene ampli cation and sequencing, and preserved in our laboratory as previously described [17].

Immunization.
irty-two female BALB/c mice were divided into two groups: immunized group and control group. Immunized group mice were immunized subcutaneously with 100 μL of antigen-adjuvant suspension containing 20 μg peptide vaccine emulsi ed in complete Freund's adjuvant (CFA) (Sigma). ree booster immunizations were given on day 21, day 31, and day 41 after rst immunization with 20 μg peptide vaccine emulsi ed in incomplete Freund's adjuvant (IFA) (Sigma). Control group mice received injections with the mixture of adjuvant (CFA or IFA) and PBS on the same schedule. Blood samples were obtained 1 day prior to the rst immunization and 14 days after the nal booster by retroorbital puncture under light anesthesia, and then sera were collected and stored at −20°C.

Enzyme-Linked Immunosorbent Assay (ELISA).
Serum antibody titers were measured by ELISA with the divalent peptide as solid-phase antigens. Polyvinyl plates (CORNING) were coated with 100 μL of 10 μg/ml divalent peptide in 50 mM carbonate coating bu er (pH 9.6) overnight at 4°C and blocked with 5% BSA in PBS for 1 h at 37°C. e plates were then washed with 0.2% Tween-20 in PBS (PBST) for three times, and serial twofold dilutions of the sera in PBS, starting at an initial dilution of 1 : 200 to a nal dilution of 1 : 2,04,800, were added to the plates (100 μL/well). After 1 h at 37°C, the plates were washed with PBST for three times and incubated with horseradish peroxidase-conjugated goat antimouse IgG at a 1 : 20,000 dilution (100 μL/well) at 37°C for 1 h. e plates were washed and then incubated with 3,3′,5,5′-tetramethylbenzidine (Beyotime) for 30 min at room temperature. e reaction was stopped with 50 μL of 2 M H 2 SO 4 , and the absorbance was measured at 450 nm with an ELISA plate reader (BioTek Eon). Titers were de ned as the reciprocal of the highest dilution of sera resulting in an optical density (OD) 2 times higher than the average OD of wells containing control group mice sera diluted at 1 : 100. e M1 peptide, the M12 peptide, and the J14 peptide were also used as ELISA solid-phase antigens to test sera antibody responses to them, respectively.

Indirect Bactericidal
Tests. Emm1 and emm12 GAS were grown, respectively, in 5 mL of Todd-Hewitt Broth overnight at 37°C and then serially diluted to 10 5 in PBS. For each individual mouse, 50 μL of serum was mixed with 50 μL of the bacterial dilution and incubated for 20 min at room temperature, and then 400 μL of nonopsonic heparinized human donor blood was added. All donor blood samples had been tested to support the growth of GAS before the assay. e mixtures were incubated on shaking table at room temperature for 3 h, and 50 μL of the mixtures was cultured on blood agar plate in duplicate overnight at 37°C. e number of colony-forming unit (CFU) on each plate was counted. e results were expressed as percent killing, which was calculated by using the following formula: (1−(mean CFU in the presence of immunized mouse serum)/(mean CFU in the presence of control mouse serum)) × 100. 2.5. Immuno uorescent Assays. Emm1 and emm12 GAS were grown, respectively, in 5 mL of Todd-Hewitt Broth overnight at 37°C, centrifuged at 3000 g for 10 min, and then resuspended in 5 mL of PBS. One hundred microliter of the bacterial suspension and 400 μL of sera of mice at a 1 : 200 dilution in PBS were mixed in EP tubes and incubated at room temperature for 1 h. e mixture was then centrifuged at 3000 g for 10 min and resuspended in 400 μL of PBS, which are repeated for three times to wash the bacteria. After the third centrifugation, PBS was removed and 400 μL of 5% bovine serum albumin was added to resuspend the bacteria. After 1 h at room temperature, the bacterial suspension was centrifuged and washed with PBS for three times. PBS was removed, and the bacteria were resuspended in DyLight 488conjugated goat antimouse IgG secondary antibody at a 1 : 400 dilution in PBS and incubated in dark at room temperature for 1 h. e bacterial suspension was centrifuged and washed and resuspended in 100 μL of PBS. Ten microliter of the bacterial suspension was smeared across microscope slides and air-dried at room temperature. Slides were examined using a uorescent microscope (Leica).

