Potential Protectivity of a Conjugated COVID-19 Vaccine against Tetanus

PastoCovac is a subunit protein vaccine against COVID-19 which contains the tetanus toxoid as a carrier conjugated to SARS-CoV-2 RBD. The primary goal of the tetanus application was to elicit a stronger specific response in the individuals. However, conjugate vaccines have the potency to generate anticarrier antibodies in addition to the target antigen. Therefore, the present study aimed to evaluate the PastoCovac vaccine in the humoral immune induction against tetanus. Six groups of individuals, including those who received one, two, or three doses of the PastoCovac vaccine, Td vaccine, and also the controls who received other COVID-19 vaccines (except PastoCovac), were investigated. The anti-tetanus IgG was assessed by an ELISA assay in all vaccinated groups. The antibody persistency against tetanus in the group who received one dose of the PastoCovac vaccine was also assessed on day 60, 90, and 180 after the last injection. The anti-tetanus antibody titer in the three groups of PastoCovac recipients was positive, though additional doses of the vaccine led to a significant antibody rise (p = 0.003). Notably, the comparison of the mean antibody titer between the Td recipients and those who received one/two doses of PastoCovac showed that the mean rise in the antibody titer before and after the injection was not significant. Although the antibody titer on day 180 decreased to a lower level than on day 21, it was still estimated to be highly positive against tetanus. Eventually, none of the PastoCovac recipients presented vaccine side-effects during the follow-up. The current data indicate that the tetanus conjugate vaccine against COVID-19, PastoCovac, could induce immune responses against tetanus, which can persist for at least 6 months. Combination vaccine formulae containing TT and DT as carriers for conjugate vaccines could be considered instead of TT and/or DT boosters in adults if they are indicated.


Introduction
SARS-CoV-2 caused the most recent pandemic, COVID-19, which led to the rapid design of vaccines and clinical trials in order to inhibit the global spread of the virus worldwide.Generally, vaccines could be categorized into five main platforms, including live-attenuated or inactivated viruses, nucleic acids (RNA or DNA), recombinant protein, viral vectors, and virus-like particles (VLPs) [1,2].Vaccines are ideally supposed to induce immune responses through both humoral and cellular arms.Therefore, different Vaccines 2024, 12, 243 2 of 10 approaches, including adjuvants, nanoparticles, targeting antigen-presenting cells, heterologous prime-boost regimens, and antigen carriers, have been explored to date against different pathogens [3,4].
Carrier proteins are applied to enhance the immunogenicity of the formulated antigens in vaccine formulae.The approved carrier proteins for human use include tetanus toxoid (TT), diphtheria toxoid (DT), Haemophilus protein D (PD), CRM197 (a nontoxic mutant of diphtheria toxin), and the outer membrane protein complex of serogroup B meningococcus (OMPC) [5][6][7].TT, as a strong carrier protein, is composed of several T-cell peptides.These universal T-cell epitopes could possibly increase the effectiveness of the carrier [8].
PastoCovac was manufactured and administered in Iran after a successful technology transfer from Cuba, and showed significant efficiency in vaccine studies [9].This vaccine is a protein subunit one, composed of conjugated RBD to the tetanus toxoid.The conjugation of RBD to TT leads to the effective induction of both humoral and cellular immune responses, owing to the fact that multiple receptor-binding motifs (RBMs) expose where the neutralizing epitopes predominate [10,11].PastoCovac was primarily administered against COVID-19 as the main-dose vaccine.PastoCovac has been used as the primary series, and also as the booster dose, after inactivated vaccines and adenovirus-based vaccines, since its safety profile was proven in the clinical trial in Iran [9].
It is clear that the carrier proteins as part of the vaccine are immunogenic and elicit a specific anti-carrier response which is measurable by in vitro assays [12].Here, we aimed to assess the ability of the PastoCovac vaccine to induce antibodies against tetanus in immunized individuals.Moreover, the durability of the humoral immune responses against tetanus was investigated 6 months after the last dose of the PastoCovac vaccine, as well as the vaccine safety.

Study Population
This study was conducted at the Pasteur Institute of Iran.All the participants were provided with the written consent form prior to participation.The study protocol was performed according to the Declaration of Helsinki (Fortaleza, 13 October 2013) and was approved by the Ethics Committee of the Pasteur Institute of Iran (ethics code number: IR.PII.REC.1402.020).
The participants were divided into 6 case groups (Figure 1) based on the number of PastoCovac (PCovac)/tetanus vaccine doses.

Vaccines
An amount of 0.5 mL of the Td vaccine [2 Lf diphtheria toxoid, 8.8 Lf tetanus toxoid, 1.5 mg aluminum phosphate adsorbed, reduced antigen(s) content, reduced thimerosal, Biological E. Limited, India] was injected intramuscularly in the deltoid.

Immunogenicity Assessment
Antitetanus IgG was assessed by an ELISA assay in all vaccinated groups (Tetanus Elisa IgG kit Vircell G1008, Granada, Spain) and an antibody index > 11 was considered positive against tetanus (OD > 1.2).The antibody persistency against tetanus in the group who received one dose of the PastoCovac vaccine was also assessed on day 60, 90, and 180 after the last injection.

Vaccine Safety
All the case participants were monitored after vaccination by Td or PastoCovac 30 min post-injection.Furthermore, any kind of adverse event was recorded in the questionnaire through the interviews via phone call.

Statistical Analysis
Shapiro-Wilk's W test was utilized to assess the normality of the numerical data.Descriptive statistics (mean and standard deviation (±SD), median and interquartile range (±IQR), minimum and maximum, or frequency and percentages) were used to represent the data.The one-way ANOVA, t-test, and Mann-Whitney U-test were used to compare the means between the groups, where appropriate.The repeated-measures ANOVA was used to assess the mean of the antitetanus Ab changes over time.Statistical analyses were

Vaccines
An amount of 0.5 mL of the Td vaccine [2 Lf diphtheria toxoid, 8.8 Lf tetanus toxoid, 1.5 mg aluminum phosphate adsorbed, reduced antigen(s) content, reduced thimerosal, Biological E. Limited, India] was injected intramuscularly in the deltoid.

Immunogenicity Assessment
Antitetanus IgG was assessed by an ELISA assay in all vaccinated groups (Tetanus Elisa IgG kit Vircell G1008, Granada, Spain) and an antibody index > 11 was considered positive against tetanus (OD > 1.2).The antibody persistency against tetanus in the group who received one dose of the PastoCovac vaccine was also assessed on day 60, 90, and 180 after the last injection.

Vaccine Safety
All the case participants were monitored after vaccination by Td or PastoCovac 30 min post-injection.Furthermore, any kind of adverse event was recorded in the questionnaire through the interviews via phone call.

Statistical Analysis
Shapiro-Wilk's W test was utilized to assess the normality of the numerical data.Descriptive statistics (mean and standard deviation (±SD), median and interquartile range (±IQR), minimum and maximum, or frequency and percentages) were used to represent the data.The one-way ANOVA, t-test, and Mann-Whitney U-test were used to compare the means between the groups, where appropriate.The repeated-measures ANOVA was used to assess the mean of the anti-tetanus Ab changes over time.Statistical analyses were performed with GraphPad Prism 8.0 for Windows (GraphPad Software, San Diego, CA, USA) and SPSS (v 18.0; SPSS Inc., Chicago, IL, USA) software.