2.6.
Challenge. Twenty-one days after the last immunization, 8 mice in the immunized group and 8 mice in the control group were challenged by intraperitoneal injection of 1.0 × 10 8 CFU M1 GAS in 100 μL PBS. Another 16 mice were challenged with the equivalent amount of M12 GAS. Mice in each group were observed for 15 days, and mortality was recorded to determine survival rates.

Histopathological Analysis.
Four mice receiving a highdose immunization and 4 mice in the control group were euthanized 6 months after the last immunization. e organs were collected, maintained in 4% formaldehyde solution, and evaluated after hematoxylin and eosin stain.

Statistical
Analysis. Antibody titer and bacterial CFU data were analyzed by Student's t-test, and survival data were analyzed by log-rank Kaplan-Meier test, both using GraphPad Prism 5 software. P values were considered signi cant at <0.05.

e Chemically Synthesized Divalent Vaccine Elicits High
Titer Antibody in Mice. ELISA assay was performed to evaluate immunogenicity of the vaccine. As shown in Figure 2, all the mice of the immunized group developed high titer antibody after three immunizing boosts, ranging from 1 : 6400 to 1 : 2,04,800, signi cantly greater than the control group mice given only PBS and adjuvant. Likewise, antibody titers of immunized group mice sera against the M1 peptide (1 : 1600 to 1 : 10,2400), the M12 peptide (1 : 800 to 1 : 51,200), and the J14 peptide (1 : 400 to 1 : 6400) are all higher than that of control group mice sera (P < 0.05, resp.) (Figure 2).

Bactericidal Activity of Antisera against Type 2
Streptococci. To test whether the antibody had the potential to kill M1 and M12 GAS, sera on day 55 were used to perform a bactericidal assay that measured GAS colonyforming units. As can be seen in Table 1, sera from immunized mice had signi cant level opsonic activity against these two type streptococci, resulting in 93.1% and 89.5% decrease of CFU, respectively.

Antibody Bindsto theSurfaceofGAS.
Immuno uorescence was performed to visualize whether the antibody could connect to the streptococci. As Figure 3 shows, uorescence appeared on the surface of emm1 and emm12 streptococci when antiserum of immunized mice were used as rst antibody, while no uorescence could be seen when antiserum of nonimmunized mice was used, which indicates that the divalent vaccine could elicit antibody that bound to the surface of the streptococci.

e Synthesized Divalent Vaccine Immunization Protects Mice against GAS Infection.
e synthesized divalent vaccine immunization elicited high titer antibody, and this antibody could bind to M1 and M12 streptococci and facilitate these streptococci to be killed, as described above. To test whether the antibody can protect mice, both immunized mice and nonimmunized mice were challenged with M1 and M12 streptococci, respectively. e immunized mice had 87.5% and 75% survival rate among 15 days, respectively (Figure 4), higher than that of nonimmunized mice (37.5% and 25%) (P < 0.05).

e Chemically Synthesized Divalent Vaccine Did Not Induce Autoimmune Reactions.
e safety of the synthesized divalent vaccine was examined by histological evaluation of tissues collected from mice 6 months after their last high-dose immunization. e results showed that no autoimmune reactions were observed in the heart and other organs ( Figure 5).