Results
The present study included 106 subjects (60 females, 46 males) with the mean age of 43.2 ± 13.4 years old.The number of participants in each defined group was as follows: PCovac-1D: n = 10; PCovac-2D: n = 15; PCovac-3D: n = 19; CT: n = 10; TC: n = 11; Tetanus group: n = 15; 21 individuals as controls.There was no significant difference between the groups regarding demographic characteristics.An anti-tetanus antibody index > 11 (OD > 1.2) was considered as positive according to the semi-quantitative ELISA test.
Three groups of the individuals who received one, two, or three doses of the PastoCovac vaccine were compared.The mean anti-tetanus antibody rise was significantly different between these groups (p = 0.004).The additional statistical test showed that this difference between the PCovac-1D and PCovac-3D groups was significant (p = 0.003).The mean rise in the antibody titer in the PCovac-3D group was 36.8 units more than the PCovac-1D group.In other words, the average increase in the antibody titer in the PCovac-3D group was twice that of the PCovac-1D group.The assessment of the anti-tetanus antibody in each group is presented in Tables 1 and 2. The comparison of the mean antibody titer between the Tetanus and Pcovac-1D groups showed that the mean rise in the antibody titer during the first (p = 0.326) and second (p = 0.728) measurements was not significant (before and after the injection, respectively).
Furthermore, comparing the mean antibody titer between the Tetanus and Pcovac-2D groups showed that the difference between the mean titer rise of the two groups was not statistically significant (p = 0.128).Nevertheless, the comparison of the Tetanus group and PCovac-3D cases showed that the mean of the antibody titer between two groups was significantly different (p < 0.001).On average, the antibody titer rise in the PCovac-3D group was about 35 units more than the Tetanus group.That is to say, the mean titer rise in the PCovac-3D group was about 1.67 times that of the Tetanus group.The comparison of the antibody mean titer between the Tetanus and CT groups showed that the antibody rise in the CT group was about 1.3 times higher than the Tetanus group, although the difference was not statistically significant (p = 0.1).
Comparing the Tetanus and TC groups indicated a significantly higher rate of antitetanus Ab rise in the TC (p = 0.004).Therefore, the average rise in the antibody titer in people who received the PastoCovac vaccine after the Td vaccine was 36.8 units higher than in the people who received the Td vaccine.In other words, the average increase in the antibody titer in the TC group is nearly1.7 times more than the Tetanus group.
Furthermore, we merged the TC and CT results to compare the anti-tetanus Ab mean titer with the Td recipients.The results indicated a statistically significant difference (p = 0.006), in which the mean antibody titer rise in people who received the PastoCovac and Td vaccines was 26.8 units more than the Td group.That is to say, the ratio of the mean of the antibody titer rise in the CT + TC groups is almost 1.5 times of the Tetanus group.
Of the PCovac-1D group, one case was Ab-negative (Ab index: 4) before vaccination, in whom the PastoCovac injection led to a high antibody titer (Ab index: 74.4).Furthermore, there was a case who remained anti-tetanus Ab-negative from the PCovac-2D group.In the Control group who had a history of Td vaccination of more than 10 years, seven (33%) individuals were anti-tetanus-negative.
In total, a high titer of anti-tetanus was achieved in the PastoCovac recipients, which was comparable with the Td recipients.The best anti-tetanus induction was recorded in those that had a history of a Td vaccine and then received the PastoCovac injection.Furthermore, the individuals who received the PastoCovac vaccine as the primary series, and also as the booster one, reached a higher mean of the Ab than Td-vaccinated cases.

Antitetanus Persistency among PastoCovac Recipients
In order to explore the persistency of the anti-tetanus antibody induced by the Pas-toCovac vaccine, sera samples of PCovac-1D cases were collected before vaccination and 21, 90, and 180 days post-vaccination.According to Figure 2, a significant antibody titer was detectable from 21 to 180 days compared to the pre-vaccination time.Furthermore, the anti-tetanus Ab level on day 180 was still significantly high compared to day 21.Although the antibody titer on day 180 decreased to a lower level than on day 21, it is still estimated to be effective in the prevention from tetanus.Of the PCovac-1D group, one case was Ab-negative (Ab index: 4) before vaccin in whom the PastoCovac injection led to a high antibody titer (Ab index: Furthermore, there was a case who remained antitetanus Ab-negative from the PC 2D group.In the Control group who had a history of Td vaccination of more than 10 seven (33%) individuals were antitetanus-negative.

Tetanus
In total, a high titer of antitetanus was achieved in the PastoCovac recipients, was comparable with the Td recipients.The best antitetanus induction was record those that had a history of a Td vaccine and then received the PastoCovac inje Furthermore, the individuals who received the PastoCovac vaccine as the primary and also as the booster one, reached a higher mean of the Ab than Td-vaccinated ca

Antitetanus Persistency among PastoCovac Recipients
In order to explore the persistency of the antitetanus antibody induced b PastoCovac vaccine, sera samples of PCovac-1D cases were collected before vacci and 21, 90, and 180 days post-vaccination.According to Figure 2, a significant an titer was detectable from 21 to 180 days compared to the pre-vaccination Furthermore, the antitetanus Ab level on day 180 was still significantly high compa day 21.Although the antibody titer on day 180 decreased to a lower level than on d it is still estimated to be effective in the prevention from tetanus.

Assessment of Adverse Events (AEs)
According to the safety monitoring results, the only case who presented some sort of AE was among the Tetanus group, with a local pain at the injection site, headache, fever, and shivering after the Td vaccination.Nevertheless, there were no complaints after PastoCovac administration of any doses or the combination of the Td and PastoCovac vaccines (CT/TC).

Discussion
Tetanus vaccination has a primary-series schedule in childhood and the second decade of life (16 to 18 years old).Then, individuals should preferably receive the booster doses every 10 years.On the contrary, a large investigation showed that booster vaccines against tetanus or diphtheria are not necessary if adults have completed the profile of childhood vaccination series.They reviewed WHO data from 31 countries for 15 years, which included 11 billion person-years.No significant difference in infection rates was found between the countries which recommend adults to obtain booster shots and those that do not.Therefore, it was suggested that childhood vaccination alone could protect sufficiently against tetanus and diphtheria [13,14].However, the question of having ongoing booster shots is really more complicated than only considering the frequency of a disease [13,15].Although there has been a lack of change in tetanus incidence, other factors could strongly affect the number of cases, including wound management, the amount of bacteria in the environment, and hygiene measures.Even though the rate of infection with tetanus might be rare, people still experience serious or deadly effects.Owing to the fact that there is no specific treatment for tetanus, and no proof of having lifelong immunity with childhood vaccinations, the CDC still recommends booster vaccines every 10 years to help the immune system against the infection.The vaccination against tetanus through Td or Tdap protect > 95% of people for approximately 10 years according to CDC guidelines [13].Not only do the booster vaccines against tetanus raise questions, coupled vaccines to the tetanus toxoid also bring concerns of immune response interference.
Recombinant subunit vaccine platforms provide us with some advantages, such as the safety, stability, the targeting the immune responses to the antigen of interest, and the ease of production [3,4].PastoCovac has been the only COVID-19 subunit vaccine which is conjugated to the tetanus toxoid.There are some benefits of the vaccination with the coupled viral antigen to the tetanus toxoid, including the predominant IgG immune response caused by the affinity maturation and durable specific B-memory cells.The orientation of the RBD which is conjugated to the tetanus toxoid leads to a greater exposure to the receptor-binding motif and the increase in the level of neutralizing antibodies [16][17][18].
The impact of tetanus-conjugated vaccines on immune responses against tetanus as the carrier protein has remained unclear.Although the immunogenicity and effectiveness of conjugate vaccines regarding the target antigens have been widely investigated, their possible protection ability against tetanus has been overshadowed and less discussed.
Since the COVID-19 pandemic, different vaccine platforms have been designed to inhibit SARS-CoV-2 spread, among which PastoCovac has been the only tetanus-conjugated vaccine.This vaccine has been approved to administer in Iran as the primary and booster doses, and so there are immunized populations among whom some individuals received one, two, or three doses.This means the immune system has not only been induced against SARS-CoV-2 RBD, but also the tetanus toxoid.A similar status also applies to conjugate pneumococcal vaccines worldwide.In the present research, the PastoCovac vaccine was assessed to evaluate the anti-tetanus antibody induction and the comparability of the induced immune response with the Td vaccine.Furthermore, the safety of the Td and PastoCovac vaccines regarding the priority of injection and number of doses was considered.
It has been stated that he presence of previous immunity to a carrier protein could possibly suppress the immune induction to the conjugate, owing to carrier-induced epitopic suppression (CIES) [19,20].One of the clinical studies regarding this issue was on the meningococcal serogroup C conjugate (MCC) vaccine, which contains TT.The results showed that protection against MenC in all three groups of children who received the DT/TT and then the MCC-TT, the co-administration of the DT/TT and MC-TT, and the DT/TT after the MC-TT was indicative, though the highest Ab rise was achieved in the first group.Bystander interference has been also proposed as a possible cause of the immune response reduction when two antigens are co-administered, which is defined as carrier-induced epitopic suppression [21].The study conducted in Australia highlighted that immunization with the Tdap (tetanus, diphtheria, and pertussis) vaccine three to four weeks prior to the co-administration of the Pneumococcal conjugate vaccine (PCV13)/Meningococcal vaccine (MCV4) in adults significantly suppressed the subsequent antibody rise to some of PCV13's serotypes.They concluded that carrier proteins may induce specific anti-carrier antibody responses which interfere with immune responses to the conjugate vaccine.They found it reasonable to assume that the Tdap antigens might negatively interact with some CRM197-conjugated PCV13 serotypes as a consequence of the similarity between the CRM197 carrier protein and the DT in the Tdap vaccine [22].