Discussion
GAS is prevailing worldwide, and GAS-related diseases remain an important public health problem in both developed and developing countries. Vaccine is a feasible way to ght against the bacterium. Many research groups are trying to develop e ective vaccines. eir strategies are based on di erent virulence factors, including GAS carbohydrate [18], serum opacity factor [19], GAS C5a peptidase [20,21], and M protein. M protein is the major virulence factor of GAS, and currently research on M protein vaccine goes ahead of other virulence factors.
We developed the divalent vaccine based on M protein of emm1 and emm12 strains. Previously, we used molecular clone technology to synthesize the 84 amino acid peptide vaccine, while in this study, the peptide was synthesized by solid-phase peptide synthesis which is used by other research groups. We assessed the immunogenicity and bactericidal activity of the chemically synthesized peptide by ELISA assay and indirectly bactericidal assay, and this peptide shows similar immunological characteristics to the biosynthetic Canadian Journal of Infectious Diseases and Medical Microbiology one used in our previous research. en, we explored how the antibody is bactericidal against GAS by immuno uorescence assay, and it is demonstrated that the antibody is able to deposit on the surface of GAS, which is a potential reason for the bactericidal activity of the antibody. e assays mentioned above were all in vitro experiments, and we also performed in vivo experiment to demonstrate that the vaccine was capable of protecting mice against GAS challenge. e result corresponded to our expectation because the indirect bactericidal assay had shown that the antibody promoted killing of GAS. One major concern about GAS vaccine based on M protein is the possibility of inducing autoimmune sequelae. Here, no autoimmune reactions and other pathological lesions were observed on mice receiving high-dose immunization, indicating that this vaccine is safe in mice.
us, the questions raised on introduction were addressed; that is, this chemically synthesized divalent vaccine protects mice against GAS challenge and does not induce deleterious reaction. However, one limitation is that we could not get human tissue to test whether the antibody is cross reactive to human tissue, although cross reaction is of little possibility, because we had excluded ve or more contiguous amino acids matching with human proteins when we designed the amino acid sequence of this divalent peptide vaccine.
We selected amino acid fragments from M protein of emm1 and emm12 GAS because these 2 serotypes are predominant strains in China according to several epidemiological surveys [11][12][13][14]. So this vaccine may prevent most streptococcal infection in our country. We note that there is a pursuit for a GAS vaccine e ective in di erent regions of the world, but because of great variability of emm types in different regions and little cross reaction among heterogeneous serotypes, such a universally valid vaccine is unlikely to achieve. An alternative strategy is region-speci c vaccine. As we know, N terminal of M protein has the highest immunogenicity. e dominant emm types of GAS in a given region can be determined through epidemiological investigation, and then safe and opsonic epitopes on N terminal of M protein of CFU of one plate more than 1000 were counted as 1000. Percent reduction in CFU � (1 − (mean CFU from immunized sera/mean CFU from control sera)) × 100. Indirect bactericidal assays were performed by adding 50 μL of M1 or M12 GAS to 50 μL of immunized sera or control sera. en, 400 of μL nonopsonic heparinized human donor blood was added. e mixture was kept shaking for 3 h at room temperature, and then 50 μL of mixture was plated on blood agar. CFU was quantitated after incubating overnight.  these dominant strains can be selected to compose a multivalent vaccine. Such a chemically synthesized multivalent vaccine is able to cover all the dominant strains and can be put into use in a short period of time. In underdeveloped areas such as Africa and the Paci c region where the existing GAS vaccines like the 26-valent vaccine, 30-valent vaccine, and StreptInCor may have a low coverage; a geographical regionspeci c multivalent vaccine is promising. (a) M1 GAS exposed to antisera of immunized group mice. (b) Brighteld view of (a). (c) M12 GAS exposed to antisera of immunized group mice. (d) Bright-eld view of (c). (e) M1 GAS exposed to antisera of control group mice. (f) Bright-eld view of (e). (g) M12 GAS exposed to antisera of control group mice. (h) Bright-eld view of (g).  Figure 4: Protection of mice against group A streptococcus challenge after immunization with the vaccine. (a) Survival curves of mice challenged with M1 GAS. Immunized group mice (n � 8, black circles) and control group mice (n � 8, black squares) were intraperitoneally injected of 1.0 × 10 8 CFU M1 GAS 21 days after the last immunization. Immunized group mice had a higher survival rate than nonimmunized group mice (87.5% versus 37.5%, P � 0.0294). (b) Survival curves of mice challenged with M12 GAS. Immunized group mice (n � 8, black circles) and control group mice (n � 8, black squares) were intraperitoneally injected of 1.0 × 10 8 CFU M12 GAS 21 days after the last immunization. Immunized group mice had a higher survival rate than nonimmunized group mice (75% versus 25%, P � 0.0366). Statistical signi cance was determined by the log-rank test.

Conclusion
In summary, we have shown that this chemically synthesized divalent vaccine is immunogenic and safe in mice, and we believe that this strategy, developing a geographical regionspeci c multivalent vaccine, is worth extending to other areas.

Ethical Approval
All procedures performed in studies involving animals were in accordance with the ethical standards of Animal Ethical and Welfare Committee of Sun Yat-sen University at which the studies were conducted.

Consent
Written informed consent was obtained from each healthy donor to use blood samples.

Conflicts of Interest
e authors declare that they have no con icts of interest.