The antibody affinity, T-cell responses, and B-cell activation have been previously investigated, and the highly immunogenic potency of the RBD_TT/Alum formula in comparison with RBD alone was proven.In fact, multiple T-cell and B-cell epitopes of tetanus as the carrier induced better cellular immunity, as well as multimeric RBD-TT, which activated several B-cell receptors [18].Further studies also confirmed the strong immune induction in immunized individuals with PastoCovac [23,24].In other words, the administration of PastoCovac could simultaneously induce immune responses against SARS-CoV-2 and tetanus with no detectable interference.Furthermore, the administration of the Td vaccine prior to PastoCovac resulted in the highest anti-tetanus Ab rise in our study.
In an investigation in Korea, the comparison of the anti-tetanus antibody between two groups of individuals, including the Td vaccine + 13-valent pneumococcal conjugate vaccine (PCV13) and the Td vaccine alone, was performed.The results showed that Td injection generated significantly higher anti-tetanus antibodies than the Td + PCV13 co-administration [25].There were two groups of individuals in the present study who received the Td vaccine before the PastoCovac vaccine, or after that.However, the two vaccines were not co-administered, and the results did not indicate any negative interaction between the two vaccines.Furthermore, the prior injection of the Td vaccine led to a greater anti-tetanus Ab induction compared to those who received PastoCovac first.
In the summaries of the product characteristics (SmPCs) and the leaflets of the conjugate vaccines, it is clearly stated that the use of these vaccines should not be considered instead of the Td vaccine [12].This warning might be due to the lack of studies for the evaluation of specific immune response to the applied carrier protein.The in vivo experiment in mice or guinea pigs to show the capability of the carrier protein immune responses showed that the conjugate carriers TT and DT protected the immunized animals against a lethal challenge by the toxins.They also observed that a total amount of 10.5 µg of TT resulted in full protection after one injection of the conjugated vaccine against the tetanus lethal challenge.Interestingly, the reduced amount of TT, at 2.1 µg, also provided the same result.Moreover, the immunized animals with one dose of a conjugated vaccine containing 5 µg DT as the carrier resulted in 100% protection against the lethal challenge [12].
The PastoCovac vaccine contains 20 µg of TT as the carrier protein, which seems to present in a highly induced level, according to the serology assay, in the present data.
The vaccine data safety during the follow-up only indicated some AEs in a case who received the Td vaccine.According to the vaccine recommendations and guidelines of the Advisory Committee on Immunization Practices (ACIP), CDC, simultaneous or sequential vaccination is recommended when both are indicated.Our follow-up results clearly proved that the PastoCovac injection of any doses and the sequential administration of the PastoCovac and Td vaccines was safe, with no AEs.

Figure 1 .
Figure 1.Study design.The recipients were divided into 6 groups according to the vaccine doses and the priority of the administered TD/PastoCovac vaccines.The other COVID-19 vaccine recipients with a history of a previous Td vaccine were considered as controls.

Figure 1 .
Figure 1.Study design.The recipients were divided into 6 groups according to the vaccine doses and the priority of the administered TD/PastoCovac vaccines.The other COVID-19 vaccine recipients with a history of a previous Td vaccine were considered as controls.

Figure 2 .
Figure 2. Antitetanus antibody persistency.The durability of the induced antitetanus Ab, whi evaluated by the comparison of the mean titer on day 90 and 180 with the day 21 level.Al the mean of the Ab titer declined over time, at 180 days post-injection, it was still at a high le

Figure 2 .
Figure 2. Antitetanus antibody persistency.The durability of the induced anti-tetanus Ab, which was evaluated by the comparison of the mean titer on day 90 and 180 with the day 21 level.Although the mean of the Ab titer declined over time, at 180 days post-injection, it was still at a high level.

Table 1 .
Classification of the individuals to assess the PastoCovac immunogenicity against tetanus.

Table 2 .
Comparison of the anti-tetanus Ab induction between the vaccinated groups.
* Significant values are shown in bold.